Synthesis, structure-activity relationships and preliminary mechanism study of N-benzylideneaniline derivatives as potential TLR2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.03.001

Toll-like receptor 2 (TLR2) can recognize pathogen-associated molecular patterns to defense against invading organisms and has been represents an attractive therapeutic target. Until today, none TLR2 small molecule antagonist have been developed in clinic

Full text of DOI:10.1016/j.bmc.2018.03.001

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure-activity relationships and preliminary mechanism
study of N-benzylideneaniline derivatives as potential TLR2 inhibitors
Shaoyi Cai a,c, Gengzheng Zhu a,c, Xiaohong Cen a,c, Jingjie Bi a, Jingru Zhang a, Xiaoshan Tang a, Kun Chen b,
,
⇑
Kui Cheng a,
⇑
a Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School
of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
b The Joint Research Center of Guangzhou University and Keele Univeristy for Gene Interference and Application, School of Life Science, Guangzhou University, Guangzhou 510006,
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Toll-like receptor 2 (TLR2) can recognize pathogen-associated molecular patterns to defense against
invading organisms and has been represents an attractive therapeutic target. Until today, none TLR2
small molecule antagonist have been developed in clinical trial. Herein, we designed and synthesized
50 N-benzylideneaniline compounds with the help of CADD. And subsequent in vitro studies leading to
Received 2 January 2018
Revised 28 February 2018
Accepted 1 March 2018
Available online xxxx
the optimized compound SMU-A0B13 with most potent inhibitory activity to TLR2 (IC50=18.21 0.87
Preliminary mechanism studies indicated that this TLR2 inhibitor can work through the NF- B signaling
pathway with high specificity and low toxicity, and can also efficiently downregulate inflammatory
cytokines, such as SEAP, TNF- and NO in HEK-Blue hTLR2, human PBMC and Raw 264.7 cell lines.
lM).
j
Keywords:
Toll-like receptor 2
Small molecule antagonist
Schiff base derivatives
Inflammatory cytokines
a
Additionally, the docking situation also indicate SMU-A0B13 can well bind to the TLR2-TIR (PDB:
1FYW) active domain, which probably explains the bioactivity.
Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction
pathogen-associated molecular pattern (PAMP) ligands and diverse
cellular positions,16 among which TLR2 is one of the most thor-
Toll-like receptors (TLRs), an essential pattern recognition
receptors, are widely expressed in various immune cells of multi-
farious tissues and organs, regulating innate and acquired immu-
nity response through myeloid differentiation primary response
88 (MyD88) dependent pathway or MyD88 independent path-
way1,2 by up-regulating the production of cytokines and costimu-
latory molecules on the antigen-presenting cell (APC) to ensure
the organism releases and maintains a suitable level of proinflam-
matory cytokine.3–5 Studies have found that TLRs play an impor-
tant role in the development of autoimmune diseases, tumors
and other intractable disorders.6–13 The activation of TLRs leads a
train of signal transduction to defense pathogen.14 However, over-
express of TLR will induce too much cytokines leading to inflam-
mation.15 So the activation of TLR is a double-edged sword and
both positive and negative regulation can be used as a target for
clinical drug development. Till now, there have been 11 TLRs iden-
tified in mammals and categorized depending on their respective
oughly studied receptors.
TLR2 forms heterodimers with TLR1 or TLR6, involving the
recognition of PAMPs from various pathogens bacteria, fungi and
other hosts,16 including substance likes Lipoprotein/lipopeptides,
Peptidoglycan, Lipoteichoic acid, Glycoinositol, Phospholipids and
so on.4 Currently, several immunomodulators of TLR2 have been
developed as follows: Pam2CSK4, Pam3CSK4, CU-T12-9, BLP,
MALP-2, SMP-105 as agonists, and OPN-305, CU-CPT22, E567,
SSL-3 as antagonists and the others are in studying.17–27 However,
none of the above compounds have been put into clinical practice.
Thus, a novel TLR2-selective antagonist with high inhibition activ-
ity as well as low toxicity still needs to be developed emergently.
2. Design rationale
As we know that as a family of highly conservative type I trans-
membrane receptors, TLRs include three constituent portions:
leucine-rich repeats’ (LRRs) extracellular domain that involves
directly the recognition of pathogen-associated molecular patterns;
transmembrane domain and intracellular Toll-interleukin 1 (IL-1)
receptor (TIR) domains, which are pivotal to signaling.4 Targeting
⇑
Corresponding authors.
E-mail addresses: Kchennju@hotmail.com (K. Chen),
chengk@smu.edu.cn
(K. Cheng).
c
These three authors equally contributed to this paper.
https://doi.org/10.1016/j.bmc.2018.03.001
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cai S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.001

2
S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
at the BB-loop region of TIR domains, Mistry et al.28 employed the
computer-aided drug design (CADD) technology to screen the
FDA for approved 1 million small molecules library and finally
found a potential TLR2 inhibition (C29). Further evaluation found
that it can specifically block the interaction of TLR2 and MyD88 pro-
tein, thereby inhibiting both the mouse and human TLR2/1 and
TLR2/6 signaling pathways. Compound C29 could consistently
decreased Pam3CSK4 or Pam2CSK4-induced IL-8 mRNA by more
than 50 percent, as well as mentioning its substrate, o-vanillin
works. Therefore, we were wondering whether we can study the
structure-activity relationship (SAR) of C29 and expected to find
the optimized structure. So, we described a series of C29 derivatives
which contained aromatic aldehydes (twelve A moiety substrates)
and aromatic primary amine (eleven B moiety substrates) (Fig. 1)
guided by the computer simulation and theory of bioisosterism.
From the docking results of C29 with above-mentioned BB-loop
of TLR2 TIR domain (pdb: 1FYW) (Fig. S1), we got the hypothesis
that the 2-hydroxyl on the aromatic aldehydes might be a potent
binding site as there were small molecule-protein interactions
observed. Meanwhile, we also found that a large gap between B
moiety and the protein were existed, which probably indicated
some large or rigid groups were tolerant in this region (Fig. S2). In
addition, the polarity, steric effect, fat-solubility and electronega-
tivity of the compounds were taken into account in designing the
TLR2 inhibitors (Table. S1). Further, the derivatives of Schiff base
have various pharmacological actions such as anti-inflammatory,
antiviral, antibacterial and antifungal properties.29 The synthetic
routes for the novel TLR2 inhibitors were outlined in Fig. 1.
duced several different aromatic or aliphatic groups and their
SEAP signaling results were illustrated in Table 1. Regrettably,
except 2, 9 (with –OH at R6 position), all of these derivatives
showed negative inhibitory activity in HEK-blue hTLR2 cells, indi-
cating the importance of the N-benzylideneaniline core structure.
Thus, the following derivatives were developed base on this core
and their bioactivity results were given in Table 2 and Fig. S3.
To confirm our hypothesis about –OH function in R1 position,
compound 21–28 were designed by either removing or methylat-
ing the hydroxy group. The experiment results indicated that
removing the –OH decreased the potency significantly, which illus-
trated that it was critical to the activity, which also consisted with
the docking result that the hydroxyl in R1 position formed two vis-
ible hydrogen bonds with TIR domain (Fig. S1). In addition, from
negligible inhibition of 21–27, we can infer that changing the R3
position’s electronic density has no significant impact on inhibitory
activity. Compared with 29 and 30, we found that the inhibitory
activity had
a slight decrease when changing the methoxy
(29/C29) to ethoxy group (30) at R2 position. It required our
attention that addition –OH in R6 may play an important role in
inhibitory effect. As shown in compounds 2, 9, 14, 31, 32, 33 and
34, which have the same B moiety, this series of compounds
tended to exhibit strong inhibitory activity and it may because of
the interaction between this –OH and other important residues
of TLR2-TIR domain.
As mentioned previously, large gap between B moiety and the
protein existed, so we introduced some big substitutions in this
part. The acetophenone substitution in R4 position (35/SMU-
A0B13, 36), and the phenyl substitution at R6 position (40, 46)
can significantly improve the activity. These results indicated that
the big/rigid substitutions on B part were tolerant and helpful for
the bioactivity. In 35 (SMU-A0B13), the inhibitory effect was
3. Results and discussion
These potential TLR2 antagonists were synthesized by conden-
sation of 1:1 M ratio of aromatic aldehydes and aromatic primary
amine in anhydrous methanol at 60 °C (Fig. 1B). Finally 50 potential
TLR2 inhibitors were prepared in 80–90% yield. All targeted com-
pounds were test for their TLR2 signaling pathway inhibition by a
95.06% at 50
(51.77% inhibition at 50
l
M, a remarkable increase compared with C29
M).
l
According to previous studies of Mistry,28 the BB loop P681H
mutation in hTLR2 plays an important role in recruiting down-
stream signaling. Interestingly, the docking result of 36 shows that
its lowest energy conformation bears a striking. resemblance to
SMU-A0B13, the distinction is simply that the chlorine atoms of
SMU-A0B13 happens to cross over I680 and are closer to P681,
so it can lead to some interaction between halogen and residues
of protein, preventing P681 from triggering downstream signaling,
which can explain why SMU-A0B13’s inhibitory activity is much
better than that of 36 (Fig. 2). Further dose-dependent assay results
secreted embryonic alkaline phosphatase (SEAP) NF-jB reporter
gene in HEK-Blue hTLR2 cells. To monitor the activation of NF-
jB,
the TLR2 agonist Pam3CSK4 was used as the positive control.
3.1. Structure-activity relationship study
Initially, we wondered whether the N-benzylideneaniline scaf-
fold was essential for inhibitory hTLR2 bioactivity. So we intro-
indicated that the IC50 of SMU-A0B13 was 18.21 0.87 lM
(Fig. 3A) and this inhibition effect was better than C29 and the neg-
ative control 15. What’s more, to verify the biological activity after
C@N been reduction, we synthesized compound HA0B13. The test
results showed that although there has been a substantial activity
decline after reduction, but HA0B13 still maintain a certain effect
(Fig. 3B).
After the SAR, we got an optimal compound SMU-A0B13, and
then we evaluated its toxicity, downstream signal inhibition and
effect on the target protein TLR2.
3.2. In vitro cytotoxicity studies
At the same time, the toxicity of these compounds were evalu-
ated by a MTT colorimetric method. From the MTT data (Fig. 4) we
can confirm that SMU-A0B13 had no cytotoxicity at concentration
up to 100 lM. Considering compound SMU-A0B13 having the low-
est IC50, high inhibitory activity (95.06%), low toxicity, as well as
appropriate molecular weight, it might own the higher drug-like
potential ability. Thus, compound SMU-A0B13 was picked out to
evaluate its selectivity and other biophysical potential.
Fig. 1. General idea for the design (A) and synthetic route of the N-benzylide-
neaniline derivatives as TLR2 inhibitors (B).
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S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Table 1
Derivative 1–20 inhibition of SEAP signaling in HEK-blue hTLR2 cells.
Comd
Generic structure
R4
R5
R6
R7
Inhibition (%)a
25
1.74
27.54
3.99
5.63
l
M
50 lM
1
2
3
4
–CH3
–H
–H
–COOH
–H
–H
–H
–OH
–OCH3
–C6H5
–H
–H
–H
–H
14.45
95.78
15.06
3.10
–H
–H
5
6
7
8
–CH3
–H
–COOH
–H
–COOH
–H
–H
–H
–OCH3
–H
–H
–H
–CF3
–H
2.39
2.11
0.33
2.35
2.58
0
5.77
3.80
–H
–C6H5
9
10
11
–H
–H
–H
–H
–H
–H
–OH
–OCH3
–C6H5
–H
–H
–H
44.01
5.21
0
98.76
1.97
0
12
13
–CH3
–COOH
–H
–H
–H
–H
–H
0
3.09
0
22.88
14
15
16
17
18
19
20
–H
–H
–COOH
–H
–COOH
–H
–H
–OH
–H
–H
–OCH3
–H
–C6H5
–H
–H
–H
–H
–H
–CF3
–H
–H
0
0
0
3.66
2.53
5.35
2.72
0
0
0
0
0
1.41
0
–COOH
–CH3
–H
–H
–H
–H
a
Each compound was tested in triplicate.
3.3. Specificity studies
TNF-
TNF-
a
a
signaling, with 10
signaling (Fig. 5D).
lM SMU-A0B13 almost inhibiting 90%
As mentioned above that TLR family can recognize a large range
of ligands, so we performed a selectivity examination to investi-
gate whether SMU-A0B13 could specifically inhibit TLR2 signaling.
The candidates included TLR1/2, TLR2/6, and TLR3, and their native
ligands were Pam3CSK4, Pam2CSK4 and Poly I:C, respectively. A
4. Conclusions
In conclusion, based on the previous research and guided by the
CADD technology, a series of Schiff base derivatives have been
designed and synthesized. Then, we made sense of the SAR discus-
sion of these 50 compounds in human HEK-Blue TLR2 cells. Com-
dose of 25 lM SMU-A0B13 was added to the HEK series cells and
then tested the stimulation of SEAP signaling. As shown in
Fig. 5A, both TLR1/2 and TLR2/6 were decreased, which had no
influence on TLR3, which suggested that SMU-A0B13 is a certain
specificity antagonist of TLR2.
pared with lead compound C29/29, we developed
a new
compound SMU-A0B13 with the most activated potential. And
subsequent experiments greatly confirmed that SMU-A0B13 had
low toxicity and specific selectivity in TLR2. This can be further
observed in the protein immunoblot assay which indicated that
SMU-A0B13 could efficiently downregulate the TLR2 protein.
Moreover, SMU-A0B13 can both work in murine Raw 264.7 macro-
phage cells and human PBMC cells to inhibit the inflammatory
3.4. Downstream signaling evaluation
Inducible nitric oxide synthase (iNOS) is responsible for the pro-
duction of NO which can blocks phosphorylation and subsequent
activation of IL-2 receptor-associated proteins, playing a major role
in mediating immunosuppressive effects. TLR2-related cellular
cytokines, including NO and TNF-a, respectively. In a manner of
speaking, our study has provided a kind of potential novel small
molecular, drug-like antagonist targeted at TLR2, which might be
further explored involving some diseases associated with inflam-
mation or tumor.
iNOS test in Raw 264.7 macrophage cells showed 40 lM SMU-
A0B13 decreased more than 70% inhibition than positive control
(Fig. 5B). With the dose increasing, we observed SMU-A0B13 can
suppress Pam3CSK4-induced SEAP signaling as well as Pam2CSK4-
induced one in HEK-Blue hTLR2 cells, which indicate SMU-A0B13
through inhibit TLR2 to downregulate the TLR1/2 and TLR2/6 sig-
naling pathway (Fig. 5B). This result also was observed in the
5. Experimental section
Western blot experiment, which showed that 10
lM SMU-A0B13
5.1. Chemistry
suppressed the expression of TLR2 protein by about 50% with
anti-hTLR2 as the primary antibody (Fig. 5C). Meanwhile, Elisa data
in human peripheral blood mononuclear cells (PBMC) showed that
SMU-A0B13 can dose dependently decrease the Pam3CSK4-triggered
5.1.1. Materials and methods
All reagents were obtained from commercial sources and used
without further purification. Reactions were followed by analytical
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4
S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 2
Derivative 21–50 inhibition of SEAP signaling in HEK-blue hTLR2 cells.
Compds
21
Structure
Inhibition (%)a
Compds
36
structure
Inhibition (%)a
25 50 lM
25
0
lM
50
0
lM
lM
1.86
30.03
22
23
0.99
0
1.61
0
37
38
0
0
0
23.66
24
0
0
39
3.77
45.64
25
26
2.39
7.27
0
40
41
23.31
35.53
75.30
70.71
0.28
27
5.07
0
0.75
0
42
6.93
19.40
28
29
43
44
2.39
8.33
27.75
35.24
14.90
51.77
30
31
5.43
38.00
91.12
45
46
24.18
3.59
64.14
48.97
14.95
32
0.74
56.41
47
5.00
9.05
33
34
5.77
7.51
75.73
62.69
48
49
15.00
17.03
21.23
44.86
35 (SMU-A0B13)
66.84
95.06
50
0
19.21
a
Each compound was tested in triplicate.
thin layer chromatography (TLC), performed on pre-coated TLC
plates. Flash column chromatography was performed on silica gel
(300–400 mesh). The 1H NMR spectra were obtained on AVANCE
III 400 MHz (Bruker) spectrometer. Chemical shift (d) was reported
in parts per million (ppm) with TMS as an internal standard and
coupling constants (J) were expressed in hertz (Hz). Low-resolution
mass spectra ESI-MS were recorded on a Waters ZQ 4000 appara-
tus. High resolution mass spectra were performed on a Bruker
Apex Qe 7T mass spectrometer.
The general condensation reaction to form Schiff base under-
went addition–elimination procedure. To a vial, 1 equiv. (1 mmol)
of aromatic aldehyde (A moiety) and 1 equiv. of aromatic primary
amine (B moiety) were added and dissolved in 6 mL of absolute
methanol. After heating and stirring at 60 °C for 3 h (or longer
based on TLC), the mixture was set for 48–72 h to generate the
solid Schiff base. Collecting the crystal from the solvent and
checked with TLC, if aromatic aldehyde or aromatic primary amine
Fig. 2. The docking results between SMU-A0B13, 36 and BB-loop of TLR2-TIR
domain. A ribbon presentation of protein and a stick representation of molecule
were shown in this picture. The green one is 36 and the yellow one is SMU-A0B13.
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S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
7.26 (s, 1H), 7.14 (d, J = 8.7 Hz, 3H), 6.80 (s, 2H). 13C NMR (101 MHz,
CDCl3) d 164.35, 157.23, 141.61, 139.24, 135.85, 129.27, 128.45,
126.53, 123.96, 120.34, 118.42. ESI-MS: m/z calcd for C15H13NO
[M+H]+ 224.3, found 224.1.
(A)
100
80
60
40
20
0
5.1.1.3. N-(4-methoxyphenyl)-3-phenylprop-2-en-1-imine (3). 1H
NMR (400 MHz, DMSO) d 8.43 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 7.2
Hz, 2H), 7.43 (s, 4H), 7.24 (d, J = 6.8 Hz, 2H), 7.14 (m, 1H), 6.97
(d, J = 8.8 Hz, 2H), 3.78 (s, 3H). 13C NMR (101 MHz, CDCl3)
d164.37, 160.43, 141.21, 139.39, 135.83, 128.10, 126.92, 126.23,
123.76, 120.44, 116.29, 56.34. ESI-MS: m/z calcd for C16H15NO
[M+H]+ 238.3, found 237.7.
10ng/ml Pam3+ 29/C29
10ng/ml Pam3+ SMU-A0B13
10ng/ml Pam3+ 15
1
10
100
5.1.1.4. N-(3-Phenylallylidene)-[1,10-biphenyl]-4-amine (4). 1H NMR
(400 MHz, DMSO) d 8.50 (s, 1H), 7.71 (s, 6H), 7.50–7.41 (m, 5H),
7.39 (s, 2H), 7.33 (s, 2H), 7.20 (d, J = 7.1 Hz, 1H). 13C NMR (101
MHz, DMSO) d 162.36, 151.12, 144.74, 140.04, 138.24, 135.92,
130.06, 129.41, 128.92, 128.04, 127.90, 127.77, 126.89, 121.98
(s). ESI-MS: m/z calcd for C21H17N [M+H]+ 284.4, found 284.2.
[compound]
/µM
(B)
10ng/ml Pam3 + SMU-A0B13
10ng/ml Pam3 + HA0B13
100
80
60
40
20
0
5.1.1.5. 3-(((1H-Indol-3-yl)methylene)amino)-2-methylbenzoic acid
(5). 1H NMR (400 MHz, DMSO) d 12.13 (s, 1H), 9.94 (s, 1H), 8.28
(s, 1H), 8.11 (s, 1H), 7.51 (s, 1H), 7.25 (m, 2H), 6.92 (m, 2H), 6.77
(dd, J = 7.6, 1.5 Hz, 1H), 5.02 (s, 1H), 2.19 (s, 3H). 13C NMR (101
MHz, DMSO) d 185.40, 170.67, 147.81, 138.79, 137.50, 133.00,
126.04, 124.57, 123.89, 122.57, 121.28, 121.16, 118.62, 117.78,
117.17, 112.84, 14.36. ESI-MS: m/z calcd for C17H14N2O2 [M+H]+
279.3, found 279.1.
0
20
40
60
80 100
[Compound]/
µM
5.1.1.6. N-((1H-Indol-3-yl)methylene)-4-methoxyaniline (6). 1H NMR
(400 MHz, DMSO) d 11.70 (s, 1H), 8.71 (s, 1H), 8.39 (s, 1H), 7.96 (s,
1H), 7.49 (s, 1H), 7.21 (d, J = 8.8 Hz, 2H), 7.17 (s, 1H), 6.96 (d, J = 8.9
Hz, 2H), 3.77 (s, 3H). 13C NMR (101 MHz, DMSO) d 157.23, 154.11,
146.56, 137.54, 133.23, 125.19, 123.11, 122.27, 122.12, 121.12,
115.54, 114.76, 112.32, 55.66. ESI-MS: m/z calcd for C16H14N2O
[M+H]+ 251.3, found 251.2.
Fig. 3. The dose-dependent inhibition of SEAP signaling triggered by Pam3SCK4. (A)
The experiment shows SMU-A0B13’s inhibitory effect is better than C29, and
compound 15 is
a negative control. (B) The experiment shows SMU-A0B13’s
inhibitory effect is better than HA0B13.
SMU-A0B13
100%
80%
60%
40%
20%
0%
5.1.1.7.
2-(((1H-Indol-3-yl)methylene)amino)-4-(trifluoromethyl)
benzoic acid (7). 1H NMR (400 MHz, DMSO) d 9.12 (s, 1H), 8.34 (s,
1H), 8.20 (d, J = 7.5 Hz, 2H), 8.03 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H),
7.67 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 6.7 Hz, 1H), 7.34 (s, 2H).
13C NMR (101 MHz, DMSO) d 185.38, 169.09, 151.76, 138.89,
138.15, 137.45, 132.99, 132.31, 124.52, 123.87, 122.94, 122.53
121.23, 120.98, 118.57, 113.46, 112.83. HRMS (ESI): m/z calcd for
C
17H11N2O2 F3 [M+H]+ 333.0845, found 333.0845.
5.1.1.8.
N-((1H-Indol-3-yl)methylene)-[1,10-biphenyl]-4-amine
0
20 40 60 80 100
SMU-A0B13 (μM)
(8). 1H NMR (400 MHz, DMSO) d 11.79 (s, 1H), 8.78 (s, 1H), 8.40
(s, 1H), 8.03 (s, 1H), 7.70 (d, J = 7.8 Hz, 4H), 7.47 (s, 3H), 7.34 (s,
3H), 7.22 (s, 2H). 13C NMR (101 MHz, DMSO) d 155.73, 152.89,
140.29, 137.66, 136.79, 134.08, 129.34, 127.79, 127.47, 126.74,
125.25, 123.32, 122.36, 121.82, 121.40, 115.59, 112.47. ESI-MS:
m/z calcd for C21H16N2 [M+H]+ 297.4, found 297.7.
Fig. 4. Cytotoxicity of SMU-A0B13 by MTT colorimetric method.
presented, removing them by column chromatography with 1:4
ethyl acetate: petroleum ether to obtain pure product (80–90%).
5.1.1.9. 4-((Naphthalen-1-ylmethylene)amino)phenol (9). 1H NMR
(400 MHz, DMSO) d 9.54 (s, 1H), 9.23 (s, 1H), 9.21 (s, 1H), 8.15 (s,
1H), 8.07 (s, 1H), 8.02 (s, 1H), 7.63 (s, 3H), 7.32 (s, 2H), 6.84 (s,
2H). 13C NMR (101 MHz, DMSO) d 157.33, 156.83, 143.58, 133.97,
131.88, 131.64, 131.27, 129.91, 129.04, 127.71, 126.63, 125.88,
125.00, 123.08, 116.19. ESI-MS: m/z calcd for C17H13NO [M+H]+
248.3, found 248.5.
5.1.1.1. 2-methyl-3-((3-phenylallylidene)amino)benzoic acid (1).
1H NMR (400 MHz, DMSO) d 12.89 (s, 1H), 8.27 (s, 1H), 7.69 (s, 2H),
7.60 (s, 1H), 7.45 (s, 4H), 7.29 (s, 1H), 7.21 (d, J = 7.1 Hz, 1H), 7.11
(s, 1H), 2.45 (s, 3H). 13C NMR (101 MHz, DMSO) d 169.57, 162.87,
152.26, 144.92, 135.77, 132.75, 132.05, 130.07, 129.36, 128.76,
128.07, 127.12, 126.76, 121.32, 15.30. HRMS (ESI): m/z calcd for
17H15NO2 [M+H]+ 266.1176, found 266.1177.
5.1.1.10. 4-Methoxy-N-(naphthalen-1-ylmethylene)aniline (10). 1H
NMR (400 MHz, DMSO) d 9.24 (d, J = 11.1 Hz, 2H), 8.17 (d, J = 7.0
Hz, 1H), 8.10 (s, 1H), 8.05 (s, 1H), 7.65 (s, 3H), 7.44 (s, 2H), 7.04
(s, 2H). 13C NMR (101 MHz, DMSO) d 158.52, 158.45, 145.04,
C
5.1.1.2. 4-((3-Phenylallylidene)amino)phenol (2). 1H NMR (400 MHz,
DMSO) d 9.50 (s, 1H), 8.39 (s, 1H), 7.66 (s, 2H), 7.40 (m, 3H),
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S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 5. The influence of SMU-A0B13 in the TLR2 signaling pathway. (A) The SEAP specificity experiment showed SMU-A0B13 can inhibit both TLR1/2 and TLR2/6, not TLR3.
The ligand for TLR1/2, TLR2/6, and TLR3 are 10 ng/mL Pam3CSK4, 10 ng/mL Pam2CSK4 and 10 g/mL Poly I:C, respectively. (B) The iNOS inhibition by SMU-A0B13 in Raw 264.7
macrophage cells. Pam3CSK4 and Pam2CSK4 are the ligands for TLR1/2 and TLR2/6. (C) The Western Blot experiment indicated SMU-A0B13 can inhibit the Pam3CSK4 (400 ng/
l
mL) upregulated TLR2 in HEK-Blue hTLR2 cells. (D) ELISA assay showed SMU-A0B13 can inhibit the TNF
a signaling which activated by Pam3CSK4 in human PBMC cells. Data
shown are mean SD of representative data of three independent experiments. *P < 0.05.
133.97, 131.88, 131.74, 131.28, 130.20, 129.07, 127.79, 126.67,
132.92, 132.58, 131.90, 129.46, 129.39, 129.33, 128.52, 127.47,
126.68, 124.13, 121.14, 121.03, 119.96, 109.62. ESI-MS: m/z calcd
for C24H16ClNO2 [M+H]+ 386.8, found 386.8.
125.87, 125.00, 122.98, 114.86, 55.74. ESI-MS: m/z calcd for
C
18H15NO [M+H]+ 262.3, found 261.7.
5.1.1.11.
N-(Naphthalen-1-ylmethylene)-[1,10-biphenyl]-4-amine
5.1.1.14.
1-(((4-Hydroxyphenyl)imino)methyl)naphthalen-2-ol
(11). 1H NMR (400 MHz, DMSO) d 9.31 (s, 1H), 9.27 (s, 1H), 8.22
(s, 1H), 8.14 (s, 1H), 8.08 (s, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.74 (d,
J = 7.2 Hz, 2H), 7.72–7.62 (m, 3H), 7.50 (t, J = 8.0 Hz, 4H), 7.39 (s,
1H). 13C NMR (101 MHz, DMSO) d 160.85, 151.49, 140.06, 138.30,
133.98, 132.41, 131.48, 131.32, 130.89, 129.38, 129.14, 127.99,
127.90, 127.76, 126.90, 126.77, 125.88, 125.02, 122.25. ESI-MS:
m/z calcd for C23H17N [M+H]+ 308.4, found 307.9.
(14). 1H NMR (400 MHz, DMSO) d 16.07 (s, 1H), 9.69 (s, 1H), 9.61
(d, J = 4.4 Hz, 1H), 8.48 (d, J = 8.5 Hz, 1H), 7.91 (s, 1H), 7.79 (d, J =
4.1 Hz, 1H), 7.51 (d, J = 8.7 Hz, 3H), 7.34 (s, 1H), 7.02 (d, J = 9.1
Hz, 1H), 6.88 (d, J = 8.7 Hz, 2H). 13C NMR (101 MHz, DMSO) d
169.30, 157.07, 154.24, 136.23, 136.15, 133.45, 129.33, 128.25,
127.04, 123.59, 122.41, 122.23, 120.63, 116.54, 108.92. ESI-MS:
m/z calcd for C17H13NO2 [M+H]+ 264.3, found 264.1.
5.1.1.12.
3-(((2-Hydroxynaphthalen-1-yl)methylene)amino)-2-
5.1.1.15. 3-(((2-Hydroxynaphthalen-1-yl)methylene)amino)benzoic
acid (15). 1H NMR (400 MHz, DMSO) d 15.63 (d, J = 3.7 Hz, 1H),
13.20 (s, 1H), 9.74 (d, J = 2.7 Hz, 1H), 8.55 (d, J = 8.5 Hz, 1H), 8.08
(s, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.89 (t, J = 9.2 Hz, 2H), 7.82 (d, J =
7.9 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.38 (s, 1H), 7.06
(d, J = 9.2 Hz, 1H). 13C NMR (101 MHz, DMSO) d 169.97, 167.34,
157.40, 145.21, 137.31, 133.49, 132.75, 130.28, 129.39, 128.51,
127.54, 127.21, 125.24, 124.00, 122.13, 121.87, 120.99, 109.18.
ESI-MS: m/z calcd for C18H13NO3 [M+H]+ 292.3, found 292.1.
methylbenzoic acid (12). 1H NMR (400 MHz, DMSO) d 15.77 (s,
1H), 13.10 (s, 1H), 9.65 (s, 1H), 8.54 (s, 1H), 7.97 (s, 1H), 7.91 (s,
1H), 7.84 (s, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 7.41 (m, 2H), 7.09 (s,
1H), 2.58 (s, 3H). 13C NMR (101 MHz, DMSO) d 169.71, 169.44,
157.43, 145.23, 137.21, 133.47, 133.43, 130.79, 129.42, 128.54,
127.83, 127.21, 127.17, 124.00, 122.02, 122.00, 120.88, 109.38,
15.17. HRMS (ESI): m/z calcd for C19H15NO3 [M+H]+ 306.1125,
found 306.1126.
5.1.1.13.
(4-Chlorophenyl)(2-(((2-hydroxynaphthalen-1-yl)methy-
5.1.1.16.
2-(((2-Hydroxynaphthalen-1-yl)methylene)amino)-6-
lene)amino)phenyl)methanone (13). 1H NMR (400 MHz, DMSO) d
14.31 (s, 1H), 9.64 (s, 1H), 8.49 (d, J = 8.6 Hz, 1H), 7.94 (s, 2H),
7.77 (t, J = 11.0 Hz, 4H), 7.63 (s, 2H), 7.53 (s, 2H), 7.47 (s, 1H),
7.38 (s, 1H), 6.97 (d, J = 9.1 Hz, 1H). 13C NMR (101 MHz, DMSO) d
195.84, 166.94, 158.62, 144.93, 139.06, 136.95, 135.93, 133.20,
methylbenzoic acid (16). 1H NMR (400 MHz, DMSO) d 15.11 (s,
1H), 13.49 (s, 1H), 9.70 (s, 1H), 8.54 (d, J = 8.6 Hz, 1H), 8.01 (s,
1H), 7.87 (s, 1H), 7.69 (s, 1H), 7.58 (s, 1H), 7.43 (m, 2H), 7.24 (s,
1H), 7.12 (s, 1H), 2.36 (s, 3H). 13C NMR (101 MHz, DMSO) d
169.81, 166.40, 158.67, 143.29, 136.62, 135.27, 133.24, 130.95,
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S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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130.33, 129.41, 128.58, 128.51, 127.51, 124.08, 121.06, 120.98,
116.57, 109.62, 19.51. HRMS (ESI): m/z calcd for C19H15NO3
[M+H]+ 306.1125, found 306.1126.
122.53, 120.74, 115.92, 112.50, 105.27, 97.50, 81.68, 56.03. ESI-
MS: m/z calcd for C15H14N2O4 [M+H]+ 287.3, found 287.1.
5.1.1.24.
2-Methoxy-4-(((4-methoxyphenyl)imino)methyl)phenol
(24). 1H NMR (400 MHz, DMSO) d 9.67 (s, 1H), 8.46 (s, 1H), 7.51
(s, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.7 Hz, 2H), 6.96 (d, J =
8.7 Hz, 2H), 6.88 (d, J = 8.1 Hz, 1H), 3.85 (s, 3H), 3.77 (s, 3H). 13C
NMR (101 MHz, CDCl3) d 181.21, 153.34, 152.57, 147.99, 132.23,
125.87, 124.14, 117.36, 115.17, 103.61, 102.44, 58.42, 55.81. ESI-
MS: m/z calcd for C15H15NO3 [M+H]+ 258.3, found 258.1.
5.1.1.17.
1-(((4-Methoxyphenyl)imino)methyl)naphthalen-2-ol
(17). 1H NMR (400 MHz, DMSO) d 15.98 (s, 1H), 9.65 (s, 1H), 8.51
(s, 1H), 7.91 (s, 1H), 7.79 (s, 1H), 7.63 (d, J = 8.9 Hz, 2H), 7.55 (s,
1H), 7.35 (s, 1H), 7.09–7.01 (m, 3H), 3.82 (s, 3H). 13C NMR (101
MHz, CDCl3) d 171.04, 159.11, 153.88, 144.36, 133.05, 132.41,
129.10, 128.02, 126.84, 129.96, 122.13, 120.56, 118.63, 115.65,
108.52, 55.83. ESI-MS: m/z calcd for C18H15NO2 [M+H]+ 278.3,
found 278.2.
5.1.1.25.
4-(([1,10-Biphenyl]-4-ylimino)methyl)-2-methoxyphenol
(25). 1H NMR (400 MHz, DMSO) d 9.77 (s, 1H), 8.52 (s, 1H), 7.71
(d, J = 8.4 Hz, 4H), 7.56 (s, 1H), 7.47 (s, 2H), 7.36 m, 4H), 6.92 (s,
1H), 3.87 (s, 3H). 13C NMR (101 MHz, DMSO) d 160.53, 151.55,
150.75, 148.45, 140.10, 137.61, 129.35, 128.36, 127.81, 127.63,
126.81, 124.68, 121.96, 115.81, 110.84, 55.97. ESI-MS: m/z calcd
for C20H17NO2 [M+H]+ 304.4, found 304.7.
5.1.1.18. 2-(((2-Hydroxynaphthalen-1-yl)methylene)amino)-4-(triflu-
oromethyl)benzoic acid (18). 1H NMR (400 MHz, DMSO) d 12.00 (s,
1H), 10.82 (s, 1H), 9.53 (s, 1H), 8.50 (d, J = 8.3 Hz, 1H), 8.33 (s, 1H),
8.10 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 9.3 Hz, 1H), 7.77 (d, J = 7.7 Hz,
1H), 7.69 (s, 1H), 7.55 (s, 1H), 7.36 (s, 1H), 6.92 (d, J = 8.9 Hz, 1H).
13C NMR (101 MHz, DMSO) d 193.31, 173.61, 167.25, 154.67,
144.84, 138.72, 133.96, 132.31, 129.48, 128.68, 127.22, 124.31,
123.59, 121.38, 117.02, 109.78. HRMS (ESI): m/z calcd for
5.1.1.26.
4-Methoxy-N-(4-(trifluoromethyl)benzylidene)aniline
(26). 1H NMR (400 MHz, DMSO) d 8.78 (s, 1H), 8.12 (s, 2H), 7.88
(s, 2H), 7.38 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 8.9 Hz, 2H), 3.80 (s,
3H). 13C NMR (101 MHz, CDCl3) d 160.05, 159.12, 144.38, 139.74,
133.37, 129.52, 125.26, 124.16, 122.13, 115.61, 55.80. ESI-MS:
m/z calcd for C15H12F3NO [M+H]+ 280.3, found 280.1.
C
19H12F3NO3 [M+H]+ 360.0842, found 360.0844.
5.1.1.19.
1-(([1,10-Biphenyl]-4-ylimino)methyl)naphthalen-2-ol
(19). 1H NMR (400 MHz, DMSO) d 15.86 (s, 1H), 9.72 (s, 1H), 8.53
(d, J = 8.5 Hz, 1H), 7.94 (d, J = 9.2 Hz, 1H), 7.82 (d, J = 8.6 Hz, 3H),
7.75 (d, J = 8.8 Hz, 4H), 7.56 (dd, J = 11.3, 4.1 Hz, 1H), 7.50 (t, J =
7.6 Hz, 2H), 7.37 (s, 2H), 7.03 (s, 1H). 13C NMR (101 MHz, DMSO)
d 171.44, 155.42, 143.38, 139.69, 138.60, 137.42, 133.61, 129.43,
129.39(s), 128.18, 127.93, 127.07, 126.92, 123.90, 122.74, 121.38,
120.78, 109.03. ESI-MS: m/z calcd for C23H17NO [M+H]+ 324.4,
found 325.1.
5.1.1.27. N-(4-(Trifluoromethyl)benzylidene)-[1,10-biphenyl]-4-amine
(27). 1H NMR (400 MHz, DMSO) d 8.84 (s, 1H), 8.17 (s, 2H), 7.92 (s,
2H), 7.76 (s, 2H), 7.71 (s, 2H), 7.47 (m, 4H), 7.38 (s, 1H). 13C NMR
(101 MHz, CDCl3)
d 158.29, 150.41, 140.47, 139.63, 139.30,
129.00, 128.87, 127.98, 127.40, 126.98, 125.78, 125.75, 121.47.
HRMS (ESI): m/z calcd for C20H14F3N [M+H]+ 326.1156, found
326.1155.
5.1.1.20. (2-(((2-Hydroxynaphthalen-1-yl)methylene)amino)phenyl)
(phenyl)methanone (20). 1H NMR (400 MHz, DMSO) d 14.41 (s,
1H), 9.64 (s, 1H), 8.49 (s, 1H), 7.94 (s, 2H), 7.79 (s, 3H), 7.76 (s,
1H), 7.67 (t, J = 7.3 Hz, 1H), 7.55 (s, 4H), 7.46 (s, 1H), 7.38 (s, 1H),
6.96 (s, 1H). 13C NMR (101 MHz, DMSO) d 196.90, 167.20, 158.39,
144.77, 137.20, 136.93, 134.12, 133.32, 133.24, 132.36, 130.11,
129.38, 129.27, 128.52, 127.42, 126.57, 124.10, 121.16, 121.08,
119.94, 109.57. ESI-MS: m/z calcd for C24H17NO2 [M+H]+ 352.4,
found 351.8.
5.1.1.28. N-(3-Methoxybenzylidene)-[1,10-biphenyl]-4-amine (28). 1H
NMR (400 MHz, DMSO) d 8.68 (s, 1H), 7.76–7.69 (m, 4H), 7.56–7.52
(m, 2H), 7.51–7.43 (m, 3H), 7.38 (d, J = 8.5 Hz, 3H), 7.13 (d, J = 8.3
Hz, 1H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO) d 160.94, 160.00,
150.95, 139.99, 138.30, 137.92, 130.36, 129.38, 127.83 (d), 126.89,
122.11, 118.21, 113.04, 55.67. HRMS (ESI): m/z calcd for C20H17NO
[M+H]+ 288.1383, found 288.1386.
5.1.1.29. 3-((2-Hydroxy-3-methoxybenzylidene)amino)-2-methylben-
zoic acid (29). 1H NMR (400 MHz, DMSO) d 13.07 (m, 2H), 8.85 (s,
1H), 7.67 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7
Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.16 (d, J = 7.9 Hz, 1H), 6.94 (t, J
= 7.9 Hz, 1H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO) d 169.51,
161.13, 160.01, 151.88, 137.87, 132.71, 132.13, 130.39, 127.31,
126.80, 121.91, 121.58, 118.08, 113.40, 55.67, 15.29. ESI-MS: m/z
calcd for C16H15NO4 [M+H]+ 286.3, found 286.1.
5.1.1.21. 2-Methyl-3-((4-(trifluoromethyl)benzylidene)amino)benzoic
acid (21). 1H NMR (400 MHz, DMSO) d 8.14 (d, J = 8.1 Hz, 3H), 7.88
(d, J = 8.3 Hz, 3H), 6.93 (m, 1H), 6.78 (m, 1H), 2.19 (s, 3H).13C NMR
(101 MHz, DMSO) d 170.57, 166.62, 147.85, 135.01, 132.87, 130.52,
129.73, 126.02, 125.99, 121.55, 121.12, 117.67, 117.12, 14.33.
HRMS (ESI): m/z calcd for C16H12NO2F3 [M+H]+ 308.0893, found
308.0890.
5.1.1.30. 3-((3-Ethoxy-2-hydroxybenzylidene)amino)-2-methylben-
zoic acid (30). 1H NMR (400 MHz, DMSO) d 13.17 (s, 1H), 13.03
(s, 1H), 8.85 (s, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.8 Hz,
1H), 7.38 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.7 Hz, 1H), 7.15 (d, J = 7.9
Hz, 1H), 6.92 (t, J = 7.8 Hz, 1H), 4.09 (d, J = 7.0 Hz, 2H), 1.37 (t, J =
6.9 Hz, 3H), 1.24 (s, 3H). 13C NMR (101 MHz, DMSO) d 169.43,
164.72, 151.18, 148.73, 147.48, 133.26, 132.14, 128.20, 127.09,
124.48, 121.96, 119.84, 119.10, 117.39, 64.50, 15.28, 15.18. HRMS
(ESI): m/z calcd for C17H17NO4 [M+H]+ 300.1230, found 300.1228.
5.1.1.22. 3-((3-Methoxybenzylidene)amino)-2-methylbenzoic acid
(22). 1H NMR (400 MHz, DMSO) d 12.86 (s, 1H), 8.47 (s, 1H), 7.62
(d, J = 7.7 Hz, 1H), 7.54 (d, J = 7.3 Hz, 2H), 7.46 (t, J = 7.8 Hz, 1H),
7.31 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.17–7.11 (m, 1H),
3.84 (s, 3H), 2.48 (s, 3H). 13C NMR (101 MHz, DMSO) d 169.51,
161.13, 160.01, 151.92, 137.87, 132.67, 132.13, 130.39, 127.31,
126.80, 121.87, 121.50, 117.95, 113.40, 55.67, 15.37. HRMS (ESI):
m/z calcd for C16H15NO3 [M+H]+ 270.1125, found 270.1126.
5.1.1.23. 3-Amino-5-((4-hydroxy-3-methoxybenzylidene)amino)ben-
zoic acid (23). 1H NMR (400 MHz, DMSO) d 9.23 (s, 1H), 6.83 (s,
1H), 6.73 (s, 1H), 6.59 (s, 1H), 6.25 (s, 1H), 6.20 (s, 1H), 6.13 (s,
1H), 5.29 (s, 2H), 4.70 (s, 2H), 3.71 (s, 3H). 13C NMR (101 MHz,
DMSO) d 171.63, 151.53, 147.94, 147.68, 144.00, 127.57, 127.42,
5.1.1.31.
2-(((4-Hydroxyphenyl)imino)methyl)-6-methoxyphenol
(31). 1H NMR (400 MHz, DMSO) d 13.60 (s, 1H), 9.68 (s, 1H), 8.89
(s, 1H), 7.33 (s, 2H), 7.17 (s, 1H), 7.08 (s, 1H), 6.89 (s, 1H), 6.85
(d, J = 8.7 Hz, 2H), 3.82 (s, 3H). 13C NMR (101 MHz, DMSO) d
160.75, 157.40, 150.82, 148.26, 139.42, 124.08, 123.04, 119.74,
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S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
118.82, 116.39, 115.46, 56.26. ESI-MS: m/z calcd for C14H13NO3 [M
7.43 (d, J = 8.9 Hz, 2H), 7.20 (s, 1H), 7.10 (s, 1H), 7.02 (s, 2H), 6.90
(s, 1H), 3.84 (s, 3H), 3.80 (s, 3H). 13C NMR (101 MHz, CDCl3) d
160.67, 159.30, 151.65, 148.79, 141.27, 123.98, 122.69, 119.67,
118.83, 114.99, 114.80, 56.53, 55.87. ESI-MS: m/z calcd for
+H]+ 244.3, found 244.1.
5.1.1.32. 4-((3-Methoxybenzylidene)amino)phenol (32). 1H NMR
(400 MHz, DMSO) d 9.50 (s, 1H), 8.59 (s, 1H), 7.47 (s, 2H), 7.40
(d, J = 7.9 Hz, 1H), 7.19 (s, 2H), 7.06 (d, J = 7.9 Hz, 1H), 6.80 (d, J =
11.9 Hz, 2H), 3.83 (s, 3H). 13C NMR (101 MHz, DMSO) d 159.55,
157.06, 156.37, 142.49, 137.94, 129.85, 122.55, 121.25, 117.11,
115.73, 112.20, 55.19. ESI-MS: m/z calcd for C14H13NO2 [M+H]+
228.3, found 227.7.
C
15H15NO3 [M+H]+ 258.3, found 258.2.
5.1.1.40.
2-(([1,10-Biphenyl]-4-ylimino)methyl)-6-methoxyphenol
(40). 1H NMR (400 MHz, DMSO) d 13.27 (s, 1H), 9.04 (s, 1H), 7.77
(s, 2H), 7.71 (s, 2H), 7.52 (m, 4H), 7.39 (s, 1H), 7.26 (s, 1H), 7.14
(s, 1H), 6.93 (s, 1H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO) d
163.81, 151.05, 148.36, 147.41, 139.75, 139.17, 129.40, 128.09,
127.97, 126.97, 124.36, 122.40, 119.69, 119.05, 116.01, 56.29.
ESI-MS: m/z calcd for C20H17NO2 [M+H]+ 304.4, found 303.9.
5.1.1.33.
2-Ethoxy-6-(((4-hydroxyphenyl)imino)methyl)phenol
(33). 1H NMR (400 MHz, DMSO) d 13.64 (s, 1H), 9.67 (s, 1H), 8.89
(s, 1H), 7.32 (d, J = 8.7 Hz, 2H), 7.18 (d, J = 7.8 Hz, 1H), 7.07 (d, J =
9.1 Hz, 1H), 6.85 (d, J = 8.7 Hz, 3H), 4.07 (d, J = 7.0 Hz, 2H), 1.35
(s, 3H). 13C NMR (101 MHz, DMSO) d 160.71, 157.42, 151.15,
147.39, 139.36, 124.25, 123.02, 119.85, 118.79, 116.85, 116.40,
64.49, 15.21. ESI-MS: m/z calcd for C15H15NO3 [M+H]+258.3, found
258.1.
5.1.1.41.
(4-Chlorophenyl)(2-((3-ethoxy-2-hydroxybenzylidene)
amino)phenyl)methanone (41). 1H NMR (400 MHz, DMSO) d 8.89
(s, 1H), 7.72 (t, J = 11.3 Hz, 3H), 7.59 (d, J = 8.6 Hz, 3H), 7.48 (m,
2H), 7.14 (s, 1H), 7.05 (s, 1H), 6.83 (s, 1H), 3.99 (q, 2H), 1.29 (t,
3H). 13C NMR (101 MHz, DMSO) d 196.34, 167.91, 150.26, 147.04,
145.82, 138.43, 137.79, 133.62, 131.15, 130.86, 130.26, 128.68,
127.41, 125.23, 120.04, 119.78, 119.07, 117.63, 65.84, 15.61. ESI-
MS: m/z calcd for C22H18ClNO3 [M+H]+380.8, found 380.7.
5.1.1.34.
4-(((4-Hydroxyphenyl)imino)methyl)-2-methoxyphenol
(34). 1H NMR (400 MHz, DMSO) d 9.61 (s, 1H), 9.38 (s, 1H), 8.43
(s, 1H), 7.49 (s, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 8.2 Hz,
2H), 6.87 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.2 Hz, 2H), 3.84 (s, 3H).
13C NMR (101 MHz, DMSO) d 157.56, 156.16, 150.13, 148.42,
143.64, 128.78, 124.01, 122.62, 116.14, 115.79, 110.51, 55.96.
ESI-MS: m/z calcd for C14H13NO3 [M+H]+ 244.3, found 244.2.
5.1.1.42. 3-((3-Ethoxy-2-hydroxybenzylidene)amino)benzoic acid
(42). 1H NMR (400 MHz, DMSO) d 13.18 (s, 1H), 13.03 (s, 1H),
9.04 (s, 1H), 7.93 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 8.2
Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.15 (d, J
= 8.0 Hz, 1H), 6.91 (t, J = 7.9 Hz, 1H), 4.09 (q, J = 6.9 Hz, 2H), 1.36
(t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3) d 170.51(s), 164.05,
151.94, 148.95, 147.93, 130.88, 129.84, 128.64, 127.55, 124.35,
122.02, 119.39, 118.93, 117.14, 64.98, 15.07. ESI-MS: m/z calcd
for C16H15NO4 [M+H]+286.3, found 286.7.
5.1.1.35. (SMU-A0B13) (4-chlorophenyl)(2-((2-hydroxy-3-methoxy-
benzylidene)amino)phenyl)methanone (35). 1H NMR (400 MHz,
DMSO) d 11.88 (s, 1H), 8.91 (s, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.69
(s, 1H), 7.60 (d, J = 8.4 Hz, 3H), 7.53–7.44 (m, 2H), 7.12 (s, 1H),
7.07 (s, 1H), 6.86 (s, 1H), 3.75 (s, 3H). 13C NMR (101 MHz, DMSO)
d 195.94, 164.83, 150.39, 148.12, 146.61, 139.04, 135.88, 134.43,
132.22, 131.72, 129.49, 128.68, 127.29, 124.30, 119.54, 119.32,
119.12, 116.28, 56.17. HRMS (ESI): m/z calcd for C21H16ClNO3 [M
+H]+ 366.0897, found 366.0895.
5.1.1.43. 2-((3-Ethoxy-2-hydroxybenzylidene)amino)-6-methylben-
zoic acid (43). 1H NMR (400 MHz, DMSO) d 13.34 (s, 1H), 13.00
(s, 1H), 8.97 (s, 1H), 7.48–7.35 (m, 2H), 7.23 (d, J = 7.2 Hz, 2H),
7.13 (d, J = 7.9 Hz, 1H), 6.90 (t, J = 7.9 Hz, 1H), 4.08 (m, 2H), 2.33
(s, 3H), 1.36 (s, 3H). 13C NMR (101 MHz, DMSO) d 169.89, 164.42,
151.09, 147.50, 144.62, 135.03, 132.14, 130.15, 128.87, 124.71,
119.61, 119.03, 117.42, 115.61, 64.46, 19.29, 15.19. HRMS (ESI):
m/z calcd for C17H17NO4 [M+H]+ 300.1230, found 300.1233.
5.1.1.36. (2-((2-Hydroxy-3-methoxybenzylidene)amino)phenyl)(phe-
nyl)methanone (36). 1H NMR (400 MHz, DMSO) d 11.94 (s, 1H),
8.90 (s, 1H), 7.74 (d, J = 7.3 Hz, 2H), 7.66 (s, 2H), 7.61 (s, 1H), 7.51
(m, 4H), 7.12 (s, 1H), 7.06 (s, 1H), 6.85 (s, 1H), 3.74 (s, 3H). 13C
NMR (101 MHz, DMSO) d 197.00, 164.80, 150.41, 148.11, 146.57,
137.12, 134.92, 134.14, 131.97, 129.91, 129.28, 128.60, 127.19,
124.34, 119.50, 119.29, 119.05, 116.16, 56.13. HRMS (ESI): m/z
calcd for C21H17NO3 [M+H]+ 332.1281, found 332.1285.
5.1.1.44.
2-Ethoxy-6-(((4-methoxyphenyl)imino)methyl)phenol
(44). 1H NMR (400 MHz, DMSO) d 13.53 (s, 1H), 8.94 (s, 1H), 7.43
(d, J = 8.8 Hz, 2H), 7.20 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 7.9 Hz, 1H),
7.03 (d, J = 8.9 Hz, 2H), 6.88 (t, J = 7.9 Hz, 1H), 4.07 (d, J = 7.0 Hz,
2H), 3.80 (s, 3H), 1.35 (s, 3H). 13C NMR (101 MHz, DMSO) d
161.75, 158.97, 151.19, 147.42, 140.88, 124.35, 122.98, 119.82,
118.85, 117.04, 115.07, 64.51, 55.77, 15.20. ESI-MS: m/z calcd for
5.1.1.37. N-(2,3-Dimethoxybenzylidene)-4-methoxyaniline (37). 1H
NMR (400 MHz, DMSO) d 8.79 (s, 1H), 7.61 (d, J = 7.4 Hz, 1H),
7.28 (d, J = 8.9 Hz, 2H), 7.18 (dd, J = 8.2, 4.8 Hz, 2H), 6.99 (d, J =
8.9 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.78 (s, 3H). 13C NMR (101
MHz, CDCl3)d 158.20, 154.32, 152.76, 149.83, 145.18, 130.05,
122.36, 124.20, 118.74, 114.61, 114.27, 61.85, 55.81, 55.40. ESI-
MS: m/z calcd for C16H17NO3 [M+H]+ 272.3, found 272.1.
C
16H17NO3 [M+H]+272.3, found 272.1.
5.1.1.45.
2-(([1,10-Biphenyl]-4-ylimino)methyl)-6-ethoxyphenol
(45). 1H NMR (400 MHz, DMSO) d 11.94 (s, 1H), 8.90 (s, 1H), 7.74
(d, J = 8.6 Hz, 2H), 7.70 (s, 1H), 7.60 (d, J = 8.6 Hz, 3H), 7.49 (m,
2H), 7.14 (d, J = 6.5 Hz, 1H), 7.08 (s, 1H), 6.84 (s, 1H), 4.01 (s, 2H),
1.32 (s, 3H). 13C NMR (101 MHz, DMSO) d 163.91, 151.36, 147.50,
147.36, 139.77, 139.20, 129.41, 128.12, 127.98, 126.98, 124.56,
122.40, 119.79, 119.04, 117.49, 64.56, 15.21. HRMS (ESI): m/z calcd
for C21H19NO2 [M+H]+ 318.1489, found 318.1490.
5.1.1.38. 3-((2-Hydroxy-3-methoxybenzylidene)amino)benzoic acid
(38). 1H NMR (400 MHz, DMSO) d 13.16 (s, 1H), 12.96 (s, 1H),
9.03 (s, 1H), 7.92 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 8.7
Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.16 (d, J
= 7.9 Hz, 1H), 6.93 (t, J = 7.9 Hz, 1H), 3.84 (s, 3H). 13C NMR (101
MHz, DMSO) d 167.36, 164.98, 150.92, 148.70, 148.32, 132.57,
130.21, 128.01, 126.40, 124.40, 122.20, 119.63, 119.11, 116.14,
56.26. ESI-MS: m/z calcd for C15H13NO4 [M+H]+ 272.3, found 272.2.
5.1.1.46. (2-((3-Ethoxy-2-hydroxybenzylidene)amino)phenyl)(phenyl)
methanone (46). 1H NMR (400 MHz, DMSO) d 12.03 (s, 1H), 8.91 (s,
1H), 7.74 (d, J = 7.3 Hz, 2H), 7.66 (s, 2H), 7.61 (s, 1H), 7.53 (s, 2H),
7.48 (s, 2H), 7.14 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.83
(s, 1H), 4.00 (s, 2H), 1.29 (s, 3H). 13C NMR (101 MHz, DMSO) d
196.94, 164.93, 150.76, 147.24, 146.62, 137.15, 134.79, 134.12,
5.1.1.39.
2-Methoxy-6-(((4-methoxyphenyl)imino)methyl)phenol
(39). 1H NMR (400 MHz, DMSO) d 13.46 (s, 1H), 8.94 (s, 1H),
Please cite this article in press as: Cai S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.001

S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
131.99, 129.96, 129.26, 128.66, 127.11, 124.56, 119.64, 119.38,
control Pam3CSK4 (final conc. 10 ng/ml), as well as indicated con-
centration compounds to 200 l totally, then incubated for 24 h.
On the third day, 20 l of 5 mg/ml MTT (in PBS) solution was added
119.05, 117.62, 64.44, 15.10. HRMS (ESI): m/z calcd for
l
C
22H19NO3 [M+H]+346.1438, found 346.1440.
l
to each well of the above 96-well plate, and then placed in 37 °C
incubator for 3 h, gently suck the medium out and buckled the
96 wells plate overnight. Next, 100 ll of DMSO was added to each
well of the 96-well plate, shaken for 1 h and measured at an
5.1.1.47. 3-((2,3-Dihydroxybenzylidene)amino)benzoic acid (47). 1H
NMR (400 MHz, DMSO) d 13.25–13.07 (s, 1H), 12.92 (s, 1H), 9.25
(s, 1H), 9.00 (s, 1H), 7.97–7.85 (m, 2H), 7.67 (d, J = 8.0 Hz, 1H),
7.60 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 7.7 Hz,
1H), 6.81 (t, J = 7.7 Hz, 1H). 13C NMR (101 MHz, DMSO) d 167.34,
165.31, 149.66, 148.75, 146.03, 132.59, 130.23, 127.92, 126.37,
123.34, 122.15, 119.83, 119.65, 119.30. HRMS (ESI): m/z calcd for
absorbance of 490 nm through a plate reader.
5.2.3. Nitric oxide assay
C
14H11NO4 [M+H]+ 258.0761, found 258.0761.
Raw 264.7 cells were cultured with a RPMI 1640 medium made
of 10% PBS, 1% penicillin/streptomycin. On day one, the culture
medium containing cells (8 ꢀ 104 cell per well) was added to the
96-well plates for 24 h at 37 °C incubator. Then replaced the media,
5.1.1.48. 2-((2,3-Dihydroxybenzylidene)amino)-6-methylbenzoic acid
(48). 1H NMR (400 MHz, DMSO) d 13.29 (s, 1H), 12.78 (s, 1H), 9.22
(s, 1H), 8.91 (s, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 7.22 (s, 1H), 7.11 (s,
1H), 6.97 (s, 1H), 6.80 (s, 1H), 2.33 (s, 3H). 13C NMR (101 MHz,
DMSO) d 169.94, 164.80, 149.46, 146.05, 144.92, 135.00, 131.96,
130.21, 128.75, 123.52, 119.78, 119.76, 119.31, 115.83, 19.33.
ESI-MS: m/z calcd for C15H13NO4 [M+H]+ 272.3, found 272.7.
added compound and RPMI 1640 only to 200
ll totally, and it was
incubated for 24 h also. 100 l supernatant and 10
l
ll DAN (2,3-
diaminonaphthalene, 0.05 mg/ml DAN dilute in 0.62 M aqueous
HCl) were drawn to flat black 96-well microfluor plates for 15
min’ incubation in the dark. 5 ll NaOH solvent (3 mol/L) was added
and read on Infinite M100 Pro (Tecan) reader with excitation at
365 nm and emission at 410 nm in 10 min.
5.1.1.49.
3-(((4-Methoxyphenyl)imino)methyl)benzene-1,2-diol
(49). 1H NMR (400 MHz, DMSO) d 13.41 (s, 1H), 9.14 (s, 1H), 8.90
(s, 1H), 7.43 (d, J = 8.8 Hz, 2H), 7.05 (dd, J = 13.5, 8.3 Hz, 3H), 6.92
(d, J = 7.6 Hz, 1H), 6.78 (t, J = 7.8 Hz, 1H), 3.80 (s, 3H). 13C NMR
(101 MHz, CDCl3) d 160.3, 159.2, 149.9, 145.4, 140.6, 122.9,
122.4, 119.0, 118.7, 117.4, 114.9, 55.8. ESI-MS: m/z calcd for
5.2.4. Cytokine ELISA assays
PBMC, peripheral blood mononuclear cells, were seeded in
twelve-well plates at a density of 2.5 ꢀ 105 cells per well with
0.5 mL of medium [RPMI 1640 medium supplemented with 10%
FBS, and 1% penicillin/streptomycin]. The cells were treated with
indicated concentrations of SMU-A0B13 and 10 ng/ml Pam3CSK4
(Invivogen) as positive control and incubated for 24 h at 37 °C in
a 5% CO2 humidified incubator. The cell culture supernatants were
collected and frozen at ꢁ80 °C until measurement. The level of
C
14H13NO3 [M+H]+ 244.2, found 244.4.
5.1.1.50. (4-Chlorophenyl)(2-((2-hydroxy-3-methoxybenzyl)amino)
phenyl)methanone (50). 1H NMR (400 MHz, DMSO) d 8.68 (s, 1H),
7.37 (m, 4H), 7.00 (m, 2H), 6.81 (d, J = 7.9 Hz, 1H), 6.62 (t, J = 7.8
Hz, 1H), 6.53 (m, 2H), 6.46 (d, J = 8.1 Hz, 1H), 6.16 (d, J = 4.3 Hz,
1H), 4.21 (s, 2H), 3.79 (s, 3H). 13C NMR (101 MHz, DMSO) d
147.59, 146.31, 144.19, 143.36, 131.55, 128.68, 128.55, 128.17,
128.14, 126.49, 120.21, 118.87, 115.78, 110.91, 110.58, 56.17,
41.52. HRMS (ESI): m/z calcd for C21H18ClNO3 [M+H]+ 368.1048,
found 368.1050.
cytokine TNF-a was determined using recombinant human-cyto-
kine standards, cytokine-specific capture antibodies and detection
antibodies according to the commercially available ELISA kit (BD
Biosciences) with each sample for triplicate.
5.2.5. Immunoblot analysis
5.2. Cellular assays
Entire proteins from antagonist-treated cells were extracted
with PIPA solution involving 1% PMSF (both from Life Sciences).
These proteins were separated using the SDS-PAGE (Life Sciences)
and transferred onto PVDF membranes (Merck Millipore). Mem-
branes were blocked with 5% (w/v) skim milk (Becton, Dickinson
and Company) in TBST and incubated with TLR2 or GAPDH primary
antibody overnight at 4 °C on the shaker. After washed for five
times with TBST, membranes were incubated with their respective
secondary antibody for 1 h in RT on the shaker. Detection was per-
formed with ECL Chemiluminescent Substrate Reagent Kits (Life
Sciences) and the FluorChem R Multifunctional Imaging Analysis
System (ProteinSimple). During this operation, the following pri-
mary and secondary antibodies were employed: rat-GAPDH
(1:2000, Life Sciences), rat-TLR2 (1:250, Boster), goat-anti-rat-
GAPDH/TLR2-HRP (1:2500, Boster).
5.2.1. Secreted embryonic alkaline phosphatase (SEAP) and specificity
experiments
HEK-TLR2 cells were cultured with a medium made of DMEM as
well as 10% PBS and 1% penicillin/streptomycin. On the first day,
200
l
l cells culture medium (4 ꢀ 104 cells per well) was added to
the 96-well plates for 24 h at 37 °C incubator. Then, the medium
was changed to DMEM with indicated concentration of compounds
as well as Pam3CSK4 (final conc. 10 ng/ml) with totally 200
ium, and incubated it at 37 °C for another 24 h. Finally, 50ul
Quanti-Blue (invivoGen) and 50 l cellular supernatant was added
ll med-
l
into a new 96-well plate. After 15–30 min, you can observe the
emergence of metachromatism, and then test the optical density
at 620 nm. As for specificity experiment, replace the positive with
respectively specific TLR agonist (10 ng/ml Pam3CSK4 for TLR1/2,
10 ng/ml Pam2CSK4 for TLR2/6, 10 lg/ml Poly I:C for TLR3). HEK-
Blue hTLR2 and HEK-Blue hTLR3 cells were employed in the speci-
5.2.6. Molecular docking modeling
fic assay.
All molecular docking calculations were performed with the
help of Autodock vina 1.1.2 based on the crystal structure of the
TLR2 TIR domain (PDB: 1FYW). All rotatable bonds of compounds
were set rotatable and the residues of the BB loop pocket (Y647,
C673, D678, F679, I680, K683, D687, N688, D691 and S692) were
chose as Flexible Residues. The box was centered on (14.249,
95.837, 13.802) and the number of points in each dimension is
50 which spaces 0.375 Å.
5.2.2. Cell toxicity assay
HEK-TLR2 cells were cultured with a DMEM medium made of
10% FBS and 1% penicillin/streptomycin. On the first day, the cul-
ture medium containing the cells (4 ꢀ 104 cell per well) was added
to the 96-flash plate at a volume of 200 ll for 24 h. The next day,
the medium was changed to DMEM only and added positive
Please cite this article in press as: Cai S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.001

10
S. Cai et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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This work was supported, in part, by the scientific research project
of high level talents (No. C1033269) in Southern Medical Univer-
sity of China, Youth Pearl River Scholar Program of Guangdong Pro-
vince (No. C1034007), Science and Technology Planning Project of
Guangdong Province, China (No. 2014750) and Innovation and
Entrepreneurship of college students in Guangdong (No.
C1033579), as well as National Natural Science Foundation of
China (No. 81773558).
U
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Supplementary data associated with this article can be found, in
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