Synthesis and discovery of asiatic acid based 1,2,3-triazole derivatives as antitumor agents blocking NF-κB activation and cell migration

Article abstract of DOI:10.1039/c8md00620b

A series of asiatic acid (AA) based 1,2,3-triazole derivatives were designed, synthesized and subjected to a cell-based NF-κB inhibition screening assay. Among the tested compounds, compound 6k displayed impressive NF-κB inhibitory activity with an IC

Full text of DOI:10.1039/c8md00620b

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DOI: 10.1039/C8MD00620B
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ARTICLE
Synthesis and discovery of asiatic acid based 1,2,3-triazoles derivatives as
antitumor agents blocking NF-κB activation and cell migration
Received 00th January 20xx,
Accepted 00th January 20xx
Ri-Zhen Huanga, b, †, Gui-Bin Liangd, †, Mei-Shan Lid, Yi-Lin Fangd, Shi-Feng Zhaoa, Mei-Mei Zhoud, Zhi-Xin Liaoa, b, Jing Sunc*,
Heng-Shan Wang d
DOI: 10.1039/x0xx00000x
A series of asiatic acid (AA) based 1,2,3-triazoles derivatives were designed, synthesized and subjected to a cell-based NF-
κB inhibition screening assay. Among the tested compounds, compound 6k displayed impressive NF-κB inhibitory activity
with IC50 value in low micromolar range. The molecular docking study was performed to reveal key interactions between
6k and NF-κB in which the 1,2,3-triazoles moiety and the hydroxyl groups of AA skeleton were important for improving
inhibitory activity. Subsequently, surface plasmon resonance analysis validated the high affinity between compound 6k
and NF-κB protein with an equilibrium dissociation constant (KD) value of 0.36 μM. Further studies showed that compound
6k observably inhibited the NF-κB DNA binding, nuclear translocation and IκBα phosphorylation. Moreover, in vitro
antitumor activity screening showed that compound 6k (IC50 = 2.67 ± 0.06 μM) exhibited the best anticancer activity
against A549 cells, at least partly, by inhibition of the activity of NF-κB. Additionally, the treatment of A549 cells with
compound 6k resulted in the apoptosis induction potency and in vitro cell migration inhibition. Thus, we conclude that AA
based 1,2,3-triazoles derivatives may be the potential NF-κB inhibitors with the ability to induce apoptosis and suppress
cells migration.
www.rsc.org/medchemcomm
considerable efforts have focused on targeting NF-κB for the
development of therapeutic drugs for cancer therapy and overcoming
drug resistance.
Introduction
Nuclear factor kappa B (NF-κB) is a crucial transcription factor
involved in regulating the transcription of multiple target genes
included in tumor processes, innate and adaptive immune responses,
inflammation, cellular growth and apoptosis.1-3 NF-κB is commonly
over-expressed and constitutively activated in different types of
cancers.4 It is well established that aberrant NF-κB activation has
been associated with tumor cell proliferation, invasion, angiogenesis,
In previous work, we have reported that aniline-derived ursolic
acid derivatives as multidrug resistance reversers by blocking NF-κB
pathway.12, 13 Triterpenoids are highly multifunctional agents owing
to their ability to interact with multiple biological targets and has
been reported to block NF-κB activation.14, 15 Asiatic acid (AA), a
pentacyclic triterpenoid isolated from the tropical medicinal plant,
Centella asiatica (Apiaceae), has been found to possess numbers of
pharmaceutical effects, including hepatoprotective16, anti-
inflammation17, antidiabetics18, and antitumor activities19.
Especially, AA exhibits potent anticancer efficacy in various types
of cancer cells through its ability to inhibit NF-κB, modulate of Bcl-
2 family as well as activate p53.20, 21 Furthermore, previous studies
also reviewed that the modification C-28 position of AA has
properties of increasing the potency of anticancer drugs.22, 23 Several
groups were able to show that the introduction of a ketone group
substituent on C-11 position increased the biological activity of the
and metastasis through its regulation of various gene products.5,
6
Additionally, increasing resistance to chemotherapy is tightly linked
to aberrant activation of NF-κB signaling.7-9 There are numbers of
evidences which suggest that the inhibition of NF-κB activation can
prevent tumor resistance to chemotherapeutic agents, shift the death-
survival balance toward apoptosis, and improve the efficacy of
current chemotherapeutic regimens.10, Because the activation of
NF-κB is regarded as an essential feature of the survival of cancer
11
cells during treatment, which contributes to cancer drug resistance,
24
compounds.22, Besides, it is generally acknowledged that 1,2,3-
a
Department of Pharmaceutical Engineering, School of Chemistry and Chemical
triazole derivatives showed remarkable binding affinity with a
variety of enzymes and receptors by non-covalent interactions, hence
resulting in versatile biological activities.25 In fact, many studies
demonstrated that 1,2,3-triazole moieties were found to be highly
useful as pharmacophores in medicinal chemistry and wide potential
as therapeutic agents such as anticancer, anti-infective, antiviral, and
antimicrobial agents.26-28 Due to the favourable therapeutic indexes,
several 1,2,3-triazole containing drugs have been development for
application in clinical trials.29 Recently, several reports revealed that
pentacyclic triterpene ursolic, betulinic and oleanolic acid based
Engineering, Southeast University, Nanjing 211189, P.R. China
b
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research ， Southeast
University, Nanjing 211189, P.R. China
c Chinese Acad Sci, Northwest Inst Plateau Biol, Qinghai Key Laboratory of Qinghai-
Tibet Plateau Biological Resources, Xining, 810000, P.R. China
d
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal
University, No. 15 Yucai Road, Guilin 541004, P. R. China.
Corresponding authors: E-mail addresses: zxliao@seu.edu.cn (Z. -X. Liao),
sunj@nwipb.cas.cn (J. S), whengshan@163.com (H. -S. Wang).
† These authors contributed equally to this work.
‡
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
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NaOH,
2
h,
r.t.
View Article Online
triazole derivatives exerted cytotoxic activity and induced apoptosis
towards a variety of cancer cell lines.30-32 However, the molecular
target or the mechanism of action of these triazole derivatives
mediated antitumor activity remains unclear and is yet to be
elucidated.
DOI: 10.1039/C8MD00620B
In vitro inhibition of TNF-α-induced NF-κB activation in A549
lung cancer cell line
The inhibitory activity of all synthesized compounds toward NF-κB
was evaluated by a cell-based luciferase assay in A549 lung cancer
cells, which specifically reported on the ability of molecules to
inhibit TNF-α-induced NF-κB transcriptional activity. The human
lung cancer A549 cell line transiently co-transfected with NF-κB-luc
was used to determine the effects of AA contained 1,2,3-triazoles
derivatives on TNF-α-induced NF-κB activation. IMD-0354, a well-
characterized IKKβ inhibitor, was used as a positive control.
Inhibitory activities of the tested compounds 5a-5h and 6a-6l from
TNF-α-induced NF-κB activation assay are presented in Table 1. As
shown in Table 1, some synthesized AA contained 1,2,3-triazoles
derivatives display highly NF-κB inhibition potency, with IC50
values mostly in the micromolar range. In addition, the investigated
compounds showed improvement inhibition of TNF-α-induced NF-
κB activation than parent compound AA, indicating introduction the
1,2,3-triazoles moieties resulted in increase in the activity.
At first, polar groups were investigated at R1 using halogen group
substitution, of which the bromine group substitution (5b) yielded a
moderate IC50 value of 15.04 μM. In contrast, in comparison to its
analogue 5b, bearing the acetyl moiety at R2, compound 6a is about
2-fold more potent. Even higher activities resulted by a substitution
with 4-fluorine (5c) and 2-fluorine (5g) at R1, with low IC50 values
of 5.39 and 4.78 μM, respectively. The 2-fluorine derivative with
hydroxyl moiety substitution at R2 (6k), which showed the highest
In continuation of our studies aiming for the discovery of more
potential compounds as NF-κB inhibitors, for present study, we
synthesized a series of 1,2,3-triazole derivatives based on the
modification at C-28 position of AA and assessed the potency of
these compounds towards TNF-α-induced NF-κB activation and the
anticancer activity against a number of cancer cell lines. The most
active compound 6k was further docked with NF-κB protein to
explore the possible binding interactions and investigated the
inhibitory potency of migration and drug resistant.
Results and discussion
Chemistry
The general procedures for the synthesis of AA bearing 1,2,3-
triazoles derivatives are outlined in Scheme 1. Compound 2 was
synthesized by the treatment of AA (1) with acetic anhydride in dry
pyridine. Compound 2 were followed by treatment of potassium
dichromate in acetic acid to obtain compound 3, which was then
reacted with propargyl amine and sodium hydride in presence of
THF at room temperature to afford compound 4. Compound 4 was
reacted with different substituted aromatic azides (9), sodium
ascorbate and CuSO4·5H2O in THF to yield compounds 5. The
aromatic azides (9) are prepared by addition of substituted phenyl
amine (7) in the presence of HCl with NaNO2, this intermediate was
then reacted with sodium azide in DMF. Hydrolyzation reaction of
compounds 5 with NaOH (aq) resulted into the formation of target
compounds 6. All the target compounds (5 and 6) were confirmed by
spectroscopic methods, including 1H NMR, 13C NMR and high
resolution mass spectroscopy (HR-MS).
activity among the investigated compounds, yielded in
a
significantly lower IC50 of 0.14 μM. Similar activities resulted from
a 2- bromine (6j, 1.88 μM) substituent. The resulting IC50 values of
compounds 5a−5h, all contained an acetyl group at R2, were
considerably higher than corresponding analogues (6a-6l), lacking
the acetyl function at R2, illustrating the beneficial impact of the
hydroxyl group.22 Replacement of the bromine group at R1 of 6a
with 4-methyl group led to compound 6b, which resulted in
decreased activity (IC50 = 13.28 μM), while a electron withdrawing
functional group (-NO2) in 6c resulted in the recovery of activity
(IC50 = 0.92 μM). Also, installation of a 2-chlorine group at R1 led to
compound 6i (IC50 = 1.06 μM), which was 3 times more potent than
its analogue 6g with 2-methyl group (IC50 = 3.32 μM). In general,
compounds containing electron withdrawing functional groups (-F, -
Cl) displayed more potent inhibitory activity compared with the
electron donor functional groups (-CH3, -OCH3). In a similar trend
for compound 6d, substituents in meta-position with methyl group
exhibited weak potency. Additionally, by the comparison of IC50
values of para-, meta- and ortho-position substituents, it also
suggested that ortho-position substituent at R1 showed better
inhibitory activity compared to meta- or para-substituent.
Extraordinarily high inhibitory activities resulted from substitutions
at R1 with 2-nitro (IC50 = 0.23 μM), 2-fluorine (IC50 = 0.14 μM) and
2-chlorine (IC50 = 1.06 μM). Substitution with 2-bromine resulted in
good activities compared with 3-bromine and 4-bromine
substitution. Unfortunately, a bulky 2-methoxy group substitution
(6h) entirely abolished activity. In shorts, the investigation of AA
containing 1,2,3-triazoles derivatives implied that the introduction of
1,2,3-triazoles moieties at C-28 position and retained hydroxyl
29
30
13
20
21
22
O
H
19
18
O
O
12
26
17
16
11
25
H
H
a
b
28
COOH
14H
1
AcO
AcO
OH
AcO
AcO
OH
9
HO
HO
H
H
150H
6
24
2
3
8
15
27
7
H
4 H
3
23
1 (AA)
AcO
AcO
HO
2
R1
O
O
H
O
c
AcO
AcO
HN
O
N
H
H
H
f
AcO
AcO
AcO
N
HN
N
H
4
AcO
5
H
g
NH2
N2Cl
N3
d
e
R1
O
H
R1
R1
R1
O
N
H
9
8
7
HO
HO
N
HN
N
H
6
HO
Scheme 1. Synthetic pathway to target compounds 5 and 6. Reagents
and conditions: (a) acetic anhydride, pyridine, 8 h, r.t, (b) acetic acid,
K2CrO7, 70 °C, 5 h, r.t, (c) sodium hydride, THF, propargyl amine,
Et3N, 8 h, r.t (d) NaNO2, 10%HCl, 3 h, r.t, (e) sodium azide, DMF,
r.t, (f) CuSO4, Sodium-L-ascorbate, t-BuOH:H2O(1:1), 48 h, r.t, (g)
2 | MedChemComm, 2018, 00, 1-14
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groups at C-2, C-3, C-23 position in AA was beneficial for
View Article Online
DOI: 10.1039/C8MD00620B
inhibitory potency on NF-κB.
Table.1 In vitro inhibition of TNF-α-induced NF-κB activation in A549 lung adenocarcinoma cell line
O
H
O
H
N
NH
R2
R2
N
H
N
R2
R1
Compd.
R1
4-Cl
4-Br
4-F
R2
IC50 (μM)a
10.61±0.59
15.04±1.21
5.39±0.71
18.84±1.13
>20
5a
5b
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
-
5c
5d
3-F
5e
3-Br
2-Cl
2-F
5f
8.17±0.68
4.78±0.32
12.15±0.87
7.86±0.44
13.28±1.05
0.92±0.14
>20
5g
5h
2-Br
4-Br
4-CH3
4-NO2
3-CH3
3-Cl
3-Br
2-CH3
2-OCH3
2-Cl
2-Br
2-F
6a
6b
6c
6d
6e
17.26±1.18
19.79±0.93
3.32±0.47
>20
6f
6g
6h
6i
1.06±0.26
1.88±0.39
0.14±0.08
0.23±0.06
>20
6j
6k
6l
2-NO2
-
AA
IMD-0354
-
-
1.27±0.31
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a IC50 values are presented as the mean ± SD (standard error of the mean) from three separated experiments.
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with the immobilized NF-κB protein (Fig. 2D),OaIs: 1d0e.1m0o39n/sCtr8aMteDd0b0y62t0hBe
concentration-dependent responses for the association and
dissociation, respectively, and by the clearly discernible exponential
curves, indicative of the binding of compound 6k to and dissociation
from the NF-κB protein, with a binding affinity, equilibrium
dissociation constant (KD) of 0.36 μM. These data provide the
definitive evidence of the interaction of 6k with NF-κB protein.
Molecular docking
As revealed in the SAR studies, 6k exhibited the most potent
inhibitory activity towards TNF-α-induced NF-κB. To further
elucidate the improved potency of 6k at the molecular level, the
molecular docking studies of 6k in the active site of NF-κB were
performed using SYBYL-X 2.0 software. The docking score results
were summarized in Table S1. The best possible interacting mode of
compound 6k (docking score 10.75) at the binding site of the NF-κB
protein was shown in Fig. 1. As shown in Fig. 1, the docking model
proposed that 6k extended tightly into the cavity of the NF-κB active
site, adopting an energetically stable conformation. The fluorine
atom of terminal aromatic group formed one hydrogen-bonding
interaction with DNA chain (DA6), while the benzene ring
appropriately positioned to give π−π interactions with PHE307.
Remarkably, 1,2,3-triazoles moiety as an acceptor established four
hydrogen bonds with amino hydrogen of LYS272 and DA5, which
confirmed that this moiety was also crucial for binding and
contributed to improve the potency of 6k. Moreover, the polar
hydrogen of the C-2 and C-3 hydroxy moiety formed two hydrogen
bonds with the DNA backbone P=O of DG2. Also, the C-23 hydroxy
of compound 6k formed two hydrogen bonds with amine group of
LYS249. Additionally, the pentacyclic ring skeleton of compound
6k was surrounded by residues LYS241, PRO243, SER246,
ASN247, LYS249, ASP271 and LYS272 mainly through the
hydrophobic interaction. We also recheck docking study on NF-κB
structure without DNA fragments. As indicated in Fig. S1, C-2 and
C-23 hydroxy moiety of 6k formed two hydrogen bonds with
ASP291 and ARG295, respectively, and the terminal aromatic group
extended into a hydrophobic groove.
Fig. 2 Biophysical binding data supports the interaction of
compound 6k with NF-κB protein. Dose response curve determined
by SPR for the binding of NF-κB with 6k is shown. The
measurement yielded an equilibrium dissociation constant (KD) of
0.36
μM.
Compound 6k inhibits the NF-κB DNA binding
Since the docking experiments suggested compound 6k bound to
NF-κB through altering NF-κB/DNA interactions, we subsequently
evaluated its effects on the NF-κB signaling pathway using ELISA
based DNA binding assay. As expected, we observed a significant
inhibition of TNF-α-induced NF-κB activation in A549 cells treated
with 6k (Fig. 3). Compound 6k reduced the TNF-α-induced NF-κB
DNA binding activity by 18.2%, 34.1%, and 61.4% as compared
with TNF-α at 5, 10, and 20 μM, respectively. These results
indicated that 6k bound to NF-κB and modulated constitutive NF-κB
activation by interfering with the NF-κB/DNA interaction.
Fig. 1 Binding modes of compound 6k in the active site of NF-κB
(PDB: 1NFK). Ligands and the important residues for binding
interactions are represented by stick and line models. The hydrogen
bonds are shown as yellow dotted lines (color figure online).
Surface plasmon resonance assay
The docking study revealed that compound 6k directly binding to
NF-κB. To further validate this possibility, a biospecific interaction
analysis was performed through surface plasmon resonance (SPR)
based binding assay using Biacore T200. This analytic technique
allows measuring kinetic and thermodynamic parameters of
ligand−protein complex formation, and it is widely utilized to
investigate several enzyme/inhibitor interactions.33 Association and
dissociation measurements were taken and the binding affinity of
compound 6k for NF-κB was determined using Biacore evaluation
software. Results showed that compound 6k efficiently interacted
Fig. 3 ELISA based DNA binding assay to evaluate NF-κB DNA
binding ability following compound 6k treatment. The data is
expressed as mean ± SD, compared with the untreated control, and
TNF-α treated control (p < 0.05).
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and NF-κB. The results revealed that compound 6k suppressed the
View Article Online
DOI: 10.1039/C8MD00620B
IKKβ activation in a concentration-dependent manner stimulated by
Compound 6k inhibits NF-κB translocation to nucleus
TNF-α. These data also demonstrated that compound 6k inhibits
TNF-α–induced NF-κB activity via impairment of IκBα
phosphorylation mediated by the IKKβ phosphorylation.
Translocation to nuclear is a symbol for TNF-α induced NF-κB
activation. Recently, extensive research has reported that the
anticancer activity of pentacyclic triterpenoids is associated with the
suppression of NF-κB translocation to the nucleus.34
Mechanistically, NF-κB nuclear translocation was examined using
immunofluorescence staining. A549 cells were treated for 24 h with
10 μM of 6k, after which NF-κB translocation was stimulated by
adding TNF-α for 30 minutes. As shown in Fig. 4, in A549 cells
treated with TNF-α, an intense nuclear fluorescence was observed,
showing the nuclear translocation of NF-κB. NF-κB nuclear
translocation in the 6k–treated cells was inhibited in comparison
with TNF-α–stimulated cells. Therefore, these results confirmed that
6k blocked the nuclear translocation resulting in the inhibitory effect
of 6k on NF-κB activation.
Fig. 5. Suppression of the NF-κB signaling cascade by compound 6k
on A549 cells. The effect of compound 6k on the expression of p-
IκBα, p-IKKβ and NF-κB stimulated with TNF-α. β-Actin was used
as loading control. Means
experiments, P < 0.05.
± SD were from three separate
In vitro cytotoxicity activity
NF-κB is a transcription factor involved in the control of various
normal cellular and organism processes, such as immune and
inflammatory responses, cellular proliferation and tumorigenesis.
Due to the pivotal role of the transcription factor NF-κB in
tumorigenesis and survival, we speculate whether the NF-κB
inhibitory activity of AA containing 1,2,3-triazoles derivatives is
translated to anticancer activity. The in vitro cytotoxicity of the
synthesized compounds was evaluated by MTT assay against a panel
of cancer cell lines including human lung cancer cells (NCI-H460),
human liver cancer cells (HepG2), human non-small cell lung cancer
cells (A549), human bladder cancer cells (T24), and human normal
cell line (HL-7702). AA and doxorubicin (DOX) were used as the
positive control. As shown in Table 2, most of newly synthesized
AA derivatives exhibited a considerable growth inhibitory activity
than AA against the tested cell lines, indicating the introduction of
1,2,3-triazoles on AA could markedly increase of anticancer activity.
Interestingly, compound 6k was the most potent compound in this
series, with IC50 values of 5.24 μM, 4.46 μM, 2.67 μM and 3.96 μM
against HepG2, NCI-H460, A549 and T24 cancer cell lines,
respectively. Moreover, 6k showed lower toxicity toward to human
normal live cells HL-7702, while it was weaker than DOX.
Fig. 4. The effects of compound 6k on the nuclear translocation of
the NF-κB protein induced by cytokine TNF-α. After serum-free
overnight, A549 cells were pretreated with 6k (10 μM) for 24 hours,
followed by TNF-α (10 ng/mL) for 30 min, and then processed for
NF-κB nuclear translocation detection by immunofluorescence
staining.
Drug resistance is a critical therapeutic problem that limited the
efficacies of most anticancer drugs for a variety of human cancer
cells. In terms of the strong inhibitory effects on the tested cancer
cell lines, compounds 5c, 5g, 6c, 6g and 6i-6l were further evaluated
against DOX-resistant NCI-H460 (NCI-H460/DOX). As shown in
Table 2, DOX displayed low activity on NCI-H460/DOX resistant
cell lines. It was significant to observe that compound 6k had a
much lower resistance factor (1.08) for resistant NCI-H460 cell line.
Moreover, except for 5c, the other selected compounds showed
potent cytotoxicity against the corresponding DOX resistant cells
comparable to that of DOX. These results implied that these
compounds might be useful in the treatment of drug refractory
tumors resistant to DOX.
Compound 6k inhibites the activity of IKKβ and IκBα
phosphorylation
TNF-α triggers the nuclear translocation of NF-κB via the
phosphorylation and activation of the IKK complex, which
subsequently phosphorylates of IκBα and result in the activation of
NF-κB.35 To determine if inhibition of TNF-α-induced NF-κB
activation and nuclear translocation is caused by inhibition IκBα
phosphorylation, the expression of phosphorylated IKKβ and IκBα
were evaluated by Western blotting assay. As shown in Fig. 5, after
treatment with the TNF-α, the phosphorylated IκBα levels were
markedly increased, while compound 6k significantly inhibited this
phosphorylation in a concentration-dependent manner. Meanwhile,
as illustrated in Fig. 5, similar trend was also observed on p-IKKβ
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Table. 2 Cytotoxicity of compounds 5a-5h and 6a-6l against different cancer cell lines
IC50(μM)a
DOI: 10.1039/C8MD00620B
Compd.
5a
T24
HepG2
29.76±0.57
37.55±0.88
25.12±1.25
43.75±1.48
>50
A549
NCI-H460
26.84±0.36
34.63±0.99
20.78±0.85
40.91±1.37
>50
NCI-H460/DOX
n.d.b
HL-7702
>50
RFc
27.39±1.15
33.82±0.63
20.27±0.54
39.28±1.26
>50
26.71±1.06
33.47±1.21
18.51±1.34
38.23±1.26
>50
-
5b
5c
n.d.
>50
-
>50.
>50
-
5d
5e
n.d
>50
-
n.d.
>50
-
5f
22.47±1.23
17.33±1.09
31.95±0.43
23.62±1.35
34.49±1.12
9.10±0.74
>50
26.93±0.91
21.48±0.78
36.84±0.96
28.42±1.03
37.29±1.07
10.32±0.45
>50
21.85±0.68
16.74±0.93
30.26±0.87
20.36±0.62
33.15±0.98
8.98±0.56
>50
23.50±0.59
19.06±0.76
34.49±1.14
24.96±1.46
34.88±0.75
9.65±0.29
>50
n.d.
>50
-
5g
27.11±1.05
n.d.
>50
1.42
5h
6a
>50
-
n.d.
>50
-
6b
6c
n.d.
>50
-
1.49
-
14.43±0.58
n.d.
>50
6d
6e
>50
37.69±0.71
41.14±2.58
14.95±1.15
>50
40.58±0.87
45.80±1.80
18.71±0.90
>50
35.68±1.25
39.87±1.55
14.78±0.32
>50
39.73±0.79
43.57±1.64
15.92±0.57
>50
n.d.
>50
-
6f
n.d.
>50
-
6g
17.41±0.34
n.d.
>50
1.09
-
6h
6i
>50
9.61±0.63
10.67±0.72
3.96±0.21
7.51±0.54
43.97±1.36
0.96±0.09
12.66±0.86
14.87±0.37
5.24±1.34
8.36±1.11
35.37±1.47
1.37±0.24
9.24±1.14
10.20±0.49
2.67±0.06
5.16±0.51
41.02±0.78
1.07±0.18
11.74±0.37
12.05±0.84
4.46±0.15
7.85±0.83
44.82±1.25
0.8±0.06
13.99±0.95
16.92±1.01
4.84±0.42
10.27±0.35
n.d.
>50
1.19
1.40
1. 08
1.31
-
6j
>50
6k
6l
>50
>50
AA
DOX
>50
30.99±1.15
10.44±0.5
38.74
b
c
a IC50 values are presented as the mean ± SD (standard error of the mean) from three separated experiments. n.d. = not detected. RF
(resistant factor) is defined as IC50 in NCI-H460/DOX /IC50 in NCI-H460.
with compound 6k, we observed a dose-dependent increase in the
percentage of apoptotic cells, at the concentrations of 5 μM, 10 μM
and 20 μM for 24 h. As shown in Fig. 6, few (1.58%) apoptotic cells
Compound 6k induces apoptosis in A549 cells
The ability of 6k to induce apoptosis was investigated by flow
cytometry. A549 cells were allowed to grow in the presence or
absence of 6k for 24 h, and co-stained with PI and Annexin-V/FITC.
Four quadrant images, Q1, Q2, Q3, and Q4, represented four
different cell states: necrotic cells, late apoptotic or necrotic cells,
early apoptotic cells and living cells, respectively. In the cells treated
were present in the control panel, in contrast, the percentage rose to
11.80% at the concentration of 5 μM after treatment with 6k for 24
h. In the presence of 6k with concentrations of 10 μM and 20 μM,
the apoptotic cells were further increased to 29.42% and 40.38%,
respectively. These data suggested that compound 6k effectively
induced apoptosis in A549 cells in a dose-dependent manner.
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number of migration cells in a dose-dependent manneVrieawftAerrtic2le4Ohnlianes
compare with control. This demonstrated thDaOtI:th10e.1N03F9-/κCB8MinDh0i0b6it2o0rBy
functionality is critical for 6k to suppress migration
Fig. 7. Compound 6k induced apoptotic in A549 cells were
determined by AO/EB staining and were photographed via
fluorescence microscopy. A549 cells were treated with 6k (5, 10 and
20 μM, for 24 h), incubated with AO/EB. Images were acquired
using a Nikon Te2000 deconvolution microscope (magnification
200×).
Fig. 6. Annexin V-FITC and PI staining to evaluate apoptosis in
A549 cells following compound 6k treatment. A549 cells were
treated with 6k (5, 10 and 20 μM, for 24 h), incubated with annexin
V-FITC and PI and analyzed using flow cytometry.
Apoptosis assay by acridine orange/ethidium bromide (AO/EB)
staining
To further characterize the cell apoptosis induced by compound 6k,
AO/EB staining was performed to evaluate the accompanying
changes in morphology. A549 cells were exposed to compound 6k at
concentrations of 5 μM, 10 μM and 20 μM for 24 h. The results (Fig.
7) showed that the morphology of A549 cells had changed
significantly in the presence of 6k. The cell nuclei were stained
yellow green or orange, and the morphology showed pycnosis,
membrane blebbing and cell budding characteristic of apoptosis. The
nearly complete absence of red-stained cells showed that 6k
treatment was associated with low toxicity. Hence, the observation
results further demonstrated that compound 6k induced apoptosis in
A549 cells.
Compound 6k inhibited the migration of A549 cell in vitro
NF-κB is constitutively activated in a number of cancer cells and
promotes tumor proliferation, invasion, and metastasis.36 Increasing
evidence has revealed that the metastatic cancer cells exerted great
capability of migration and invasion.37 Migration plays a key role in
later period of cancer progression and is known to connect with the
tumor progression and metastatic cascade. Since cell migration is
connected with the metastatic activity of cancer cells, transwell
migration assays were carried out to investigate whether compound
6k could suppress the migration of A549 cells. The migration of
A549 cells was recorded by microscopic observations at the
concentrations of 5 μM, 10 μM and 20 μM of compound 6k for 24 h.
Results from Fig. 8 clearly showed that 6k strongly decrease the
Fig. 8 Transwell assay detected migration inhibition of A549 cells
after incubation with the compound 6k at the concentrations of 5, 10
and 20 μM for 24 h, respectively. The experiments were performed
three times, and the results of the representative experiments are
shown.
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View Article Online
DOI: 10.1039/C8MD00620B
Conclusions
A series of AA containing 1,2,3-triazoles derivatives were rationally 8. Then, the mixture was diluted with ethyl acetate (20 mL) and
designed and synthesized as anticancer agents which targeted NF-κB washed with water (10 mL × 5). The organic phase was dried over
with good IC50 values in low micromolar range. The cell-based anhydrous sodium sulfate, and the solvent was evaporated under
luciferase assay led to the identification of compound 6k, possessing reduced pressure. The crude product was purified by silica gel
high affinity and potency, as a potent inhibitor of TNF-α-induced chromatography with a gradient elution of CH2Cl2/MeOH (V:V =
NF-κB activation. A docking study of the most active compound 6k 30:1) to yield compound 3. Compound 3 (125.6 mg, 0.2 mmol) was
revealed key interactions between 6k and NF-κB in which the 1,2,3- allowed to react with NaH (80% in oil, 0.019 g, 0.8 mmol) in dry
triazoles moiety and the hydroxyl groups of AA skeleton were THF (20 mL) under nitrogen atmosphere for 8 h before a solution of
important for improving inhibitory activity. Subsequently, surface propargyl amine (0.025 g; 0.45 mmol in 5 mL THF) was slowly
plasmon resonance analysis validated the interaction between added. After the reaction was completed, the reaction mixture was
compound 6k and NF-κB protein with an equilibrium dissociation extracted with ethyl acetate. The combined organic layer was dried
constant (KD) value of 0.36 μM. Further studies revealed that over anhydrous Na2SO4 and concentrated under reduced pressure to
compound 6k observably inhibited the NF-κB DNA binding, nuclear give crude product, which was purified by column chromatography
translocation and IκBα phosphorylation. Notably, further antitumor using 5% EtOAc:Hexane to give the propargylated product 4. The
activity screening showed that compound 6k (IC50 = 2.67 ± 0.06 title compounds (5) were prepared by the reaction of substituted aryl
μM) exhibited the best anticancer activity against the A549 cell line, azides (1.5 mmol), intermediate 4 (1.0 mmol), CuSO4·5H2O (0.1
while displayed slightly weaker inhibitory activity than DOX. The mmol) in t-BuOH : H2O (1:1) mixture. Sodium ascorbate (0.1 mmol)
exposure of compound 6k to A549 cells resulted in inducing was added to the reaction mixture and the contents stirred vigorously
apoptosis evidenced by flow cytometry and AO/EB staining assay. at room temperature for 48 h till completion of the reaction
In addition, transwell migration assays indicated that compound 6k (monitored by TLC analysis). The reaction was worked up by
inhibition of in vitro cell migration by blockage of NF-κB signaling dilution of the contents with water and extraction with ethyl acetate
pathway. Consequently, the rational design of AA containing 1,2,3- (3 times). The combined ethyl acetate extract was washed with brine
triazoles derivatives offers significant potential for the discovery of a solution, dried over anhydrous Na2SO4 and evaporated under
new class of NF-κB inhibitors with the ability to suppress cancer reduced pressure. The crude product was purified by
cells migration and induce apoptosis.
chromatography on silica gel eluted with ethyl acetate/Hexane (V: V
= 3:1) to offer compounds 5a-5k. The structures were confirmed by
1H NMR, 13C NMR and HR-MS (see Supporting information).
Experimental methods
Compound 5a: Yields 88%, as a white solid. Mp: 164.7−165.8 °C.
1H NMR (400 MHz, CD3OD) δ 8.31 (s, 1H, H-33), 7.84‒7.81 (m,
2H, Ar-H), 7.59‒7.57 (m, 2H, Ar-H), 5.57 (s, 1H, H-12), 5.23‒5.16
(m, 1H, H-2), 4.99 (d, J = 11.2 Hz, 1H, H-3), 4.49‒4.37 (m, 2H, H-
31), 3.79 (d, J = 9.6 Hz, 1H, H-23), 3.62 (d, J = 10.8 Hz, 1H, H-23),
3.07 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.46 (d, J = 10.8 Hz, 1H, H-
16), 2.44 (s, 1H, H-9), 2.25‒2.18 (m, 1H, H-20), 2.04 (s, 3H,
CH3CO), 2.01 (s, 3H, CH3CO), 1.95 (s, 3H, CH3CO), 1.79‒1.09 (m,
15H), 1.31 (s, 3H, CH3-27), 1.04 (s, 3H, CH3-24), 0.97 (d, J = 6.4
Hz, 3H, CH3-29), 0.87 (d, J = 6.4 Hz, 3H, CH3-30), 0.85 (s, 3H,
CH3-25), 0.48 (s, 3H, CH3-26). 13C NMR (100 MHz, CD3OD) δ
200.94, 178.95, 172.37, 172.19, 172.00, 166.45, 147.21, 136.96,
135.48, 131.17, 131.05, 123.04, 122.74, 76.11, 70.47, 66.17, 61.99,
54.04, 48.42, 48.31, 45.74, 45.04, 44.87, 43.12, 39.98, 39.86, 38.92,
38.01, 35.54, 33.39, 31.49, 29.18, 24.72, 21.34, 21.00, 20.76, 19.54,
18.24, 17.97, 17.64, 14.13. HR-MS (m/z) (ESI): calc for
C45H59ClN4O8 [M + Na]+ : 841.3914, founded: 841.3893.
Compound 5b: Yields 85%, as a white solid. Mp: 177.1−179.0 °C.
1H NMR (400 MHz, CD3OD) δ 8.32 (s, 1H, H-33), 7.78‒7.71 (m,
4H, Ar-H), 5.57 (s, 1H, H-12), 5.23‒5.16 (m, 1H, H-2), 4.99 (d, J =
11.2 Hz, 1H, H-3), 4.49‒4.37 (m, 2H, H-31), 3.79 (d, J = 9.6 Hz,
1H, H-23), 3.62 (d, J = 10.8 Hz, 1H, H-23), 3.07 (dd, J = 12.8, 4.4
Hz, 1H, H-19), 2.45 (d, J = 10.8 Hz, 1H, H-16), 2.44 (s, 1H, H-9),
2.25‒2.17 (m, 1H, H-20), 2.04 (s, 3H, CH3CO), 2.01 (s, 3H,
CH3CO), 1.95 (s, 3H, CH3CO), 1.79‒1.09 (m, 15H), 1.31 (s, 3H,
CH3-27), 1.03 (s, 3H, CH3-24), 0.97 (d, J = 6.4 Hz, 3H, CH3-29),
General
Asiatic acid purchase from biological technology of wuhan, China.
All the chemical reagents and solvents used were of analytical grade.
All the chemical reagents and solvents used were of analytical grade.
Silica gel (200-300 mesh) used in column chromatography was
provided by Tsingtao Marine Chemistry Co. Ltd. H NMR and 13C
1
NMR spectra were recorded on a BRUKER AV-400 spectrometer
with TMS as an internal standard in CDCl3. Mass spectra were
determined on an FTMS ESI spectrometer.
Synthesis: general procedure for compounds 5a-5h
AA (1, 200 mg, 0.4 mmol) was added to pyridine (10 mL) following
acetic anhydride (0.5 mL, 5.0 mmol) and the mixture was stirred at
o
20 C for 8 h. After the reaction, the mixture was diluted with ethyl
acetate (25 mL), washed with aqueous 1 M HCl (10 mL×5),
saturated CuSO4 (15 mL× 2) and saturated NaCl solution (20 mL).
The organic phase was dried with anhydrous sodium sulfate, and the
solvent was evaporated under reduced pressure. The crude product
was purified by column chromatography on silica gel eluted with
petroleum ether/ethyl acetate (V:V = 3:1) to give 2. Compound 2
(100 mg, 0.16 mmol) and K2Cr2O7·2H2O (150 g, 0.5 mmol) were
added to acetic acid (20 mL) and refluxed for 5 h. The mixture was
cooled to 20 oC and neutralized with 10% NaHCO3 solution to pH 7-
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0.87 (d, J = 6.4 Hz, 3H, CH3-30), 0.85 (s, 3H, CH3-25), 0.47 (s, 3H, 0.94 (d, J = 6.4 Hz, 3H, CH3-29), 0.85 (s, 3H, CH3-25), 0.83 (d, J =
View Article Online
CH3-26). 13C NMR (100 MHz, CD3OD) δ 200.92, 178.93, 172.37, 6.4 Hz, 3H, CH3-30), 0.62 (s, 3H, CH3-26). C NMR (100 MHz,
13
DOI: 10.1039/C8MD00620B
172.19, 171.99, 166.45, 147.24, 137.40, 134.09, 131.16, 123.25, CDCl3) δ 198.68, 176.78, 170.86, 170.53, 170.17, 163.40, 145.67,
123.00, 122.93, 76.11, 70.47, 66.17, 61.97, 54.03, 48.41, 48.30, 137.88, 131.94, 131.22, 130.45, 123.67, 123.46, 121.32, 118.89,
45.73, 45.03, 44.86, 43.12, 39.97, 39.84, 38.91, 38.01, 35.53, 33.38, 74.92, 69.06, 65.30, 61.14, 52.99, 47.38, 47.22, 44.61, 44.20, 43.94,
31.50, 29.17, 24.72, 21.34, 21.02, 20.80, 20.77, 19.54, 18.26, 17.97, 41.96, 39.05, 38.68, 37.73, 37.09, 34.98, 32.52, 30.56, 28.14, 24.39,
17.65, 14.17. HR-MS (m/z) (ESI): calc for C45H59BrN4O8 [M + Na]+ 21.09, 20.98, 20.95, 20.86, 18.73, 17.76, 17.18, 17.10, 13.95. HR-
: 885.3408, founded: 885.3391.
MS (m/z) (ESI): calc for C45H59BrN4O8 [M + Na]+ : 885.3408,
Compound 5c: Yields 84%, as a white solid. Mp: 180.3−182.2 °C. founded: 885.3397.
1H NMR (400 MHz, CD3OD) δ 8.27 (s, 1H, H-33), 7.85‒7.82 (m,
Compound 5f: Yields 83%, as a white solid. Mp: 166.4−168.3 °C.
2H, Ar-H), 7.35‒7.31 (m, 2H, Ar-H), 5.58 (s, 1H, H-12), 5.23‒5.17 1H NMR (400 MHz, CD3OD) δ 8.14 (s, 1H, H-33), 7.70‒7.67 (m,
(m, 1H, H-2), 5.00 (d, J = 11.2 Hz, 1H, H-3), 4.48‒4.37 (m, 2H, H- 2H, Ar-H), 7.60‒7.52 (m, 2H, Ar-H), 5.59 (s, 1H, H-12), 5.26‒5.19
31), 3.79 (d, J = 9.6 Hz, 1H, H-23), 3.62 (d, J = 10.8 Hz, 1H, H-23), (m, 1H, H-2), 5.02 (d, J = 11.2 Hz, 1H, H-3), 4.53‒4.40 (m, 2H, H-
3.07 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.47 (d, J = 10.8 Hz, 1H, H- 31), 3.82 (d, J = 9.6 Hz, 1H, H-23), 3.63 (d, J = 10.8 Hz, 1H, H-23),
16), 2.46 (s, 1H, H-9), 2.26‒2.19 (m, 1H, H-20), 2.05 (s, 3H, 3.09 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.49 (s, 1H, H-9), 2.47 (d, J =
CH3CO), 2.01 (s, 3H, CH3CO), 1.96 (s, 3H, CH3CO), 1.81‒1.12 (m, 10.8 Hz, 1H, H-16), 2.27‒2.19 (m, 1H, H-20), 2.05 (s, 3H, CH3CO),
15H), 1.33 (s, 3H, CH3-27), 1.07 (s, 3H, CH3-24), 0.98 (d, J = 6.4 2.01 (s, 3H, CH3CO), 1.96 (s, 3H, CH3CO), 1.83‒1.12 (m, 15H),
Hz, 3H, CH3-29), 0.88 (d, J = 6.4 Hz, 3H, CH3-30), 0.85 (s, 3H, 1.35 (s, 3H, CH3-27), 1.19 (s, 3H, CH3-24), 0.98 (d, J = 6.4 Hz, 3H,
CH3-25), 0.52 (s, 3H, CH3-26). 13C NMR (100 MHz, CD3OD) CH3-29), 0.89 (s, 3H, CH3-25), 0.88 (d, J = 6.4 Hz, 3H, CH3-30),
δ200.99, 178.99, 172.40, 172.21, 172.03, 166.46, 163.87 (1J(C,F) = 0.70 (s, 3H, CH3-26). 13C NMR (100 MHz, CD3OD) δ 200.98,
246.0 Hz), 147.08, 134.73 (4J(C,F) = 4.0 Hz), 131.18, 123.59 179.01, 172.39, 172.20, 172.01, 166.43, 146.27, 136.02, 132.52,
(3J(C,F) = 9.0 Hz), 123.23, 117.72 (2J(C,F) = 23.0 Hz), 76.12, 70.49, 131.98, 131.18, 129.95, 129.34, 129.01, 126.67, 76.14, 70.47, 66.19,
66.16, 62.03, 54.06, 48.44, 48.34, 45.78, 45.08, 44.88, 43.14, 40.00, 62.09, 53.95, 48.45, 45.87, 45.12, 44.90, 43.15, 39.97, 39.86, 38.96,
39.88, 38.94, 38.03, 35.55, 33.41, 31.50, 29.19, 24.71, 21.34, 21.31, 38.02, 35.63, 33.47, 31.49, 29.23, 24.69, 21.39, 21.34, 21.01, 20.80,
20.98, 20.75, 20.73, 19.52, 18.23, 17.99, 17.62, 14.10. (1J(C,F) = 20.77, 19.82, 18.36, 18.07, 17.66, 14.18. HR-MS (m/z) (ESI): calc
249.3 Hz). HR-MS (m/z) (ESI): calc for C45H59FN4O8 [M + Na]+ : for C45H59ClN4O8 [M + Na]+ : 841.3914, founded: 841.3895.
825.4209, founded: 825.4191.
Compound 5g: Yields 86%, as a white solid. Mp: 187.8−189.6 °C.
Compound 5d: Yields 86%, as a white solid. Mp: 182.4−183.1 °C. 1H NMR (400 MHz, CD3OD) δ 8.35 (s, 1H, H-33), 7.67‒7.57 (m,
1H NMR (400 MHz, CD3OD) δ 8.20 (s, 1H, H-33), 7.86‒7.81 (m, 3H, Ar-H), 7.27‒7.22 (m, 1H, Ar-H), 5.57 (s, 1H, H-12), 5.23‒5.16
1H, Ar-H), 7.59‒7.54 (m, 1H, Ar-H), 7.46‒7.39 (m, 2H, Ar-H), 5.58 (m, 1H, H-2), 4.99 (d, J = 11.2 Hz, 1H, H-3), 4.49‒4.37 (m, 2H, H-
(s, 1H, H-12), 5.24‒5.17 (m, 1H, H-2), 5.00 (d, J = 11.2 Hz, 1H, H- 31), 3.78 (d, J = 9.6 Hz, 1H, H-23), 3.62 (d, J = 10.8 Hz, 1H, H-23),
3), 4.52‒4.38 (m, 2H, H-31), 3.80 (d, J = 9.6 Hz, 1H, H-23), 3.63 (d, 3.07 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.46 (d, J = 10.8 Hz, 1H, H-
J = 10.8 Hz, 1H, H-23), 3.08 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.47 16), 2.44 (s, 1H, H-9), 2.26‒2.18 (m, 1H, H-20), 2.04 (s, 3H,
(s, 1H, H-9), 2.46 (d, J = 10.8 Hz, 1H, H-16), 2.28‒2.19 (m, 1H, H- CH3CO), 2.01 (s, 3H, CH3CO), 1.95 (s, 3H, CH3CO), 1.80‒1.18 (m,
20), 2.05 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 1.96 (s, 3H, 15H), 1.32 (s, 3H, CH3-27), 1.04 (s, 3H, CH3-24), 0.98 (d, J = 6.4
CH3CO), 1.81‒1.13 (m, 15H), 1.34 (s, 3H, CH3-27), 1.11 (s, 3H, Hz, 3H, CH3-29), 0.88 (d, J = 6.4 Hz, 3H, CH3-30), 0.84 (s, 3H,
CH3-24), 0.99 (d, J = 6.4 Hz, 3H, CH3-29), 0.89 (d, J = 6.4 Hz, 3H, CH3-25), 0.48 (s, 3H, CH3-26). 13C NMR (100 MHz, CD3OD) δ
CH3-30), 0.87 (s, 3H, CH3-25), 0.57 (s, 3H, CH3-26). 13C NMR (100 200.98, 179.00, 172.41, 172.22, 172.03, 166.49, 164.50 (1J(C,F) =
MHz, CD3OD) δ 201.07, 179.09, 172.46, 172.27, 172.08, 166.51, 245.3 Hz), 147.20, 139.56 (3J(C,F) = 10.1 Hz), 132.81 (3J(C,F) = 9.0
155.25 (1J(C,F) = 249.3 Hz), 146.75, 132.16 (1J(C,F) = 8.0 Hz), Hz), 131.17, 123.21, 116.92 (4J(C,F) = 3.2 Hz), 116.58 (2J(C,F) =
131.20, 126.54 (1J(C,F) = 3.8 Hz), 126.35 (1J(C,F) = 9.0 Hz), 21.3 Hz), 108.81 (2J(C,F) = 26.6 Hz), 76.12, 70.47, 66.17, 62.01,
126.31, 126.05 (1J(C,F) = 6.4 Hz), 118.26 (1J(C,F) = 10.0 Hz), 54.05, 48.43, 48.33, 45.75, 45.06, 44.88, 43.13, 39.99, 39.87, 38.93,
76.15, 70.51, 66.17, 62.09, 54.03, 48.47, 48.38, 45.80, 45.11, 44.92, 38.04, 35.50, 33.40, 31.50, 29.18, 24.72, 21.33, 21.31, 20.98, 20.76,
43.16, 40.01, 39.90, 38.96, 38.07, 35.53, 33.44, 31.51, 29.21, 24.70, 20.74, 19.50, 18.21, 17.97, 17.62, 14.08. HR-MS (m/z) (ESI): calc
21.33, 21.30, 20.96, 20.74, 20.72, 19.48, 18.23, 18.01, 17.60, 14.12. for C45H59FN4O8 [M + Na]+ : 825.4209, founded: 825.4194.
HR-MS (m/z) (ESI): calc for C45H59FN4O8 [M + Na]+ : 825.4209,
founded: 825.4195.
Compound 5h: Yields 84%, as a white solid. Mp: 182.2−184.6 °C.
1H NMR (400 MHz, CD3OD) δ 8.10 (s, 1H, H-33), 7.87‒7.84 (m,
Compound 5e: Yields 87%, as a white solid. Mp: 172.5−174.3 °C. 1H, Ar-H), 7.61‒7.56 (m, 1H, Ar-H), 7.54‒7.49 (m, 2H, Ar-H), 5.60
1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H, H-33), 7.91‒7.90 (m, 1H, (s, 1H, H-12), 5.26‒5.20 (m, 1H, H-2), 5.02 (d, J = 11.2 Hz, 1H, H-
Ar-H), 7.62‒7.60 (m, 1H, Ar-H), 7.56‒7.54 (m, 1H, Ar-H), 3), 4.53‒4.41 (m, 2H, H-31), 3.82 (d, J = 9.6 Hz, 1H, H-23), 3.64 (d,
7.39‒7.35 (m, 1H, Ar-H), 5.63 (s, 1H, H-12), 5.27‒5.20 (m, 1H, H- J = 10.8 Hz, 1H, H-23), 3.10 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.50
2), 5.01 (d, J = 11.2 Hz, 1H, H-3), 4.57‒4.52 (m, 1H, H-31), (s, 1H, H-9), 2.48 (d, J = 10.8 Hz, 1H, H-16), 2.28‒2.20 (m, 1H, H-
4.41‒4.36 (m, 1H, H-31), 3.79 (d, J = 9.6 Hz, 1H, H-23), 3.55 (d, J = 20), 2.06 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 1.96 (s, 3H,
10.8 Hz, 1H, H-23), 3.14 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.37 (d, J CH3CO), 1.83‒1.12 (m, 15H), 1.36 (s, 3H, CH3-27), 1.21 (s, 3H,
= 10.8 Hz, 1H, H-16), 2.32 (s, 1H, H-9), 2.19‒2.10 (m, 1H, H-20), CH3-24), 0.99 (d, J = 6.4 Hz, 3H, CH3-29), 0.90 (s, 3H, CH3-25),
2.05 (s, 3H, CH3CO), 1.99 (s, 3H, CH3CO), 1.93 (s, 3H, CH3CO), 0.89 (d, J = 6.4 Hz, 3H, CH3-30), 0.74 (s, 3H, CH3-26). 13C NMR
1.75‒1.10 (m, 15H), 1.25 (s, 3H, CH3-27), 1.14 (s, 3H, CH3-24), (100 MHz, CD3OD) δ 201.08, 179.07, 172.44, 172.25, 172.06,
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166.50, 146.21, 137.72, 135.17, 132.87, 131.19, 129.89, 129.38, 2.97 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.45 (d, J = 10.8 Hz, 1H, H-
View Article Online
DOI: 10.1039/C8MD00620B
126.73, 119.74, 76.16, 70.51, 66.19, 62.13, 53.98, 48.48, 48.41, 16), 2.37 (s, 1H, H-9), 2.26‒2.18 (m, 1H, H-20), 1.79‒1.15 (m,
45.92, 45.15, 44.92, 43.17, 39.99, 39.90, 38.99, 38.05, 35.65, 33.49, 15H), 1.31 (s, 3H, CH3-27), 0.99 (d, J = 6.4 Hz, 3H, CH3-29), 0.92
31.49, 29.25, 24.69, 21.38, 21.32, 20.99, 20.78, 20.75, 19.92, 18.38, (s, 3H, CH3-24), 0.89 (d, J = 6.4 Hz, 3H, CH3-30), 0.59 (s, 3H, CH3-
18.09, 17.63, 14.18. HR-MS (m/z) (ESI): calc for C45H59BrN4O8 [M 25), 0.46 (s, 3H, CH3-26). 13C NMR (100 MHz, CD3OD) δ 201.79,
+ Na]+ : 885.3408, founded: 885.3387.
179.12, 166.40, 148.68, 147.72, 142.54, 131.27, 126.53, 123.28,
121.72, 77.58, 69.28, 65.76, 62.38, 54.11, 48.39, 48.14, 47.63,
45.86, 45.16, 44.16, 40.03, 39.89, 38.94, 38.08, 35.51, 33.34, 31.51,
29.18, 24.74, 21.38, 21.29, 19.65, 18.19, 18.01, 17.60, 13.80. HR-
MS (m/z) (ESI): calc for C39H53N5O7 [M + Na]+ : 726.3837, founded:
726.3826.
Synthesis: general procedure for compounds 6a-6l
To a solution of 5 (0.12 mmol) in MeOH (4 mL) and THF (6 mL), 4
N NaOH (1.2 mL) were added dropwise, and this reaction mixture
was stirred for 2 h at room temperature. The mixture was acidified
with hydrochloric acid and extracted with ethyl acetate. The organic
phase was washed with sodium bicarbonate solution and brine in
sequence, and concentrated under reduced pressure. The crude
product was purified column chromatography with a gradient elution
of CH2Cl2/MeOH (V:V = 20:1) to give a white solid of 6a-6l. The
Compound 6d: Yields 78%, as a white solid. Mp: 224.8−226.5 °C.
1H NMR (400 MHz, CD3OD) δ 8.23 (s, 1H, H-33), 7.67‒7.65 (m,
2H, Ar-H), 7.38‒7.36 (m, 2H, Ar-H), 5.56 (s, 1H, H-12), 4.48‒4.35
(m, 2H, H-31), 3.72‒3.66 (m, 1H, H-2), 3.49 (d, J = 11.2 Hz, 1H, H-
3), 3.33 (d, J = 9.6 Hz, 1H, H-23), 3.21 (d, J = 10.8 Hz, 1H, H-23),
2.99 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.45 (d, J = 10.8 Hz, 1H, H-
16), 2.41 (s, 3H, Ar-CH3), 2.37 (s, 1H, H-9), 2.25‒2.17 (m, 1H, H-
20), 1.79‒1.14 (m, 15H), 1.30 (s, 3H, CH3-27), 0.98 (d, J = 6.4 Hz,
3H, CH3-29), 0.95 (s, 3H, CH3-24), 0.88 (d, J = 6.4 Hz, 3H, CH3-
30), 0.62 (s, 3H, CH3-25), 0.45 (s, 3H, CH3-26). 13C NMR (100
MHz, CD3OD) δ 201.81, 179.07, 166.44, 146.87, 140.39, 136.05,
131.35, 131.27, 123.12, 121.30, 77.59, 69.31, 65.78, 62.40, 54.04,
48.17, 47.66, 45.82, 45.14, 44.17, 40.01, 39.87, 38.96, 38.09, 35.52,
33.34, 31.52, 29.16, 24.75, 21.37, 21.30, 21.06, 19.67, 18.22, 17.99,
17.60, 13.83. HR-MS (m/z) (ESI): calc for C40H56N4O5 [M + Na]+ :
695.4143, founded: 695.4136.
Compound 6e: Yields 80%, as a white solid. Mp: 219.7-220.9 °C.
1H NMR (400 MHz, CD3OD) δ 8.35 (s, 1H, H-33), 7.89‒7.88 (m,
1H, Ar-H), 7.77‒7.74 (m, 1H, Ar-H), 7.57‒7.53 (m, 1H, Ar-H),
7.49‒7.47 (m, 1H, Ar-H), 5.55 (s, 1H, H-12), 4.49‒4.37 (m, 2H, H-
31), 3.73‒3.66 (m, 1H, H-2), 3.49 (d, J = 11.2 Hz, 1H, H-3), 3.33 (d,
J = 9.6 Hz, 1H, H-23), 3.21 (d, J = 10.8 Hz, 1H, H-23), 2.99 (dd, J =
12.8, 4.4 Hz, 1H, H-19), 2.44 (d, J = 10.8 Hz, 1H, H-16), 2.36 (s,
1H, H-9), 2.24‒2.16 (m, 1H, H-20), 1.79‒1.13 (m, 15H), 1.29 (s, 3H,
CH3-27), 0.97 (d, J = 6.4 Hz, 3H, CH3-29), 0.96 (s, 3H, CH3-24),
0.87 (d, J = 6.4 Hz, 3H, CH3-30), 0.63 (s, 3H, CH3-25), 0.41 (s, 3H,
CH3-26). 13C NMR (100 MHz, CD3OD) δ 201.73, 179.03, 166.36,
147.22, 139.26, 136.53, 132.40, 131.23, 129.84, 123.30, 121.45,
119.55, 77.58, 69.28, 65.77, 62.35, 54.02, 48.32, 48.14, 47.63,
45.79, 45.11, 44.15, 39.98, 39.84, 38.93, 38.07, 35.46, 33.33, 31.52,
29.13, 24.75, 21.37, 21.33, 19.61, 18.29, 17.99, 17.62, 13.88. HR-
MS (m/z) (ESI): calc for C39H53ClN4O5 [M + Na]+ : 715.3597,
founded: 715.3586.
1
structures were confirmed by H NMR, 13C NMR and HR-MS (see
Supporting information).
Compound 6a: Yields 76%, as a white solid. Mp: 212.5−214.1 °C.
1H NMR (400 MHz, CD3OD) δ 8.31 (s, 1H, H-33), 7.78‒7.72 (m,
4H, Ar-H), 5.56 (s, 1H, H-12), 4.48‒4.36 (m, 2H, H-31), 3.73‒3.66
(m, 1H, H-2), 3.49 (d, J = 11.2 Hz, 1H, H-3), 3.33 (d, J = 9.6 Hz,
1H, H-23), 3.22 (d, J = 10.8 Hz, 1H, H-23), 2.98 (dd, J = 12.8, 4.4
Hz, 1H, H-19), 2.45 (d, J = 10.8 Hz, 1H, H-16), 2.37 (s, 1H, H-9),
2.26‒2.18 (m, 1H, H-20), 1.79‒1.14 (m, 15H), 1.31 (s, 3H, CH3-27),
0.99 (d, J = 6.4 Hz, 3H, CH3-29), 0.94 (s, 3H, CH3-24), 0.88 (d, J =
6.4 Hz, 3H, CH3-30), 0.63 (s, 3H, CH3-25), 0.43 (s, 3H, CH3-26).
13C NMR (100 MHz, CD3OD) δ 201.85, 179.11, 166.47, 147.25,
137.45, 134.09, 131.27, 123.31, 123.11, 122.97, 77.59, 69.32, 65.76,
62.40, 54.09, 48.17, 47.66, 45.84, 45.15, 44.19, 40.03, 39.89, 38.96,
38.10, 35.50, 33.34, 31.52, 29.17, 24.75, 21.36, 21.29, 19.68, 18.21,
18.00, 17.60, 13.86. HR-MS (m/z) (ESI): calc for C39H53BrN4O5 [M
+ Na]+ : 759.3092, founded: 759.3082.
Compound 6b: Yields 72%, as a white solid. Mp: 224.8−226.4 °C.
1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1H, H-33), 7.61‒7.56 (m,
2H, Ar-H), 7.45‒7.41 (m, 1H, Ar-H), 7.31‒7.29 (m, 1H, Ar-H), 5.56
(s, 1H, H-12), 4.49‒4.35 (m, 2H, H-31), 3.72‒3.65 (m, 1H, H-2),
3.49 (d, J = 11.2 Hz, 1H, H-3), 3.33 (d, J = 9.6 Hz, 1H, H-23), 3.21
(d, J = 10.8 Hz, 1H, H-23), 2.99 (dd, J = 12.8, 4.4 Hz, 1H, H-19),
2.46 (d, J = 10.8 Hz, 1H, H-16), 2.45 (s, 3H, Ar-CH3), 2.37 (s, 1H,
H-9), 2.25‒2.17 (m, 1H, H-20), 1.79‒1.13 (m, 15H), 1.30 (s, 3H,
CH3-27), 0.99 (d, J = 6.4 Hz, 3H, CH3-29), 0.95 (s, 3H, CH3-24),
0.88 (d, J = 6.4 Hz, 3H, CH3-30), 0.62 (s, 3H, CH3-25), 0.43 (s, 3H,
CH3-26). 13C NMR (100 MHz, CD3OD) δ 201.78, 179.07, 166.40,
146.88, 141.42, 138.27, 131.27, 130.76, 130.65, 123.26, 121.91,
118.52, 77.59, 69.30, 65.78, 62.40, 54.05, 48.16, 47.64, 45.81,
45.15, 44.17, 40.01, 39.87, 38.95, 38.12, 35.50, 33.35, 31.53, 29.15,
24.74, 21.45, 21.35, 21.30, 19.64, 18.24, 17.99, 17.60, 13.85. HR-
MS (m/z) (ESI): calc for C40H56N4O5 [M + Na]+ : 695.4143, founded:
695.4133.
Compound 6f: Yields 78%, as a white solid. Mp: 223.8−225.3 °C.
1H NMR (400 MHz, CD3OD) δ 8.35 (s, 1H, H-33), 8.04‒8.03 (m,
1H, Ar-H), 7.81‒7.79 (m, 1H, Ar-H), 7.64‒7.62 (m, 1H, Ar-H),
7.51‒7.47 (m, 1H, Ar-H), 5.55 (s, 1H, H-12), 4.49‒4.36 (m, 2H, H-
31), 3.73‒3.66 (m, 1H, H-2), 3.49 (d, J = 11.2 Hz, 1H, H-3), 3.33 (d,
J = 9.6 Hz, 1H, H-23), 3.21 (d, J = 10.8 Hz, 1H, H-23), 2.99 (dd, J =
12.8, 4.4 Hz, 1H, H-19), 2.44 (d, J = 10.8 Hz, 1H, H-16), 2.36 (s,
1H, H-9), 2.24‒2.16 (m, 1H, H-20), 1.79‒1.12 (m, 15H), 1.29 (s, 3H,
CH3-27), 0.97 (d, J = 6.4 Hz, 3H, CH3-29), 0.96 (s, 3H, CH3-24),
0.87 (d, J = 6.4 Hz, 3H, CH3-30), 0.63 (s, 3H, CH3-25), 0.40 (s, 3H,
CH3-26). 13C NMR (100 MHz, CD3OD) δ 201.71, 179.02, 166.34,
147.21, 139.32, 132.85, 132.60, 131.23, 124.32, 124.18, 123.30,
120.00, 77.58, 69.28, 65.78, 62.35, 54.02, 48.32, 48.14, 47.63,
Compound 6c: Yields 74%, as a white solid. Mp: 228.3−230.2 °C.
1H NMR (400 MHz, CD3OD) δ 8.47 (s, 1H, H-33), 7.46‒7.43 (m,
2H, Ar-H), 7.15‒7.12 (m, 2H, Ar-H), 5.56 (s, 1H, H-12), 4.50‒4.41
(m, 2H, H-31), 3.71‒3.64 (m, 1H, H-2), 3.48 (d, J = 11.2 Hz, 1H, H-
3), 3.31 (d, J = 9.6 Hz, 1H, H-23), 3.20 (d, J = 10.8 Hz, 1H, H-23),
10 | MedChemComm, 2018, 00, 1-14
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45.79, 45.11, 44.16, 39.98, 39.85, 38.93, 38.07, 35.46, 33.33, 31.53, (s, 1H, H-12), 4.53‒4.41 (m, 2H, H-31), 3.77‒3.70 (m, 1H, H-2),
View Article Online
29.13, 24.77, 21.36, 21.33, 19.61, 18.34, 18.00, 17.63, 13.94. HR- 3.51 (d, J = 11.2 Hz, 1H, H-3), 3.45 (d, J =D9O.6I: 1H0z.1,013H9/,CH8M-2D30)0,632.02B4
MS (m/z) (ESI): calc for C39H53BrN4O5 [M + Na]+ : 759.3092, (d, J = 10.8 Hz, 1H, H-23), 3.02 (dd, J = 12.8, 4.4 Hz, 1H, H-19),
founded: 759.3080.
2.46 (d, J = 10.8 Hz, 1H, H-16), 2.43 (s, 1H, H-9), 2.26‒2.19 (m,
Compound 6g: Yields 77%, as a white solid. Mp: 213.5−215.5 °C. 1H, H-20), 1.82‒1.22 (m, 15H), 1.34 (s, 3H, CH3-27), 1.13 (s, 3H,
1H NMR (400 MHz, CD3OD) δ 7.99 (s, 1H, H-33), 7.49‒7.43 (m, CH3-24), 0.98 (d, J = 6.4 Hz, 3H, CH3-29), 0.88 (d, J = 6.4 Hz, 3H,
2H, Ar-H), 7.39‒7.35 (m, 1H, Ar-H), 7.34‒7.32 (m, 1H, Ar-H), 5.58 CH3-30), 0.72 (s, 3H, CH3-25), 0.68 (s, 3H, CH3-26). 13C NMR (100
(s, 1H, H-12), 4.52‒4.39 (m, 2H, H-31), 3.76‒3.69 (m, 1H, H-2), MHz, CD3OD) δ 201.78, 179.09, 166.34, 146.22, 137.70, 135.16,
3.51 (d, J = 11.2 Hz, 1H, H-3), 3.35 (d, J = 9.6 Hz, 1H, H-23), 3.24 132.85, 131.26, 129.85, 129.36, 126.73, 119.74, 77.60, 69.28, 65.80,
(d, J = 10.8 Hz, 1H, H-23), 3.02 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 62.47, 53.94, 48.40, 48.16, 47.67, 45.97, 45.20, 44.19, 39.98, 39.86,
2.47 (d, J = 10.8 Hz, 1H, H-16), 2.43 (s, 1H, H-9), 2.28‒2.20 (m, 38.98, 38.07, 35.64, 33.42, 31.50, 29.22, 24.70, 21.45, 21.34, 20.04,
1H, H-20), 2.19 (s, 3H, Ar-CH3), 1.83‒1.22 (m, 15H), 1.35 (s, 3H, 18.40, 18.12, 17.64, 13.93. HR-MS (m/z) (ESI): calc for
CH3-27), 1.11 (s, 3H, CH3-24), 0.99 (d, J = 6.4 Hz, 3H, CH3-29), C39H53BrN4O5 [M + Na]+ : 759.3092, founded: 759.3081.
0.89 (d, J = 6.4 Hz, 3H, CH3-30), 0.69 (s, 3H, CH3-25), 0.67 (s, 3H,
Compound 6k: Yields 73%, as a white solid. Mp: 212.4−214.6 °C.
CH3-26). 13C NMR (100 MHz, CD3OD) δ 201.84, 179.17, 166.41, 1H NMR (400 MHz, CD3OD) δ 8.20 (s, 1H, H-33), 7.85‒7.81 (m,
146.27, 137.68, 134.75, 132.68, 131.30, 131.17, 128.03, 126.93, 1H, Ar-H), 7.59‒7.53 (m, 1H, Ar-H), 7.45‒7.38 (m, 2H, Ar-H), 5.57
126.29, 77.63, 69.32, 65.81, 62.51, 54.01, 48.45, 48.20, 47.70, (s, 1H, H-12), 4.52‒4.38 (m, 2H, H-31), 3.74‒3.67 (m, 1H, H-2),
45.99, 45.23, 44.21, 40.02, 39.90, 39.01, 38.10, 35.67, 33.44, 31.52, 3.50 (d, J = 11.2 Hz, 1H, H-3), 3.33 (d, J = 9.6 Hz, 1H, H-23), 3.23
29.23, 24.73, 21.42, 21.31, 20.01, 18.33, 18.10, 17.99, 17.62, 13.89. (d, J = 10.8 Hz, 1H, H-23), 3.00 (dd, J = 12.8, 4.4 Hz, 1H, H-19),
HR-MS (m/z) (ESI): calc for C40H56N4O5 [M + Na]+ : 695.4143, 2.46 (d, J = 10.8 Hz, 1H, H-16), 2.40 (s, 1H, H-9), 2.26‒2.20 (m,
founded: 695.4135.
1H, H-20), 1.81‒1.20 (m, 15H), 1.32 (s, 3H, CH3-27), 1.03 (s, 3H,
Compound 6h: Yields 72%, as a white solid. Mp: 214.4−217.1 °C. CH3-24), 0.99 (d, J = 6.4 Hz, 3H, CH3-29), 0.89 (d, J = 6.4 Hz, 3H,
1H NMR (400 MHz, CD3OD) δ 8.19 (s, 1H, H-33), 7.67‒7.64 (m, CH3-30), 0.65 (s, 3H, CH3-25), 0.55 (s, 3H, CH3-26). 13C NMR (100
1H, Ar-H), 7.51‒7.47 (m, 1H, Ar-H), 7.28‒7.25 (m, 1H, Ar-H), MHz, CD3OD) δ 201.81, 179.14, 166.37, 155.25 (1J(C,F) = 249.2
7.13‒7.09 (m, 1H, Ar-H), 5.57 (s, 1H, H-12), 4.51‒4.35 (m, 2H, H- Hz), 146.77, 132.13 (2J(C,F) = 7.9 Hz), 131.30, 126.50 (3J(C,F) =
31), 3.91 (s, 3H, Ar-OCH3), 3.74‒3.67 (m, 1H, H-2), 3.50 (d, J = 3.8 Hz), 126.41, 126.30, 126.05 (3J(C,F) = 6.5 Hz), 118.25 (2J(C,F)
11.2 Hz, 1H, H-3), 3.33 (d, J = 9.6 Hz, 1H, H-23), 3.23 (d, J = 10.8 = 19.9 Hz), 77.62, 69.31, 65.80, 62.46, 54.02, 48.40, 48.18, 47.68,
Hz, 1H, H-23), 3.00 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.46 (d, J = 45.86, 45.19, 44.19, 40.02, 39.88, 38.97, 38.10, 35.53, 33.38, 31.52,
10.8 Hz, 1H, H-16), 2.39 (s, 1H, H-9), 2.26‒2.18 (m, 1H, H-20), 29.19, 24.73, 21.39, 21.31, 19.62, 18.23, 18.04, 17.61, 13.86. HR-
1.81‒1.18 (m, 15H), 1.32 (s, 3H, CH3-27), 1.02 (s, 3H, CH3-24), MS (m/z) (ESI): calc for C39H53FN4O5 [M + H]+ : 677.4073,
0.98 (d, J = 6.4 Hz, 3H, CH3-29), 0.88 (d, J = 6.4 Hz, 3H, CH3-30), founded: 677.4078.
0.65 (s, 3H, CH3-25), 0.53 (s, 3H, CH3-26). 13C NMR (100 MHz,
Compound 6l: Yields 76%, as a white solid. Mp: 218.3−219.1 °C.
CD3OD) δ 201.79, 179.09, 166.34, 152.78, 145.83, 131.72, 131.30, 1H NMR (400 MHz, CD3OD) δ 8.16 (s, 1H, H-33), 8.15‒8.12 (m,
127.23, 126.85, 126.21, 122.04, 113.87, 77.64, 69.31, 65.83, 62.45, 1H, Ar-H), 7.91‒7.87 (m, 1H, Ar-H), 7.82‒7.78 (m, 1H, Ar-H),
56.66, 53.99, 48.39, 48.18, 47.70, 45.85, 45.19, 44.18, 40.01, 39.87, 7.71‒7.69 (m, 1H, Ar-H), 5.60 (s, 1H, H-12), 4.53‒4.40 (m, 2H, H-
38.98, 38.12, 35.61, 33.39, 31.53, 29.18, 24.73, 21.38, 21.31, 19.70, 31), 3.75‒3.69 (m, 1H, H-2), 3.51 (d, J = 11.2 Hz, 1H, H-3), 3.35 (d,
18.27, 18.06, 17.60, 13.86. HR-MS (m/z) (ESI): calc for J = 9.6 Hz, 1H, H-23), 3.24 (d, J = 10.8 Hz, 1H, H-23), 3.02 (dd, J =
C40H56ClN4O6 [M + Na]+ : 711.4092, founded: 711.4079.
12.8, 4.4 Hz, 1H, H-19), 2.47 (d, J = 10.8 Hz, 1H, H-16), 2.44 (s,
Compound 6i: Yields 74%, as a white solid. Mp: 201.5−202.9 °C. 1H, H-9), 2.28‒2.20 (m, 1H, H-20), 1.82‒1.21 (m, 15H), 1.35 (s, 3H,
1H NMR (400 MHz, CD3OD) δ 8.31 (s, 1H, H-33), 7.84‒7.81 (m, CH3-27), 1.11 (s, 3H, CH3-24), 0.99 (d, J = 6.4 Hz, 3H, CH3-29),
2H, Ar-H), 7.60‒7.56 (m, 2H, Ar-H), 5.56 (s, 1H, H-12), 4.48‒4.37 0.90 (d, J = 6.4 Hz, 3H, CH3-30), 0.71 (s, 3H, CH3-25), 0.66 (s, 3H,
(m, 2H, H-31), 3.73‒3.67 (m, 1H, H-2), 3.49 (d, J = 11.2 Hz, 1H, H- CH3-26). 13C NMR (100 MHz, CD3OD) δ 201.96, 179.20, 166.50,
3), 3.32 (d, J = 9.6 Hz, 1H, H-23), 3.26 (d, J = 10.8 Hz, 1H, H-23), 146.99, 145.94, 135.11, 132.25, 131.30, 130.96, 128.53, 126.69,
2.99 (dd, J = 12.8, 4.4 Hz, 1H, H-19), 2.45 (d, J = 10.8 Hz, 1H, H- 125.85, 77.67, 69.33, 65.84, 62.54, 54.04, 48.45, 48.20, 47.73,
16), 2.37 (s, 1H, H-9), 2.25‒2.17 (m, 1H, H-20), 1.79‒1.14 (m, 46.02, 45.25, 44.20, 40.03, 39.90, 39.01, 38.03, 35.55, 33.39, 31.52,
15H), 1.31 (s, 3H, CH3-27), 0.98 (d, J = 6.4 Hz, 3H, CH3-29), 0.95 29.24, 24.73, 21.43, 21.31, 19.89, 18.32, 18.08, 17.62, 13.88. HR-
(s, 3H, CH3-24), 0.88 (d, J = 6.4 Hz, 3H, CH3-30), 0.63 (s, 3H, CH3- MS (m/z) (ESI): calc for C39H53ClN5O7 [M + Na]+ : 726.3837,
25), 0.45 (s, 3H, CH3-26). 13C NMR (100 MHz, CD3OD) δ 201.80, founded: 726.3826.
179.08, 166.42, 147.22, 136.98, 135.53, 131.27, 131.03, 123.12,
122.76, 77.58, 69.30, 65.77, 62.39, 54.07, 48.17, 47.65, 45.83,
45.14, 44.17, 40.02, 39.88, 38.95, 38.09, 35.51, 33.34, 31.52, 29.16,
24.74, 21.38, 21.30, 19.66, 18.21, 18.00, 17.61, 13.84. HR-MS (m/z)
The A549 cells were cultured in 12-well plates and transiently co-
(ESI): calc for C39H53ClN4O5 [M + Na]+ : 715.3597, founded:
In vitro inhibition of TNF-a-induced NF-κB activation in A549
lung cells
transfected with 0.2 mg of a pNF-κB-Luc vector (Stratagene, La
Jolla, CA) and 0.2 mg of pSV-b-galactosidase dissolved in 3 ml
lipofectamine or lipofectamine 2000 (Invitrogen, Carlsbad, CA) as
the internal control. The plasmids were transfected according to the
715.3602.
Compound 6j: Yields 79%, as a white solid. Mp: 203.7−205.6 °C.
1H NMR (400 MHz, CD3OD) δ 8.10 (s, 1H, H-33), 7.86‒7.84 (m,
1H, Ar-H), 7.59‒7.55 (m, 1H, Ar-H), 7.53‒7.48 (m, 2H, Ar-H), 5.58
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manufacturer’s instructions. After 6 h, the medium was changed and
To study the binding mode of the inhibitors in theViaewctAivrteiclesiOtenlionef
cells were cultured for 6 h. Cells were then treated with TNF-α (15 NF-κB protein, molecular docking was perfDoOrmI:e1d0.1u0s3in9g/Ct8hMeDS0u0r6fl2e0xB-
ng/mL) and test compounds simultaneously for 7 h. The A549 cells Dock module in SYBYL-X 2.0. The crystal structure of the NF-κB
treated with TNF-α alone served as positive controls, while cells complex was retrieved from the RCSB Protein Data Bank (PDB
40
without TNF-α treatment served as negative controls. Luciferase entry code: 1NFK, 1IKN).39,
Ligands were docked in the
activities from these cells were then measured by using the Bright- corresponding protein’s binding site by an empirical scoring function
Glo Luciferase Assay kit from Promega (Madison, WI), following and a patented search engine in Surflex-Dock. Before the docking
the manufacturer’s protocol. The relative NF-κB activities of the process, the natural ligand was extracted; the water molecules were
cells treated by test compounds were obtained as the ratio of its removed from the crystal structure. Subsequently, the protein was
luciferase activity to that from the positive controls, both of which prepared by using the Biopolymer module implemented in Sybyl.
have been corrected with background (signals from negative The polar hydrogen atoms were added. The automated docking
controls) and cell viability. In these experimental conditions, none of manner was applied in the present work. Other parameters were
the test compounds induced significant toxicity to A549 cells (<5% established by default in the software. Surflex-Dock total scores,
reduction of cell viability). The IC50 of each fraction was determined which were expressed in -log10 (Kd) units to represent binding
by fitting the relative NF-κB activity to the drug concentration by affinities, were applied to estimate the ligand-receptor interactions of
using a sigmoidal dose eresponse model of varied slope in GraphPad newly designed molecules.
Prism 6. The IC50 reported herein is the average of at least three
replicates.
Immunofluorescence Staining
Studies were performed as previously described.41 Briefly, A549
cells were cultured in six-well plates (2 × 105 cells per well) for 24 h
SPR Analysis
Surface plasmon resonance experiments were performed using a and then pretreated with compound 6k at 10 μM for 24 h, followed
Biacore T200 instrument at 25 °C. Sensor chips, buffer stock by TNF-α for another 30 minutes. The cells were then washed and
solutions, and immobilization reagents were from GE Healthcare. fixed with anhydrous methanol for 15 min at room temperature,
Recombinant human NF-κB was Novoprotein. Other reagents were permeabilized with 0.1% Triton X-100 for 15 min, and blocked with
obtained from Sigma. Immobilization: PBS was used as the running 1% BSA for 10 min at room temperature. Next, cells were incubated
buffer. The four flow cells were treated in the same way to optimize with primary NF-κB antibody in PBS overnight at 4 °C, followed by
throughput. In summary, using a CM5 chip spots 1 and 2 were FITC-conjugated antibody secondary antibodies. After being washed
activated with the coupling reagents EDC and NHS for 10 min. NF- with PBS three times, cells were incubated in 10 μM DAPI for 15
κB at a concentration of 20 μg/mL in 10 mM sodium acetate pH 5 min in the dark. The fluorescence of the NF-κB protein and nucleus
was injected onto the surface for 10 and 5 min in spots 1 and 2, were green and blue, respectively, and live cell imaging was
respectively, to generate surfaces with high and low density. The simultaneously accomplished using
unmodified spot 3 was used as a reference. Kinetics and affinity (Olympus BX-51, Toyko, Japan).
measurements: PBS (containing 0.05% DMSO) was used as the
a fluorescent microscope
running buffer and sample dilution buffer. Dose−responses were
obtained using a 2-fold sample dilution from 0 to 500 μM, using an
Western blot assay
Total cell lysates from cultured A549 cells after compound 6k
treatments as mentioned earlier were obtained by lysing the cells in
ice-cold RIPA buffer with protease and phosphatase inhibitor and
stored at -20 °C for future use. The protein concentrations were
quantified by Bradford method (BIO-RAD) using Multimode
varioscan instrument (Thermo Fischer Scientifics). Equal amounts of
protein per lane was applied in 12% SDS polyacrylamide gel for
electrophoresis and transferred to polyvinylidine difluoride (PVDF)
injection time of 60 s. Data processing: Binding curves were
corrected for variations in DMSO concentration and normalized by
molecular weight. KDs reported are derived from steady-state
binding responses and therefore correspond to the equilibrium
binding affinity of the compounds.
NF-κB DNA binding assay
To determine NF-κB activation, we performed DNA binding assay membrane (Amersham Biosciences). After the membrane was
using TransAM NF-κB Kit according to the manufacturer’s blocked at room temperature for 2 h in blocking solution, primary
instructions and as previously described.38 Briefly, 20 μg of nuclear antibody was added and incubated at 4 °C overnight. IκBα, IKKβ
proteins was added into 96-well plate coated with an unlabeled and NF-κB antibodies were purchased from Imgenex, USA. After
oligonucleotide containing the consensus binding site for NF-κB (5’- three TBST washes, the membrane was incubated with
GGGACTTTCC-3’) and incubated for 1 h. The wells were washed corresponding horseradish peroxidase-labeled secondary antibody
and incubated with antibodies against NF-κB. An HRP conjugated (1:2000) (Santa Cruz) at room temperature for 1 h. Membranes were
secondary antibody was then applied to detect the bound primary washed with TBST three times for 15 min and the protein blots were
antibody and provided the basis for colorimetric quantification. The detected with chemiluminescence reagent (Thermo Fischer
enzymatic product was measured at 450 nm by microplate reader Scientifics Ltd.). The X-ray films were developed with developer
(TECAN Systems).
and fixed with fixer solution.
Molecular docking
Cytotoxicity assay
12 | MedChemComm, 2018, 00, 1-14
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The human cell lines HepG2, NCI-H460, A549, T24, A549, Statistics
View Article Online
The data were processed by the Student' st -DteOsIt:w10i.t1h03th9e/Cs8igMnDi0fi0c6a2n0cBe
A549/DOX and HL-7702 were obtained from the Shanghai Cell
Bank in the Chinese Academy of Sciences. HepG2, NCI-H460,
A549, T24, A549, A549/DOX and HL-7702 cell lines were grown
on 96-well microtitre plates at a cell density of 10× 105 cells/well in
DMEM medium with 10% FBS. DMEM and FBS were obtained
from Gibco-Thermo (BRL Co. Ltd., USA). The plates were
incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air
for overnight. Therewith, the cells were exposed to different
concentrations of target compounds and DOX, and incubated for
another 48 h. The cells were stained with 10 μl of MTT at incubator
for about 4 h. The medium was thrown away and replaced by 100
mL DMSO. The O. D. Value was read at 570/630 nm enzyme
labeling instrument.
level P≤0.05 using SPSS.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (No. 21431001, 81760626), the Qinghai key R
& D and transformation project (Qinghai science and Technology
Department) (2017-NK-C25), Project of State Key Laboratory for
Chemistry and Molecular Engineering of Medicinal Resources,
Ministry of Science and Technology of China (No. CMEMR2016-
B06), A Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (No.
1107047002), The Open Project of Qinghai Key Laboratory of
Qinghai-Tibet Plateau Biological Resources (2017-ZJ-Y10), the
Ministry of Education Innovation Team Fund (IRT_16R15,
2016GXNSFGA380005), the Fundamental Research Funds for the
Central Universities and the Innovation Program for Graduate
Students in Jiangsu Province (KYCX17_0133), Scientific Research
Foundation of Graduate School of Southeast University (YBJJ1786).
Apoptosis analysis
A549 cells were seeded at the density of 2× 106 cells/mL of the
DMEM medium with 10% FBS on 6-well plates to the final volume
of 2 mL. The plates were incubated for overnight and then treated
with different concentrations compound 6k for 24 h. Briefly, after
treatment with compound 6k for 24 h, cells were collected and
washed with PBS twice, and then re-suspend cells in 1×Binding
Buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at
a concentration of 1× 106 cells /mL. The cells were subjected to 5 μL
of FITC Annexin V and 5 μL propidium iodide (PI) staining using
annexin-V FITC apoptosis kit (BD, Pharmingen) followed the 100
μL of the solution was transfer to a 5 mL culture tube and incubate
for 30 min at RT (25 °C) in the dark. The apoptosis ratio was
quantified by system software (CellQuest; BD Biosciences).
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DOI: 10.1039/C8MD00620B
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