Discovery of antitumor ursolic acid long-chain diamine derivatives as potent inhibitors of NF-κB

Article abstract of DOI:10.1016/j.bioorg.2018.05.005

A series of inhibitors of NF-κB based on ursolic acid (UA) derivatives containing long-chain diamine moieties were designed and synthesized as well as evaluated the antitumor effects. These compounds exhibited significant inhibitory activity to the NF-κB with IC₅₀ values at micromolar concentrations in A549 lung cancer cell line. Among them, compound 8c exerted potent activity against the test tumor cell lines including multidrug resistant human cancer lines, with the IC₅₀ values ranged from 5.22 to 8.95 μM. Moreover, compound 8c successfully suppressed the migration of A549 cells. Related mechanism study indicated compound 8c caused cell cycle arrest at G1 phase and triggered apoptosis in A549 cells through blockage of NF-κB signalling pathway. Molecular docking study revealed that key interactions between 8c and the active site of NF-κB in which the bulky and strongly electrophilic group of long-chain diamine moieties were important for improving activity.

Full text of DOI:10.1016/j.bioorg.2018.05.005

Accepted Manuscript
Discovery of antitumor ursolic acid long-chain diamine derivatives as potent
inhibitors of NF-κB
Wei Jiang, Ri-Zhen Huang, Jing Zhang, Tong Guo, Meng-Ting Zhang, Xiao-
Chao Huang, Bin Zhang, Zhi-Xin Liao, Jing Sun, Heng-Shan Wang
PII:
S0045-2068(18)30166-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.005
YBIOO 2358
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 February 2018
4 May 2018
7 May 2018
Please cite this article as: W. Jiang, R-Z. Huang, J. Zhang, T. Guo, M-T. Zhang, X-C. Huang, B. Zhang, Z-X. Liao,
J. Sun, H-S. Wang, Discovery of antitumor ursolic acid long-chain diamine derivatives as potent inhibitors of NF-
κB, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.05.005
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Discovery of antitumor ursolic acid long-chain diamine derivatives as potent
inhibitors of NF-κB
Wei Jiang a, 1, Ri-Zhen Huang a, 1, Jing Zhang a, Tong Guo a, Meng-Ting Zhang a, Xiao-Chao
Huang a, Bin Zhang a, Zhi-Xin Liao a, Jing Sunb*, Heng-Shan Wang c
a Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research，School of Chemistry and
Chemical Engineering, Southeast University, Nanjing 211189, P.R. China
b Chinese Acad Sci, Northwest Inst Plateau Biol, Qinghai Key Laboratory of Qinghai-Tibet
Plateau Biological Resources, Xining, 810000, P.R. China
c State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
(Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi
Normal University, Guilin 541004, PR China
* Corresponding author.
**Corresponding author at: Department of Pharmaceutical Engineering, School of Chemistry and
Chemical Engineering, Southeast University, Nanjing 211189, China
E-mail addresses: zxliao@seu.edu.cn (Z. -X. Liao), sunj@nwipb.cas.cn (J. S),
whengshan@163.com (H. -S. Wang).
1 These authors contributed equally to this work.

Abstract
A series of inhibitors of NF-κB based on ursolic acid (UA) derivatives containing long-chain
diamine moieties were designed and synthesized as well as evaluated the antitumor effects. These
compounds exhibited significant inhibitory activity to the NF-κB with IC50 values at micromolar
concentrations in A549 lung cancer cell line. Among them, compound 8c exerted potent activity
against the test tumor cell lines including multidrug resistant human cancer lines, with the IC50
values ranged from 5.22 to 8.95 μM. Moreover, compound 8c successfully suppressed the
migration of A549 cells. Related mechanism study indicated compound 8c caused cell cycle arrest
at G1 phase and triggered apoptosis in A549 cells through blockage of NF-κB signalling pathway.
Molecular docking study revealed that key interactions between 8c and the active site of NF-κB in
which the bulky and strongly electrophilic group of long-chain diamine moieties were important
for improving activity.
Keywords: Ursolic acid; long-chain diamine; NF-κB; Drug resistance; Migration

Introduction
Lung cancer accounts for more than one-quarter of cancerrelated deaths worldwide, and the
5-year relative survival is currently less than 20%. Non-small-cell lung cancer (NSCLC) is the
most common subtype of lung cancer, which accounts for about 80−85%, with metastasis and
drug resistance responsible for >70% of deaths [1, 2]. Therefore, in order to improve the quality of
life of patients, it is urgent to develop new anticancer drugs that can inhibit the metastasis and
drug resistance of primary non-small cell lung cancer.
Transcription factor nuclear factor-kappa B (NF-κB), which is known to be the master
regulators of a wide variety of anti-apoptotic proteins, is commonly over-expressed and
constitutively activated in different types of hematologic cancers and solid tumors [3-5]. NF-κB
influences the gene expression events that impact a large number of biological processes including
immune response, cell survival, differentiation, and proliferation [6]. Constitutive activation of
NF-κB promotes tumor proliferation, invasion, and metastasis. Recent studies have revealed
blockade of NF-κB activity is associated with suppression of angiogenesis, invasion, and
metastasis in certain cancer cells [7, 8]. Moreover, NF-κB activation allows malignant cells to
escape apoptosis and therefore, contributes to the development of radiation and chemotherapy
resistance in cancer cells [9, 10]. Increasing evidences suggested that the suppression of NF-κB
activation may prevent tumor resistance to chemotherapeutic agents, shift the death-survival
balance toward apoptosis, and improve the efficacy of current anticancer drugs [11, 12].
Therefore, designing of NF-κB inhibitors is a promising approach to develop the new cancer
therapeutics and overcome drug resistance.
Recently, pentacyclic triterpenoids ursolic acid (UA) served as one of the major effective
elements of many traditional chinese medicines has attracted a lot of interest because of its
anticancer properties [13-15]. UA and its derivatives exhibited potent cytotoxic effects against
various types of cancer cell lines, as well as to induce tumor cell differentiation and apoptosis, and
were found to be potent inhibitors of NF-κB [16-18]. Our previous work reported that
aniline-derived ursolic acid derivatives as multidrug resistance reversers by blocking NF-κB
pathway [19]. The C-28 amide moiety and the C-3 ester group played an important role in the
interaction of NF-κB. In addition, previous studies also reviewed that the modification in position

3 and/or 28 of UA has properties of increasing the inhibition activity of anticancer drugs [13, 20].
In this paper, we synthesised a series of UA derivatives containing long-chain amide moieties as
inhibitors of NF-κB. These derivatives were evaluated for their in vitro anticancer activity and
TNF-α-induced NF-κB activation against lung cancer cell line A549. The most active compound
8c was further docked with the crystal structure of NF-κB to predict the possible binding mode of
the congener and evaluated the inhibition activity of migration and drug resistant.
Results and discussion
2.1 Chemistry
A series of UA derivatives containing long-chain diamide gallic acid moieties were synthesized
as outlined in Schemes 1. As shown in Scheme 1, UA (1) was conjugated with galloyl groups at
the C-28 position of UA through a flexible spacer. Firstly, the C-3 hydroxy group was protected
with an acetyl group by acetic anhydride to afford compound 2 in dry pyridine in the presence of
4-dimethylaminopyridine (DMAP). Subsequently, treatment of 2 with oxalyl chloride in
dichloromethane at room temperature gave acyl chloride 3. Coupling with butane-1,4-diamines,
pentane-1,5-diamines, hexane-1,6-diamines and heptane-1,7-diamines formed amides 4a-4d,
respectively. Saponification of 4a-4d with sodium hydroxide in a mixture of tetrahydrofuran and
methanol yielded 5a-5d, respectively.
Condensation of 4a-4d, 5a-5d with gallic acid, 3,4,5-trimethoxybenzoic acid or
3,4,5-triacetoxybenzoic acid in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) in tetrahydrofuran afforded
compounds 6a–6d, 7a-7d, 8a-8d, 9a-9d,10a-10d and 11a-11d in good to high yields . All the new
compounds were confirmed by 1H NMR, 13C NMR and high resolution mass spectroscopy.
2.2 In vitro cytotoxic activity
The growth inhibitory effects of the synthesized compounds against a panel of four human
cancer cell lines including human ovarian cancer cells (SKOV3), human liver cancer cells
(HepG2), human non-small cell lung cancer cells (A549), and human bladder cancer cells (T24),

were evaluated by MTT assay, with 10-hydroxycamptothecin (HCPT) as the positive control. The
results are shown in Table 1.
As shown in Table 1, UA suppressed proliferation of the above four cancer cell lines in
different extents (from 37.88 to 45.53 μM). From the results obtained (Table 1), it is clear that,
most of the title compounds (6, 8, 9 and 11) which modified from UA exhibited a considerable
growth inhibitory effect and were active in the micromolar range against different cell lines. Of all
tested compounds, compounds 8c showed the highest potency, inhibiting the growth of T24,
A549, HepG2 and SKOV3 cells with IC50 values of 6.01, 5.22, 6.82 and 8.95 μM, respectively.
These values are from 5-fold to 8-fold lower than UA and similar or potent activity than HCPT.
The results suggested that the modification of C-3 and C-28 position of UA with O-acetyl and
diamides moieties may improve the antitumor activity of UA.
The SAR study reveals that, diamide linkers at C-28 position are playing an important role.
Comparing the inhibitory concentrations of the pairs (with similar substitution and variation in n)
6a-6d, 8a-8d, 11a-11d clearly indicated that, longer diamide side chain (n = 6) showed relatively
enhanced activity than shorter diamide side chain (n = 4 and 5). However, the effect was
significantly reduced as the length of the carbon chain (n = 7) was increased. Compounds 6c, 8c
and 11c exhibited good inhibitory activity for all the tested carcinoma cell lines. In particular,
O-acetyl substitutions on phenyl ring at C-28 of the aniline ring were more active than the
hydroxyl substituted derivatives. Compound 8c, obtained by inserting the O-acetyl moiety in C-3,
C-4, and C-5 positions of aniline ring showed IC50 values 5.22 μM against A549, while 6c with
hydroxyl group substitutions exerted moderate anticancer activity against A549. On the other hand,
the presence of a weakly polar substituent (-OCH3) in aniline ring was harmful to the anticancer
effect: the derivatives 7 and 10 were not active against the tested cell lines, which suggested that
the polar as well as steric property were essential for anticancer activity. The previous study [21]
reported that significant further improvement of anticancer activity was observed when an acetyl
group was introduced at the 3-OH position and amino alkyl groups were introduced at the
17-COOH position. Similar results were seen in our study, where compounds 6 and 8 had more
potent antitumor effects than compounds 9 and 11, respectively, which had a free 3-OH group.
Interestingly, compounds 7 and 10 were exceptions. Replacement of 3-OH with 3-O-acetyl to
yield compounds 7 exhibited little anticancer effects, while 3-OH group in compounds 10

displayed moderate to high activity. These results prompted us to conclude that the modifications
of C-28 position by diamide group with polar and bulky substituents were the main factor on the
anticancer activity.
Potential side effects of test compounds could arise by affecting cell viability in non-cancerous
tissues. Therefore, the effect of the most active compounds was evaluated against human liver cell
line (HL-7702), as an in vitro model of normal cells. As shown in Table 1, the cytotoxicity
activities of most compounds against cancer cells was much higher than that against HL-7702
normal cells, making them good candidates as anticancer drugs.
2.3 In vitro inhibition of TNF-α-induced NF-κB activation in A549 lung cancer cell line
Next, we evaluated the impact of UA derivatives on TNF-α-induced NF-κB transcriptional
activity, which is considered as a relevant therapeutic target in hematological cancer and
malignancies [22] as well as in lung cancer [23]. The human lung cancer A549 cell line transiently
co-transfected with NF-κB -luc was used to monitor the effects of UA derivatives on tumor
necrosis factor-alpha (TNF-α)-induced NF-κB activation. IMD-0354 was used as a positive
control. The title compounds were evaluated in a dose-dependent manner to determine the
concentration needed to inhibit 50% of TNF-α-induced NF-κB activation (IC50). The results of
inhibition of TNF-α-induced NF-κB activation in A549 lung cancer cell line by tested compounds
are shown in Table 2. As shown in Table 2, the newly synthesized UA derivatives are potent
NF-κB inhibitors, with IC50 values mostly in the micromolar range. The synthetic compounds
showed improvement inhibition of TNF-α-induced NF-κB activation than parent compound UA,
supporting introduction the long-chain diamide moieties led to increase in the activity. In
comparing compounds with the same substituent on the aniline ring in terms of the average IC50,
analogues having six carbon linkers were more activity than longer amide side chain
(6c>6b>6a>6d). Compound 6c, which possessed trihydroxyl substituted aniline ring, displayed
moderate activity. Notably, introduction of a trimethoxy group, a stronger electron-donating
group, resulted in compound 7c, which caused a loss of activity relative to 6c, with the least
effects within this subset. On the other hand, the inhibition potency was markedly increased when
the hydroxyl of aniline of compound 6c was esterified (8c), which suggested that essential polar
and steric capacity of substitutents is important for inhibitory activity. Furthermore, the

esterification of 3-OH group of compounds 9 and 11 into C-3 ester group of compounds 6 and 8
exhibited significantly improved activities. This was consistent with the cytotoxicity assay results
and our previous work [19]. Intriguingly, the inhibitory effect of 8c was more potent than
IMD-0354.The results further confirmed the importance of bulky and strongly electrophilic group
at C-28 positions that played an important role in binding of compounds to the receptor site.
2.4 Molecular docking
To understand the interactions between the most active compound 8c and the target of interest
(NF-κB), we performed molecular docking calculations on the active site of NF-κB (PDB ID:
1IKN) [24] using SYBYL-X 2.0 software (Fig. 1) and the score results are summarized in Table 3.
The interaction mode of compound 8c (docking score = 9.53), with the best docking score and
interaction in the binding site of the NF-κB receptor, is described and shown in Fig. 1. Some key
residues, such as ASN182, SER205, CYS215, ARG252 and ARG253, as well as hydrogen bonds
between the selected compound and the residues are also labeled. Our docking study revealed that
the C-3 carbonyl moiety of 8c formed hydrogen bonding interactions with ASN182 and
contributed to the inhibition against NF-κB. Furthermore, the triacetylester group extended into
the deep groove and made additional hydrophobic interactions with HIS184, ARG201, GLU211,
GLU213, CYS215, LEU223, ARG252, and ARG253, which would also contribute to the
improved potency of 8c. Moreover, as shown in Fig. 1, the triacetylester moiety interacted with
amine hydrogens of ARG252 and ARG253 by means of hydrogen bonding, which further
confirmed that this moiety is also crucial for binding. Apart from hydrogen bonding, the UA
scaffold also showed hydrophobic and van der Waals interactions with GLN154, ASN182,
HIS193, GLY209 and HIS296 residues of the target protein, while the C28-amide side chain
demonstrated hydrophobic and van der Waals interactions with SER205, ASP291, ASP294 and
GLU253 residues of the active site. However, the trimethoxybenzene moiety unable embedded the
deep cave formed by HIS184, ARG201, GLU211, GLU213, CYS215, LEU223, ARG252, and
ARG253 (Fig. S1).
2.5 Compound 8c inhibits NF-κB DNA binding

Because of the pivotal role of the transcription factor NF-κB in tumorigenesis and survival [25],
it was speculated that 8c might mediate its anticancer effects by modulating the NF-κB activation
pathway. To further determine this hypothesis, we next investigated the effect of 8c on
constitutive NF-κB activation in A549 cells using ELISA-based DNA binding assay. Results of
ELISA-based DNA binding assay clearly showed that treatment with various concentrations (5, 10
and 20 μM) of 8c significantly suppressed TNF-α-induced NF-κB activation (Fig. 2a). 8c reduced
the NF-κB DNA binding ability by nearly 2.3-fold as compared with TNF-α and these results
indicate that 8c can modulate constitutive NF-κB activation in A549 cells. Nevertheless, the
positive control IMD-0354 (20 μM) inhibited the DNA-binding activity of NF-κB by 1.6-fold as
compared with TNF-α (Fig. 2b).
2.6 Reduction effect of compound 8c on NF-κB pathway
It has been reported that polyphenols prevented NF-κB translocation to the nucleus by
inhibition the activity of IκB kinase, where NF-κB induces the expression of genes related to cell
growth, invasion, and metastasis [26]. To further determine whether the effect of compound 8c on
the NF-κB pathway is involved in the compound 8c-induced inhibition of A549 cells, the relative
levels of NF-κB, IκBα and phosphorylated IKKβ were examined by Western blotting assay. As
shown in Fig. 7, consistent with expectations, compound 8c caused dose-dependent decreases in
the levels of NF-κB of A549 cells. Meanwhile, phosphorylated IκBα was significantly inhibited in
a concentration-dependent manner, but no obvious effect was observed in the IκBα protein. As
depicted in Fig. 7, incubation of the cells with compound 8c for 24 h markedly suppressed the
phosphorylation of IKKα/β. These results indicated that compound 8c suppressed NF-κB
activation, mainly through inhibition of IκBα phosphorylation mediated by the IKKα/β
phosphorylation.
2.7 Effects of selected compounds on drug-resistant cell lines
Drug resistance is a critical therapeutic problem that limits the efficacies of most anticancer
drugs for a variety of human cancer cells. According to the above biological results, we further
evaluated the sensitivity to the selected compounds of cisplatin (CDDP)-resistant cancer cell lines
(A549/CDDP), with CDDP as the positive control. As shown in Table 4, CDDP exhibited

moderate activity against the A549/CDDP resistant cell lines. It was significant to observe that
compound 8c had a much lower resistance factor (1.59 for the resistant A549 cell line). Moreover,
except for 9b and 9c, the other selected compounds exhibited potent cytotoxicity against parental
cells and the corresponding CDDP-resistant cells comparable to that of CDDP, and had smaller
resistance factors, suggesting that these compounds might be useful in the treatment of
drug-refractory tumors resistant to CDDP.
2. 8. Compound 8c inhibited the migration of A549 cell in vitro
Metastasis plays an important role in later period of cancer progression. Thus, the inhibition of
metastasis is vital for efficient cancer treatment. Migration of cells is a key attribute of practically
each biological process, particularly cancer cells where it is known to connect with the tumor
progression and metastatic cascade [27]. As cell migration is connected with the metastatic
activity of cancer cells, therefore we have investigated the effect of compound 8c on A549 cells
using wound healing assay. The migration of A549 cells was recorded by microscopic
observations at 0 h and 24 h after treatment with 5 μM, 10 μM and 20 μM of compound 8c.
Results from Fig. 4 clearly showed that there was almost complete healing of wound in control
after 24 h, however healing was strongly suppressed in the cells treated with 8c.
2.9 Compound 8c induces apoptosis in A549 cells
In order to confirm whether 8c-induced reduction in cell viability was responsible for the
induction of apoptosis, A549 cells were co-stained with PI and Annexin-V/FITC, and the number
of apoptotic cells was estimated by flow cytometry (Fig. 5). Four quadrant images were observed
by flow cytometry analysis: the Q1 area represented damaged cells which appeared during the
process of cell collection, the Q2 region showed necrotic cells and later stage apoptotic cells; early
apoptotic cells were located in the Q3 area and the Q4 area showed normal cells. A
dose-dependent increase in the percentage of apoptotic cells was noted after the cells were treated
with compound 8c at the concentrations of 5, 10 and 20 μM for 24 h. As shown in Fig. 5, few
(0.57%) apoptotic cells were present in the control panel, in contrast, the percentage rose to 6.65%
at the concentration of 5 μM after treatment with 8c for 24 h. At concentrations of 20 μM, there
was a further increase to 31.2% after treatment with 8c. These results clearly confirmed that

compared with the control, compound 8c effectively induced apoptosis in A549 cells in a
dose-dependent manner.
2.10 Morphological characterization of A549 cell apoptosis using Hoechst 33258
Apoptosis of A549 cells treated with compound 8c were further confirmed by analyzing the
nuclear morphology. Nuclei of A549 cells treated with 0, 5, 10, and 20 μM of compound 8c for 24
h were stained by membrane permeable blue Hoechst 33258 to detect apoptotic cells
morphologically. Our experimental observation showed that, in the control group, nuclei of the
A549 cancer cells appeared as regular round contours, and the A549 cells with smaller nuclei and
condensed chromatin were rarely observed (Fig. 6). After the treatment of compound 8c, some
cells emitted brilliant blue fluorescence, and nuclei of more A549 cells were slightly changed.
However, when the concentration of compound 8c was increased to 20 μM, more nuclei of the
A549 cells appeared hyper condensed (brightly stained) (Fig. 6). Remarkably, the numbers of
apoptotic nuclei containing condensed chromatin increased significantly after the A549 cells were
treated with compound 8c for 24 h, suggesting that apoptosis of the A549 cells was induced by
compound 8c in a concentration-dependent manner.
2.11. Assessment on the caspase-3 activation for cell apoptosis
Caspases, in general, are a family of cysteine proteases that play an essential role in apoptosis,
necrosis, and inflammation [28]. It is generally accepted that the loss of mitochondrial membrane
potential leads to the leakage of cytochrome c, which would cause the caspase cascade, including
the consequent activation of caspase-9 and caspase-3 [29, 30]. To investigate the potential
molecular mechanisms involved, colorimetric was used to measure caspase-3 activity upon
treatment with 5, 10, and 20 μM of compound 8c for 24 h. As shown in Fig. 7, additional peaks
for actived-caspase-3 (FITC DEVD-FMK probes) cells were observed in compound 8c treated the
cells compared to the control group. The proportion of actived-caspase-3 cells after compound 8c
treatment was 3.53%, 7.05% and 11.27% of total cells, respectively. Therefore, these results
indicated that compound 8c induced cell apoptosis by triggering caspase-3 activity in A549 cells.
2.12 Cell Cycle Distribution

The mechanism of the anti-proliferative effect of the UA derivatives was investigated by flow
cytometric assay of the cell cycle distribution of A549 cells 24 h following treatment with 0, 5, 10
or 20 μM of analogue. As shown in Fig. 8, treatment of A549 cells with compound 8c at each
concentration enhanced cell cycle arrest at the G1 phase, resulting in concomitant increase in the
population in the G1 phase (to 42.75%, 50.12% and 54.66%) compared with the control cells
(38.36%). The S-phase population of A549 cells decreased by 44.12%, 39.25% and 36.49%
compared with the control cells (51.55%). These results suggested that compound 8c arrested the
cell cycle of A549 cells in the G1 stage.
3. Conclusion
A series of UA derivatives containing long-chain diamine moieties were designed and
synthesized as NF-κB inhibitors with marked cytotoxicity. These compounds exhibited significant
inhibition activity toward the TNF-a-induced activation of NF-κB in lung cancer cell line A549. A
docking study of the most active compound 8c revealed key interactions between 8c and NF-κB
receptor in which the bulky and strongly electrophilic group of long-chain diamine moieties at
C-28 positions were important for improving activity. In vitro antitumor activity screening
revealed that some compounds exhibited better inhibitory activity than parent compound UA,
even the commercial anticancer drug HCPT. It is noteworthy that compound 8c (IC50 = 5.22 ±
0.65 μM) exhibited the best anticancer activity against the A549 cell line and displayed more
potent inhibitory activity than HCPT. Moreover, the synthetic compounds markedly reversed
multidrug-resistance in CDDP-resistant lung cancer cells. Simultaneous molecular mechanism
studies suggested that target compound 8c may inhibit the growth of A549 cells by blocking the
NF-κB signaling pathway. In particular, compound 8c successfully suppressed the migration of
A549 cells. Furthermore, cell cycle analysis indicated that compound 8c arrested the A549 cell
line in G1 phase. Consequently, the rational design of UA derivatives containing long-chain
diamine moieties offers significant potential for the discovery of a new class of NF-κB inhibitors
with the ability to suppress migration and reverse multidrug resistance in A549 lung cacncer cell
line. The precise mechanism of this action requires further investigation.

4. Experimental methods
4.1 General
Ursolic acid (UA) with more than 98% purity was purchased from Shang He Biological
Technology Co.Ltd, Changsha, Hunan province, China. Compound 2 was synthesized according
to the literature [31]. All the chemical reagents and solvents used were of analytical grade. Silica
gel (300-400 mesh) used in column chromatography was provided by Tsingtao Marine Chemistry
Co. Ltd. 1H NMR and 13C NMR were recorded on a BRUKER AV-400 spectrometer with TMS as
an internal standard in CDCl3 or [D6]DMSO. Mass spectra were determined on an FTMS ESI
spectrometer.
4.2 General procedure for the synthesis of 6a–6d, 7a-7d, 8a-8d, 9a-9d,10a-10d and 11a-11d
Oxalyl chloride (12 mmol) was dripped into a solution of compound 2 (4 mmol) in dry CH2Cl2
(20 mL). The resulting mixture was stirred at room temperature for 6 h. The solvent was removed
by evaporation under reduced pressure, and dry CH2Cl2 (3 × 5 mL) was added to the residue,
concentrated to dryness to give compound 3. Then, butane-1,4-diamines, pentane-1,5-diamines,
hexane-1,6-diamines or heptane-1,7-diamines (12 mmol) and triethylamine were added to the
mixture and stirred at room temperature for 4 h. After the solvent was removed by evaporation
under reduced pressure, the resultant residue was dispersed in water, acidified to pH 3-4 with 2N
HCl and extracted with CH2Cl2. Then, the organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated in vacuo. The crude products were purified by silica gel column
chromatography and eluted with methylene dichloride/methyl alcohol (v:v=1:7) to give white
powders 4a-4d.
The compounds 4a-4d and aqueous NaOH (4 N, 1mL) were dissolved in 10 mL methyl
alcohol/tetrahydrofuran (v:v =1:1.5), and the solution was stirred at room temperature for 5 h. A
small amount of water was added to the reaction mixture. The solution was adjusted to pH 3-4
with 2 N HCl and extracted with ethyl acetate. The organic layer was washed with saturated
sodium chloride, dried over Na2SO4, and purified via silica gel column chromatography with
methylene dichloride/methyl alcohol (v:v=5:1) to offer white compounds 5a-5d.

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDCI,
1.2
mmol),
1-hydroxybenzotriazole (HOBt, 1.2mmol) and 3 drops of triethylamine was added to a solution of
gallic acid (1.2 mmol), 3,4,5-trimethoxybenzoic acid (1.2 mmol) or 3,4,5-triacetoxybenzoic acid
(1.2 mmol) in anhydrous THF (10 mL). The resulting mixture was stirred at 0 ℃for 0.5 h. Then, a
solution of compound 4a-4d or 5a-5d (1 mmol) in THF (5 mL) was added dropwise, and stirring
was continued for another 8 h at room temperature. The reaction mixture was poured onto ice
water (100 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was washed with
brine and water, dried with anhydrous sodium sulfate, filtered and concentrated in vacuo. The
crude products were purified by silica gel column chromatography to afford compounds 6a–6d,
7a-7d, 8a-8d, 9a-9d, 10a-10d and 11a-11d as white powders.
Compound 6a. Yield 77.6%; 1H NMR (400 MHz, CDCl3) δ 6.94 (s, 2H, galloyl-H), 6.32 (s, 1H,
CONH), 5.30 (s, 1H, H-12), 4.47 (m, 1H, H-3), 3.27 (br, s, 3H, CONH-CH2), 2.97 (br, s, 1H,
CONH-CH2), 2.04 (s, 3H, C3-COOCH3), 1.06 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.87-0.83 (m, 12H,
4CH3), 0.70 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 176.0, 170.1, 166.1, 145.3, 138.5,
136.0, 125.0, 124.5, 106.6, 80.0, 54.5, 51.9, 46.8, 46.5, 41.6, 38.6, 38.4, 37.6, 37.2, 37.0, 36.3,
32.5, 30.4, 29.0, 27.8, 27.3, 26.9, 26.4, 23.5, 23.2, 22.8, 22.1, 21.1, 20.9, 17.7, 17.1, 16.8, 16.6,
15.0. HR-MS (m/z) (ESI): calcd for C43H64N2O7 [M+Na+]: 743.4611; found: 743.4594.
Compound 6b. Yield 78.1%; 1H NMR (400 MHz, CDCl3) δ 6.98 (s, 2H, galloyl-H), 6.71 (s, 1H,
CONH), 6.24 (s, 1H, CONH), 5.30 (s, 1H, H-12), 4.50-4.46 (m, 1H, H-3), 3.30 (br, s, 3H,
CONH-CH2), 2.96 (br, s, 1H, CONH-CH2), 2.05 (s, 3H, C3-COOCH3), 1.07 (s, 3H, CH3), 0.92
(br, s, 6H, 2CH3), 0.85 (br, d, J=5.2 Hz, 9H, 3CH3), 0.73 (s, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 179.5, 171.1, 168.3, 145.0, 139.6, 135.7, 125.8, 125.6, 107.3, 80.8, 55.2, 53.7, 47.8,
47.4, 42.4, 40.1, 39.7, 39.5, 38.9, 38.3, 37.7, 36.8, 32.6, 30.8, 29.7, 29.0, 28.7, 28.1, 27.8, 24.8,
24.1, 23.5, 23.4, 23.2, 21.3, 21.2, 18.1, 17.2, 16.8, 16.7, 15.6. HR-MS (m/z) (ESI): calcd for
C44H66N2O7 [M+H+]: 735.4948; found: 735.4954.
Compound 6c. Yield 76.2%; 1H NMR (400 MHz, CDCl3) δ 6.98 (s, 2H, galloyl-H), 6.44 (s, 1H,
CONH), 6.19 (s, 1H, CONH), 5.32 (s, 1H, H-12), 4.50-4.46 (m, 1H, H-3), 3.35 (br, s, 3H,
CONH-CH2), 3.00 (br, s, 1H, CONH-CH2), 2.05 (s, 3H, C3-COOCH3), 1.09 (s, 3H, CH3), 0.94
(br, d, J=5.0 Hz, 6H, 2CH3), 0.88 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.75 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3) δ 179.2, 171.1, 168.3, 145.0, 139.7, 135.7, 125.8, 125.6,

107.2, 80.9, 55.2, 53.8, 49.2, 47.7, 47.4, 42.5, 40.0, 39.7, 39.5, 39.0, 38.3, 37.7, 37.1, 36.8, 33.8,
32.6, 30.8, 29.7, 29.2, 29.0, 28.1, 27.8, 26.3, 26.2, 25.6, 24.9, 23.5, 23.4, 23.2, 21.3, 21.2, 18.1,
17.2, 16.9, 16.7, 15.6. HR-MS (m/z) (ESI): calcd for C45H68N2O7 [M+H+]: 749.5105; found:
749.5241.
Compound 6d. Yield 77.4%; 1H NMR (400 MHz, CDCl3) δ 6.97 (s, 2H, galloyl-H), 6.52 (s, 1H,
CONH), 6.22 (s, 1H, CONH), 5.31 (s, 1H, H-12), 4.50-4.46 (m, 1H, H-3), 3.31 (br, s, 3H,
CONH-CH2), 2.97 (br, s, 1H, CONH-CH2), 2.05 (s, 3H, C3-COOCH3), 1.08 (s, 3H, CH3), 0.93
(br, s, 6H, 2CH3), 0.87 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.75 (s, 3H, CH3). 13
C
NMR (100 MHz, CDCl3) δ 179.5, 171.1, 168.4, 144.8, 139.7, 135.6, 125.9, 125.7, 107.1, 80.9,
55.2, 53.9, 47.8, 47.4, 42.5, 40.1, 39.8, 39.5, 39.0, 38.3, 37.7, 37.0, 36.8, 32.6, 30.8, 29.1, 28.9,
28.4, 28.1, 27.8, 26.5, 24.8, 23.5, 23.4, 23.2, 21.3, 21.2, 18.1, 17.2, 16.8, 16.7, 15.6. HR-MS (m/z)
(ESI): calcd for C46H70N2O7 [M+H+]: 763.5261; found: 763.5252.
1
Compound 7a. Yield 83.6%; H NMR (400 MHz, CDCl3) δ 7.14 (s, 2H, galloyl-H), 6.99 (t,
J=5.2 Hz, 1H, CONH), 6.08 (t, J=5.6 Hz, 1H, CONH), 5.31 (t, J=3.0 Hz, 1H, H-12), 4.51-4.47 (m,
1H, H-3), 3.92 (s, 6H, 2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.52-3.42 (m, 3H,
CONH-CH2), 3.07-3.00 (m, 1H, CONH-CH2), 2.05 (s, 3H, C3-COOCH3), 1.09 (s, 3H, CH3), 0.95
(s, 3H, CH3), 0.91 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.74 (s,
3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.7, 170.9, 167.1, 153.1, 139.9, 130.0, 125.6, 104.8,
80.8, 60.8, 56.4, 55.3, 53.9, 47.8, 47.5, 42.5, 39.9, 39.8, 39.6, 39.1, 38.7, 38.3, 37.7, 37.4, 36.8,
32.7, 30.9, 28.1, 27.8, 27.6, 25.9, 24.9, 23.5, 23.4, 23.2, 21.2, 21.2, 18.1, 17.3, 17.0, 16.7, 15.5.
HR-MS (m/z) (ESI): calcd for C46H70N2O7 [M+H+]: 763.5261; found: 763.5250.
Compound 7b. Yield 82.7%; 1H NMR (400 MHz, CDCl3) δ 7.02 (s, 2H, galloyl-H), 6.39 (t, J=5.2
Hz, 1H, CONH), 5.95 (t, J=5.8 Hz, 1H, CONH), 5.29 (t, J=3.2 Hz, 1H, H-12), 4.51-4.47 (m, 1H,
H-3), 3.91 (s, 6H, 2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.46-3.36 (m, 3H, CONH-CH2),
3.02-2.94 (m, 1H, CONH-CH2), 2.05 (s, 3H, C3-COOCH3), 1.07 (s, 3H, CH3), 0.94 (s, 3H, CH3),
0.92 (s, 3H, CH3), 0.87-0.84 (m, 9H, 3CH3), 0.76 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
178.4, 170.9, 167.5, 153.2, 141.0, 140.0, 130.3, 125.5, 104.7, 80.8, 60.9, 56.4, 55.3, 54.0, 47.8,
47.5, 42.5, 40.0, 39.8, 39.6, 39.1, 38.8, 38.3, 37.7, 37.2, 36.9, 32.7, 30.8, 29.3, 28.8, 28.1, 27.8,
24.8, 24.0, 23.6, 23.4, 23.2, 21.2, 18.2, 17.2, 16.9, 16.7, 15.6. HR-MS (m/z) (ESI): calcd for
C47H72N2O7 [M+H+]: 777.5418; found: 777.5422.

Compound 7c. Yield 83.2%; 1H NMR (400 MHz, CDCl3) δ 7.09 (s, 2H,galloyl-H), 6.61 (t, J=5.2
Hz, 1H, CONH), 5.95 (t, J=5.6 Hz, 1H, CONH), 5.31 (t, J=3.0 Hz, 1H, H-12), 4.51-4.47 (m, 1H,
H-3), 3.91 (s, 6H, 2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.55-3.40 (m, 2H, CONH-CH2),
3.36-3.28 (m, 1H, CONH-CH2), 2.99-2.92 (m, 1H, CONH-CH2), 2.05 (s, 3H, C3-COOCH3), 1.09
(s, 3H, CH3), 0.94 (br, s, 6H, 2CH3), 0.88 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.77
(s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.3, 170.9, 167.1, 153.2, 140.1, 130.3, 125.5,
104.7, 80.8, 60.9, 56.4, 55.3, 54.0, 47.8, 47.5, 42.6, 39.8, 39.6, 39.4, 39.1, 38.6, 38.4, 37.7, 37.3,
36.9, 32.7, 30.9, 29.4, 28.1, 27.9, 25.8, 24.9, 23.5, 23.4, 23.2, 21.2, 18.2, 17.3, 17.0, 16.7, 15.6.
HR-MS (m/z) (ESI): calcd for C48H74N2O7 [M+H+]: 791.5574; found: 791.5566.
Compound 7d. Yield 83.0%; 1H NMR (400 MHz, CDCl3) δ 7.00 (s, 2H,galloyl-H), 6.16 (t, J=5.1
Hz, 1H, CONH), 5.90 (t, J=5.1 Hz, 1H, CONH), 5.30 (br, s, 1H, H-12), 4.51-4.47 (m, 1H, H-3),
3.91 (s, 6H, 2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.46-3.41 (m, 2H, CONH-CH2),
3.37-3.28 (m, 1H, CONH-CH2), 3.02-2.94 (m, 1H, CONH-CH2), 2.05 (s, 3H, C3-COOCH3), 1.09
(s, 3H, CH3), 0.95 (br, s, 6H, 2CH3), 0.88 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.78
(s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.0, 170.9, 167.2, 153.2, 140.2, 130.3, 125.4,
104.6, 80.9, 60.9, 56.4, 55.3, 54.1, 47.8, 47.5, 42.6, 40.1, 39.8, 39.6, 39.4, 39.2, 38.4, 37.7, 37.3,
36.9, 32.8, 30.9, 29.6, 29.3, 28.9, 28.1, 27.9, 26.9, 24.9, 23.6, 23.5, 23.2, 21.2, 18.2, 17.3, 17.0,
16.7, 15.6. HR-MS (m/z) (ESI): calcd for C49H76N2O7 [M+H+]: 805.5731; found: 805.5728.
1
Compound 8a.Yield 76.3%; H NMR (400 MHz, CDCl3) δ 7.67 (s, 2H, galloyl-H), 6.10 (s, 1H,
CONH), 5.33 (s, 1H, H-12), 4.51-4.47 (m, 1H, H-3), 3.49-3.42 (m, 3H, CONH-CH2), 3.02 (br, s,
1H, CONH-CH2), 2.30 (s, 3H, CH3-COO-Ar), 2.29 (s, 6H, 2CH3-COO-Ar), 2.05 (s, 3H,
C3-COOCH3), 1.09 (s, 3H, CH3), 0.95 (d, J=6.2 Hz, 6H, 2CH3), 0.88 (s, 3H, CH3), 0.87 (s, 3H,
CH3), 0.86 (s, 3H, CH3), 0.77 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.8, 171.1, 167.7,
166.6, 165.0, 143.4, 139.9, 137.0, 132.9, 125.7, 119.9, 80.9, 55.2, 53.8, 47.7, 47.5, 42.5, 39.8,
39.7, 39.5, 39.0, 38.7, 38.3, 37.7, 37.2, 36.8, 32.6, 31.9, 31.4, 30.9, 30.2, 29.7, 28.1, 27.8, 27.3,
26.0, 24.9, 23.5, 23.4, 23.2, 22.7, 21.3, 21.2, 20.6, 20.2, 18.1, 17.3, 16.9, 16.7, 15.6. HR-MS (m/z)
(ESI): calcd for C49H70N2O10 [M+H+]: 847.5109; found: 847.5100.
Compound 8b. Yield 75.1%; 1H NMR (400 MHz, CDCl3) δ 7.59 (s, 2H, galloyl-H), 6.58 (m, 1H,
CONH), 5.98 (t, J=5.6 Hz, 1H, CONH), 5.31 (s, 1H, H-12), 4.51-4.47 (m, 1H, H-3), 3.44-3.33 (m,
3H, CONH-CH2), 3.03-2.95 (m, 1H, CONH-CH2), 2.29 (br, s, 9H, CH3-COO-Ar), 2.05 (s, 3H,

C3-COOCH3), 1.09 (s, 3H, CH3), 0.94 (d, J=3.6 Hz, 6H, 2CH3), 0.88 (s, 3H, CH3), 0.87 (s, 3H,
CH3), 0.86 (s, 3H, CH3), 0.77 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.6, 171.1, 167.7,
166.5, 165.2, 143.4, 140.0, 137.0, 133.0, 125.5, 119.7, 80.9, 55.2, 53.9, 47.7, 47.5, 42.5, 40.1,
39.8, 39.5, 39.1, 38.9, 38.3, 37.7, 37.2, 36.8, 32.6, 31.4, 30.8, 30.2, 29.7, 29.3, 28.7, 28.1, 27.8,
24.9, 24.0, 23.5, 23.4, 23.2, 22.7, 21.3, 21.2, 20.6, 20.2, 18.1, 17.3, 16.9, 16.7, 15.6. HR-MS (m/z)
(ESI): calcd for C50H72N2O10 [M+H+]: 861.5265; found: 861.5242.
1
Compound 8c. Yield 75.4%; H NMR (400 MHz, CDCl3) δ 7.61 (s, 2H, galloyl-H), 6.75 (br, s,
1H, CONH), 5.99 (t, J=5.1 Hz, 1H, CONH), 5.32 (s, 1H, H-12), 4.51-4.47 (m, 1H, H-3), 3.48-3.29
(m, 3H, CONH-CH2), 3.01-2.93 (m, 1H, CONH-CH2), 2.30 (s, 3H, CH3-COO-Ar), 2.29 (s, 6H,
2CH3-COO-Ar), 2.05 (s, 3H, C3-COOCH3), 1.09 (s, 3H, CH3), 0.95 (s, 6H, 2CH3), 0.88 (s, 3H,
CH3), 0.87 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.78 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
178.4, 171.1, 167.8, 166.6, 165.1, 143.4, 140.0, 137.0, 133.1, 125.5, 119.8, 80.9, 55.2, 53.9, 47.7,
47.5, 42.5, 39.8, 39.6, 39.5, 39.1, 38.8, 38.3, 37.7, 36.8, 32.7, 30.9, 29.7, 29.4, 29.3, 28.1, 27.8,
25.9, 25.9, 24.9, 23.5, 23.4, 23.2, 21.3, 21.2, 20.6, 20.2, 18.1, 17.3, 16.9, 16.7, 15.6. HR-MS (m/z)
(ESI): calcd for C51H74N2O10 [M+H+]: 875.5422; found: 875.5419.
Compound 8d. Yield 74.9%; 1H NMR (400 MHz, CDCl3) δ 7.53 (s, 2H, galloyl-H), 6.26 (m, 1H,
CONH), 5.91 (s, 1H, CONH), 5.31 (s, 1H, H-12), 4.51-4.47 (m, 1H, H-3), 3.40-3.31 (m, 3H,
CONH-CH2), 3.00 (br, s, 1H, CONH-CH2), 2.30 (s, 9H, CH3-COO-Ar), 2.05 (s, 3H,
C3-COOCH3), 1.09 (s, 3H, CH3), 0.95 (br, s, 6H, 2CH3), 0.88 (s, 3H, CH3), 0.87 (s, 3H, CH3),
0.86 (s, 3H, CH3), 0.78 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.1, 171.1, 167.7, 166.6,
165.1, 143.5, 140.1, 137.0, 133.1, 125.5, 119.6, 80.9, 55.2, 54.0, 47.7, 47.5, 42.5, 40.2, 39.8, 39.5,
39.4, 39.1, 38.3, 37.7, 37.2, 36.8, 32.7, 30.9, 29.7, 29.5, 29.2, 28.9, 28.1, 27.8, 26.9, 26.8, 24.9,
23.5, 23.4, 23.2, 21.3, 21.2, 20.6, 20.2, 18.2, 17.3, 16.9, 16.7, 15.6. HR-MS (m/z) (ESI): calcd for
C52H76N2O10 [M+H+]: 889.5578; found: 889.5554.
1
Compound 9a. Yield 73.2%; H NMR (400 MHz, DMSO-d6) δ 8.00 (t, J=4.9 Hz, 1H, CONH),
7.08 (t, J=4.9 Hz, 1H, CONH), 6.80 (s, 2H, galloyl-H), 5.19 (s, 1H, H-12), 3.15-3.13 (m, 2H,
CONH-CH2), 3.00-2.98 (m, 2H, CONH-CH2), 2.15 (d, J=11.0 Hz, 1H, C3-OH), 1.01 (s, 3H,
CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.81(d, J=6.2 Hz, 6H, 2CH3), 0.66 (d, J=2.1 Hz, 6H,
2CH3). 13C NMR (100 MHz, DMSO-d6) δ 176.0, 166.2, 145.3, 138.4, 136.0, 125.0, 124.6, 106.6,
79.1, 76.9, 54.8, 54.8, 51.9, 47.0, 46.5, 41.5, 38.6, 38.4, 38.3, 38.2, 37.0, 36.4, 32.7, 31.3, 31.1,

30.4, 29.8, 29.0, 28.7, 28.2, 27.3, 26.9, 26.8, 26.4, 23.5, 23.3, 22.8, 22.1, 21.1, 17.9, 17.1, 16.8,
16.0, 15.1. HR-MS (m/z) (ESI): calcd for C41H62N2O6 [M+H+]: 679.4686; found: 679.4777.
1
Compound 9b. Yield 72.9%; H NMR (400 MHz, DMSO-d6) δ 7.97 (t, J=5.1 Hz, 1H, CONH),
7.07 (t, J=5.2 Hz, 1H, CONH), 6.80 (s, 2H, galloyl-H), 5.19 (s, 1H, H-12), 4.28 (s, 1H, H-3),
3.17-3.12 (m, 2H, CONH-CH2), 2.97-2.96 (m, 2H, CONH-CH2), 2.15 (d, J=10.5 Hz, 1H, C3-OH),
1.02 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.81(d, J=6.3 Hz, 3H,
CH3), 0.67 (d, J=8.1 Hz, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 176.0, 166.2, 145.3,
138.5, 135.9, 125.1, 124.5, 106.6, 76.8, 54.8, 51.9, 47.0, 46.5, 41.6, 38.4, 38.3, 38.2, 37.0, 36.5,
32.7, 31.3, 31.1, 30.4, 29.8, 29.0, 28.7, 28.2, 27.3, 27.0, 24.0, 23.5, 23.3, 22.8, 22.1, 21.1, 21.0,
17.9, 17.1, 16.8, 16.0, 15.1. HR-MS (m/z) (ESI): calcd for C42H64N2O6 [M+H+]: 693.4843; found:
693.4931.
1
Compound 9c. Yield 74.1%; H NMR (400 MHz, DMSO-d6) δ 8.00 (t, J=5.4 Hz, 1H, CONH),
7.09 (t, J=5.4 Hz, 1H, CONH), 6.80 (s, 2H, galloyl-H), 5.20 (s, 1H, H-12), 4.27 (s, 1H, H-3),
3.17-3.12 (m, 2H, CONH-CH2), 2.98-2.93 (m, 2H, CONH-CH2), 2.16 (d, J=10.2 Hz, 1H, C3-OH),
1.02 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.84-0.81 (m, 6H, 2CH3), 0.67 (d, J=10.8
Hz, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 176.0, 166.2, 145.3, 138.5, 136.0, 125.1, 124.5,
106.6, 76.8, 54.8, 51.9, 47.0, 46.5, 41.6, 38.7, 38.4, 38.3, 38.2, 37.0, 36.5, 32.7, 31.3, 30.4, 29.8,
29.3, 29.0, 28.7, 28.2, 27.3, 26.9, 26.3, 26.3, 23.5, 23.3, 22.8, 22.1, 21.1, 21.0, 17.9, 17.1, 16.8,
16.0, 15.1. HR-MS (m/z) (ESI): calcd for C43H66N2O6 [M+H+]: 707.4999; found: 707.5102.
1
Compound 9d. Yield 72.6%; H NMR (400 MHz, DMSO-d6) δ 8.00 (t, J=5.3 Hz, 1H, CONH),
7.09 (t, J=5.3 Hz, 1H, CONH), 6.79 (s, 2H, galloyl-H), 5.19 (s, 1H, H-12), 4.28 (s, 1H, H-3),
3.17-3.12 (m, 2H, CONH-CH2), 3.00-2.93 (m, 2H, CONH-CH2), 2.16 (d, J=10.3 Hz, 1H, C3-OH),
1.02 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.81 (d, J=6.3 Hz, 3H,
CH3), 0.68 (s, 3H, 2CH3), 0.66 (s, 3H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 175.9, 166.2,
145.3, 138.5, 135.9, 125.1, 124.5, 106.6, 76.8, 54.8, 51.9, 47.0, 46.5, 41.6, 38.4, 38.3, 38.2, 37.0,
36.5, 32.7, 31.3, 31.1, 30.4, 29.8, 29.2, 29.0, 28.7, 28.2, 27.3, 26.9, 26.6, 26.6, 23.5, 23.2, 22.8,
22.1, 21.1, 17.9, 17.1, 16.8, 16.0, 15.1. HR-MS (m/z) (ESI): calcd for C44H68N2O6 [M+H+]:
721.5156; found: 721.5156.
1
Compound 10a. Yield 82.9%; H NMR (400 MHz, CDCl3) δ 7.14 (s, 2H, galloyl-H), 7.02 (t,
J=5.3 Hz, 1H, CONH), 6.09 (t, J=5.8 Hz, 1H, CONH), 5.31 (t, J=3.3 Hz, 1H, H-12), 3.92 (s, 6H,

2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.52-3.41 (m, 3H, CONH-CH2), 3.23-3.19 (m, 1H,
H-3), 3.06-3.00 (m, 1H, CONH-CH2), 1.09 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.95 (s, 3H, CH3),
0.89-0.86 (m, 6H, 2CH3), 0.78 (s, 3H, CH3), 0.74 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
178.7, 167.1, 153.1, 139.9, 130.0, 125.7, 104.8, 79.0, 60.8, 56.4, 55.2, 53.9, 47.8, 47.6, 42.6, 39.9,
39.8, 39.6, 39.1, 38.8, 37.4, 36.9, 32.8, 30.9, 28.1, 27.9, 27.6, 27.2, 25.9, 24.9, 23.4, 23.2, 21.2,
18.3, 17.2, 17.0, 15.6. HR-MS (m/z) (ESI): calcd for C44H68N2O6 [M+H+]: 721.5156; found:
721.5151.
1
Compound 10b. Yield 80.3%; H NMR (400 MHz, CDCl3) δ 7.02 (s, 2H, galloyl-H), 6.40 (t,
J=5.4 Hz, 1H, CONH), 5.95 (t, J=5.8 Hz, 1H, CONH), 5.29 (t, J=3.2 Hz, 1H, H-12), 3.91 (s, 6H,
2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.46-3.36 (m, 3H, CONH-CH2), 3.23-3.19 (m, 1H,
H-3), 3.02-2.94 (m, 1H, CONH-CH2), 1.08 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.91 (br, s, 6H,
2CH3), 0.85 (d, J=6.4 Hz, 3H, CH3), 0.78 (s, 3H, CH3), 0.76 (s, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 178.4, 167.4, 153.2, 141.0, 140.0, 130.3, 125.6, 104.7, 79.0, 60.9, 56.4, 55.2, 54.0, 47.8,
47.6, 42.6, 40.0, 39.8, 39.6, 39.1, 38.8, 38.7, 37.2, 37.0, 32.8, 30.9, 29.3, 28.8, 28.2, 27.9, 27.2,
24.9, 24.0, 23.4, 23.2, 21.2, 18.3, 17.2, 16.9, 15.6, 15.5. HR-MS (m/z) (ESI): calcd for
C45H70N2O6 [M+H+]: 735.5312; found: 735.5302.
1
Compound 10c. Yield 81.5%; H NMR (400 MHz, CDCl3) δ 7.09 (s, 2H, galloyl-H), 6.61 (t,
J=5.2 Hz, 1H, CONH), 5.95 (t, J=5.6 Hz, 1H, CONH), 5.31 (t, J=3.1 Hz, 1H, H-12), 3.91 (s, 6H,
2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.55-3.40 (m, 2H, CONH-CH2), 3.36-3.28 (m, 1H,
CONH-CH2), 3.23-3.19 (m, 1H, H-3), 2.99-2.92 (m, 1H, CONH-CH2), 1.09 (s, 3H, CH3), 0.99 (s,
3H, CH3), 0.95 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.87 (d, J=6.4 Hz, 3H, CH3), 0.78 (s, 3H, CH3),
0.77 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.3, 167.4, 153.2, 140.1, 130.3, 125.6, 104.7,
79.0, 60.9, 56.4, 55.2, 54.0, 47.8, 47.6, 42.6, 39.9, 39.6, 39.4, 39.1, 38.8, 38.6, 37.3, 37.0, 32.8,
30.9, 29.4, 28.2, 27.9, 27.2, 25.8, 24.9, 23.4, 23.3, 21.2, 18.3, 17.2, 17.0, 15.6, 15.5. HR-MS (m/z)
(ESI): calcd for C46H72N2O6 [M+H+]: 749.5469; found: 749.5466.
1
Compound 10d. Yield 80.9%; H NMR (400 MHz, CDCl3) δ 7.00 (s, 2H, galloyl-H), 6.21 (t,
J=5.5 Hz, 1H, CONH), 5.91 (t, J=5.1 Hz, 1H, CONH), 5.30 (t, J=3.1 Hz, 1H, H-12), 3.91 (s, 6H,
2galloyl-OCH3), 3.88 (s, 3H, galloyl-OCH3), 3.46-3.41 (m, 2H, CONH-CH2), 3.37-3.28 (m, 1H,
CONH-CH2), 3.23-3.3.20 (m, 1H, H-3), 3.02-2.94 (m, 1H, CONH-CH2), 1.09 (s, 3H, CH3), 0.99
(s, 3H, CH3), 0.95 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.87 (d, J=6.4 Hz, 3H, CH3), 0.78 (br, s, 6H,

2CH3). 13C NMR (100 MHz, CDCl3) δ 178.0, 167.2, 153.2, 141.0, 140.1, 130.3, 125.6, 104.6,
79.0, 60.9, 60.3, 56.4, 55.2, 54.1, 47.7, 47.6, 42.6, 40.2, 39.8, 39.6, 39.4, 39.1, 38.8, 37.3, 37.0,
32.8, 30.9, 29.6, 29.3, 28.9, 28.1, 27.9, 27.2, 26.9, 24.9, 23.5, 23.3, 21.2, 18.3, 17.3, 17.0, 15.6,
15.5. HR-MS (m/z) (ESI): calcd for C47H74N2O6 [M+H+]: 763.5625; found: 763.5629.
Compound 11a. Yield 72.9%; 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 2H, galloyl-H), 7.14 (s, 1H,
CONH), 6.11 (s, 1H, CONH), 5.32 (s, 1H, H-12), 3.54-3.37 (m, 3H, CONH-CH2), 3.23-3.21 (m,
1H, H-3), 3.04-3.00 (m, 1H, CONH-CH2), 2.29 (s, 9H, CH3-COO-Ar), 1.10 (s, 3H, CH3), 0.99 (s,
3H, CH3), 0.95 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.87 (d, J=6.5 Hz, 3H, CH3), 0.77 (d, J=5.6 Hz,
6H, 2CH3). 13C NMR (100 MHz, CDCl3) δ 178.5, 167.7, 166.4, 165.2, 143.4, 139.9, 137.1, 132.9,
125.8, 119.9, 79.0, 55.1, 53.8, 47.7, 47.5, 42.5, 39.8, 39.7, 39.5, 39.0, 38.8, 38.7, 36.9, 31.9, 31.4,
30.2, 29.7, 29.4, 28.1, 27.3, 24.9, 23.2, 22.7, 21.2, 20.6, 20.2, 18.2, 17.2, 16.9, 15.6, 15.5. HR-MS
(m/z) (ESI): calcd for C47H68N2O9 [M+H+]: 805.5003; found: 805.4982.
Compound 11b. Yield 71.8%; 1H NMR (400 MHz, CDCl3) δ 7.59 (s, 2H, galloyl-H), 6.66 (s, 1H,
CONH), 6.00 (t, J=4.7 Hz, 1H, CONH), 5.31 (s, 1H, H-12), 3.41-3.33 (m, 3H, CONH-CH2),
3.23-3.19 (m, 1H, H-3), 3.02-2.97 (m, 1H, CONH-CH2), 2.29 (s, 9H, CH3-COO-Ar), 1.09 (s, 3H,
CH3), 0.99 (s, 3H, CH3), 0.93 (d, J=7.9 Hz, 6H, 2CH3), 0.86 (d, J=6.4 Hz, 3H, CH3), 0.77 (br, s,
6H, 2CH3). 13C NMR (100 MHz, CDCl3) δ 178.6, 167.7, 166.5, 165.2, 143.4, 140.0, 137.0, 133.0,
125.6, 119.7, 79.0, 55.1, 53.9, 47.7, 47.5, 42.5, 40.1, 39.8, 39.5, 39.1, 38.9, 38.8, 38.6, 37.2, 36.9,
32.7, 30.8, 29.2, 28.7, 28.1, 27.9, 27.2, 24.9, 24.1, 23.4, 23.2, 21.2, 20.6, 20.2, 18.3, 17.2, 16.9,
15.6, 15.5. HR-MS (m/z) (ESI): calcd for C48H70N2O9 [M+H+]: 819.5160; found: 819.5152.
1
Compound 11c. Yield 72.3%; H NMR (400 MHz, CDCl3) δ 7.60 (s, 2H, galloyl-H), 6.60 (t,
J=5.8 Hz, 1H, CONH), 5.96 (t, J=5.8 Hz, 1H, CONH), 5.32 (s, 1H, H-12), 3.51-3.31 (m, 3H,
CONH-CH2), 3.23-3.19 (m, 1H, H-3), 3.01-2.96 (m, 1H, CONH-CH2), 2.30 (s, 3H,
CH3-COO-Ar), 2.29 (s, 6H, 2CH3-COO-Ar), 1.10 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.95 (s, 3H,
CH3), 0.91 (s, 3H, CH3), 0.87 (d, J=6.6 Hz, 3H, CH3), 0.78 (d, J=4.6 Hz, 6H, 2CH3). 13C NMR
(100 MHz, CDCl3) δ 178.4, 167.7, 166.5, 165.0, 143.4, 140.0, 137.0, 133.0, 125.6, 119.7, 79.0,
55.1, 54.0, 47.7, 47.5, 42.5, 39.8, 39.5, 39.1, 38.8, 37.2, 36.9, 32.7, 30.9, 29.7, 29.3, 28.1, 27.8,
27.2, 25.9, 25.8, 23.4, 23.2, 22.7, 21.2, 20.6, 20.2, 18.3, 17.3, 16.9, 15.6, 15.5. HR-MS (m/z)
(ESI): calcd for C49H72N2O9 [M+H+]: 833.5316; found: 833.5308.

Compound 11d. Yield 71.2%; 1H NMR (400 MHz, CDCl3) δ 7.53 (s, 2H, galloyl-H), 6.22 (s, 1H,
CONH), 5.91 (s, 1H, CONH), 5.31 (s, 1H, H-12), 3.42-3.31 (m, 3H, CONH-CH2), 3.23-3.19 (m,
1H, H-3), 3.00 (br, s, 1H, CONH-CH2), 2.30 (s, 9H, CH3-COO-Ar), 1.09 (s, 3H, CH3), 0.99 (s,
3H, CH3), 0.95 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.87 (d, J=6.5 Hz, 3H, CH3), 0.77 (br, s, 6H,
2CH3). 13C NMR (100 MHz, CDCl3) δ 178.1, 167.7, 166.6, 165.1, 143.5, 140.1, 137.0, 133.1,
125.6, 119.6, 79.0, 55.1, 54.0, 47.7, 47.5, 42.5, 40.2, 39.8, 39.5, 39.3, 39.1, 38.8, 38.6, 37.2, 36.9,
32.8, 31.4, 30.9, 30.2, 29.7, 29.5, 29.2, 28.9, 28.1, 27.9, 27.2, 26.9, 26.8, 24.9, 23.4, 23.2, 21.2,
20.6, 20.2, 18.3, 17.3, 16.9, 15.6, 15.5. HR-MS (m/z) (ESI): calcd for C50H74N2O9 [M+H+]:
847.5473; found: 847.5464.
4.3 Cytotoxicity assay
The cell lines T24, HepG2, SKOV3, A549 and HL-7702 were obtained from the Shanghai Cell
Bank in the Chinese Academy of Sciences. T24, HepG2, SKOV3, A549 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 HCPT, 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.
4.4 In vitro inhibition of TNF-a-induced NF-κB activation in A549 lung cancer cells
The A549 cells were cultured in 12-well plates and transiently co-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 manufacturer’s instructions. After 6 h, the medium was
changed and cells were cultured for 6 h. Cells were then treated with TNF-α (15 ng/ml) and test

compounds simultaneously for 7 h. The A549 cells treated with TNF-α alone served as positive
controls, while cells without TNF-α treatment served as negative controls. Luciferase activities
from these cells were then measured by using the Bright-Glo Luciferase Assay kit from Promega
(Madison, WI), following the manufacturer’s protocol. The relative NF-κB activities of the cells
treated by test compounds were obtained as the ratio of its luciferase activity to that from the
positive controls, both of which have been corrected with background (signals from negative
controls) and cell viability. In these experimental conditions, none of the test compounds induced
significant toxicity to A549 cells (<5% reduction of cell viability). The IC50 of each fraction was
determined by fitting the relative NF-κB activity to the drug concentration by using a sigmoidal
dose eresponse model of varied slope in GraphPad Prism 6. The IC50 reported herein is the
average of at least three replicates.
4.5 NF-κB DNA binding assay
To determine NF-κB activation, we performed DNA binding assay using TransAM NF-κB Kit
according to the manufacturer’s instructions and as previously described [32]. Briefly, 20 μg of
nuclear proteins was added into 96-well plate coated with an unlabeled oligonucleotide containing
the consensus binding site for NF-κB (5’-GGGACTTTCC-3’) and incubated for 1 h. The wells
were washed and incubated with antibodies against NF-κB p65 subunit. An HRP conjugated
secondary antibody was then applied to detect the bound primary antibody and provided the basis
for colorimetric quantification. The enzymatic product was measured at 450 nm by microplate
reader (TECAN Systems).
4.6 Molecular docking
All the docking studies were carried out using Sybyl-X 2.0 on a windows workstation. The
crystal structure of the NF-κB proteins were retrieved from the RCSB Protein Data Bank (PDB:
1IKN) [24]. The synthetic analogueswere selected for the docking studies. The 3D structures of
these selected compounds were first built using Sybyl-X 2.0 sketch followed by energy
minimization using the MMFF94 force field and Gasteiger-Marsili charges. Before the docking

process, the natural ligand was extracted; the water molecules were removed from the crystal
structure. Subsequently, the protein was prepared by using the Biopolymer module implemented
in Sybyl. The polar hydrogen atoms were added. The automated docking manner was applied in
the present work. Other parameters were established by default to estimate the binding affinity
characterized by the Surflex-Dock scores in the software. Surflex-Dock total scores, which were
expressed in -log10 (Kd) units to represent binding affinities, were applied to estimate the
ligand-receptor interactions of newly designed molecules. A higher score represents stronger
binding affinity. The optimal binding pose of the docked compounds was selected based on the
Surflex scores and visual inspection of the docked complexes.
4.7 In vitro cell migration assay/wound healing assay
A549 cells (5×105 cells/well) were cultured in 6 well plates as confluent monolayers for 24 h.
Then artificial scratch on the monolayers were created with 200 mL sterile pipette tip and washed
twice with PBS to remove non-adherent cells. The media containing 5, 10and 20 μM of compound
8c or without compound 8c were added to each well. The migration of cells across the scratched
area were photographed by using phase contrast microscope (Nikon) at 0 h (immediately) and 24
h time interval after treatment in three or more randomly selected fields.
4.8 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 8c for 24 h. Briefly, after treatment with
compound 8c for 24 h, cells were collected and washed with PBS twice, and then resuspend 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 (Cell Quest; BD Biosciences).
4.9 Hoechst 333258 assay
A549 cells (2× 106 cells) were seeded in six-well tissue culture plates and exposed to compound
8c (5, 10and 20 μM) for 24 h. The cells were fixed in 4% paraformaldehyde for 10 min followed
by the medium was discarded. The cells were then washed twice with cold PBS and incubated
with 0.5 mL of Hoechst 33258 (Beyotime, China) at dark for 5 min. After 5 min incubation, the
cells were washed twice with cold PBS and the results were analysis by a Nikon
ECLIPSETE2000-S fluorescence microscope using 350 nm excitation and 460 nm emissions.
4.10 Cell cycle analysis
The A549 cells line were treated with different concentrations of compound 8c. After 24 h of
incubation, cells were washed twice with ice-cold PBS, fixed and permeabilized with ice-cold
70% ethanol at -20°C overnight. The cells were treated with 100 μg /ml RNase A at 37°C for 30
min after washed with ice-cold PBS, and finally stained with 1 mg/ml propidium iodide (PI) (BD,
Pharmingen) in the dark at 4°C for 30 min. Analysis was performed with the system software
(Cell Quest; BD Biosciences).
4.11 Determination of caspase-3 activity
The measurement of caspase-3 activity was performed by CaspGLOW™ Fluorescein Active
Caspase-3 Staining Kit. 1×106 of A549 cells were cultured for 24 h. After a treatment with 5, 10
and 20 μM compound 8c for 24 h, cells were harvested and washed 3 times with cold PBS and
were then mixed with 300 mL culture. 1 mL of FITC-DEVD-FMK was consequently added and
incubated for 1.0 h at 37 °C with 5% CO2. The cells were then examined by a FACSAria II flow
cytometer equipped with a 488 nm argon laser and results were represented as the percent change
on the activity comparing with the untreated control.

4.12 Statistics
The data were processed by the Student' st -test with the significance level P≤0.05 using SPSS.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (No. 21431001,
81760626), Project of State Key Laboratory for Chemistry and Molecular Engineering of
Medicinal Resources, Ministry of Science and Technology of China (No. CMEMR2016-B06), the
Development Project of Qinghai Key Laboratory (No. 2017-ZJ-Y10), A Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions (No.
1107047002), Natural Science Foundation of Guangxi Province (2017AB44061), 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), the National Students’ Innovation and
Entrepreneurship Training Program (NO. 201710286088).
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Schemes
Scheme 1. General synthetic route for target compounds 6-11. Reagents and conditions: (a)
Ac2O,DMAP, Py, r.t; (b) (CO)2Cl2, CH2Cl2, r.t; (c) H2N(CH2)nNH2, Et3N, CH2Cl2, r.t; (d)NaOH,
CH3OH, THF, r.t; (e) EDCI, HOBt, Et3N, THF, r.t.

Tables
Table 1 Effect of target compounds against cell viability of different cell Lines
Compd.
IC50 (μM)a
T24
A549
23.74±1.03
17.27±0.82
10.36±0.97
30.25±1.11
>50
HepG2
28.35±1.35
19.07±0.90
18.18±1.08
35.32±1.24
>50
SKOV3
29.78±0.94
20.33±1.06
13.05±0.69
41.25±0.86
>50
HL-7702
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25.55±1.36b
18.52±0.64
11.24±1.31
34.10±1.27
>50
6a
6b
6c
6d
7a
>50
>50
>50
>50
7b
7c
>50
>50
>50
>50
>50
>50
>50
>50
7d
8a
13.37±1.13
7.74±1.25
6.01±0.87
16.31±1.39
45.27±1.09
36.16±1.13
37.51±2.03
>50
12.19±1.43
7.23±1.28
5.22±0.65
13.38±1.14
40.09±1.54
34.01±1.01
31.48±1.29
>50
12.89±1.17
9.45±1.45
6.82±1.07
17.52±1.51
47.37±1.46
41.48±1.12
40.20±1.35
>50
15.02±1.60
10.58±1.34
8.95±1.26
20.47±0.79
>50
8b
8c
8d
9a
45.14±1.27
39.38±1.10
>50
9b
9c
9d
10a
10b
10c
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50

>50
>50
>50
>50
>50
>50
10d
11a
18.14±1.32
10.73±1.16
7.27±1.24
20.13±0.95
14.69±1.75
8.17±0.92
6.83±1.38
16.78±1.04
18.41±1.06
13.4±1.08
7.72±1.25
21.69±1.19
21.37±1.11
12.94±1.26
9.23±0.86
25.37±1.53
>50
11b
>50
11c
>50
11d
UA
37.88 ± 1.12 40.91 ± 0.92 42.37 ± 0.87 45.53 ± 1.21
11.69 ± 0.75 2.73 ± 1.02 3.19±0.56 6.01±0.67
>50
HCPT
14.83±1.01
a IC50 values are presented as the mean ± SD (standard error of the mean) from five
independent experiments. b Significant difference from the viability of control (pꢀ≤0.05).
Table 2 In vitro inhibition of TNF-α-induced NF-κB activation in the A549 lung cancer cell
line
Compd.
6a
IC50 (μM)
13.45±0.43d
9.18±0.14
Compd.
9a
IC50 (μM)
18.08±0.46
15.32±0.85
6b
9b