Glycyrrhetinic acid derivatives containing aminophosphonate ester species as multidrug resistance reversers that block the NF-κB pathway and cell proliferation

Article abstract of DOI:10.1016/j.bmcl.2018.10.025

Novel NF-κB inhibitors based on Glycyrrhetinic acid (GA) derivatives containing aminophosphonate ester moieties were rationally designed and synthesized as well as evaluated their antitumor activities using MTT assay. Many target compounds showed potent antitumor activities against the tested human cancer cell lines including cisplatin-resistant cells, and exhibited significant inhibitory activity to the NF-κB with IC₅₀ values at micromolar concentrations in A549 cells, respectively. Among them, compound 12e possessed excellent antitumor activities against the tested human cancer cell lines and showed low cytotoxicity toward to human normal liver cells. Moreover, 12e caused obvious loss of MMP and significantly induced ROS production, and displayed inhibition of cell migration against A549 cells in vitro. Importantly, 12e arrested the cell cycle at the S phases and ultimately induced cell apoptosis in A549 cells through blockage of NF-κB signaling pathway. Our research provided an efficient strategy for targeting NF-κB antitumor drug development.

Full text of DOI:10.1016/j.bmcl.2018.10.025

Accepted Manuscript
Glycyrrhetinic acid derivatives containing aminophosphonate ester species as
multidrug resistance reversers that block the NF-κB pathway and cell prolifer-
ation
Le Jin, Bin Zhang, Shixian Hua, Min Ji, Xiaochao Huang, Rizhen Huang,
Hengshan Wang
PII:
S0960-894X(18)30827-8
https://doi.org/10.1016/j.bmcl.2018.10.025
BMCL 26084
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 June 2018
1 October 2018
17 October 2018
Please cite this article as: Jin, L., Zhang, B., Hua, S., Ji, M., Huang, X., Huang, R., Wang, H., Glycyrrhetinic acid
derivatives containing aminophosphonate ester species as multidrug resistance reversers that block the NF-κB
pathway and cell proliferation, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/
j.bmcl.2018.10.025
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Bioorganic & Medicinal Chemistry Letters
Glycyrrhetinic acid derivatives containing aminophosphonate ester species as
multidrug resistance reversers that block the NF-κB pathway and cell
proliferation
a,1
b,1
b
a,

b
b
c,

Le Jin , Bin Zhang , Shixian Hua , Min Ji , Xiaochao Huang , Rizhen Huang , and Hengshan Wang
a School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
b Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China.
c State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi
Normal University, Guilin 541004, PR China.
ARTICLE INFO
ABSTRACT
Novel NF-κB inhibitors based on Glycyrrhetinic acid (GA) derivatives containing
aminophosphonate ester moieties were rationally designed and synthesized as well as evaluated
their antitumor activities using MTT assay. Many target compounds showed potent antitumor
activities against the tested human cancer cell lines including cisplatin-resistant cells, and
exhibited significant inhibitory activity to the NF-κB with IC50 values at micromolar
concentrations in A549 cells, respectively. Among them, compound 12e possessed excellent
antitumor activities against the tested human cancer cell lines and showed low cytotoxicity
toward to human normal liver cells. Moreover, 12e caused obvious loss of MMP and
significantly induced ROS production, and displayed inhibition of cell migration against A549
cells in vitro. Importantly, 12e arrested the cell cycle at the S phases and ultimately induced cell
Keywords:
Glycyrrhetinic acid
Aminophosphonate ester
NF-κB
Drug resistance
Migration
apoptosis in A549 cells through
efficient strategy for targeting NF-κB antitumor drug development.
2009 Elsevier Ltd. All rights reserved.
κ
Our research provided an
Transcription factor nuclear factor-kappa B (NF-κB) is a transcript factor discovered by Sen and co-workers in 1986, which is usually
over-expressed and constitutively activated in many types of malignancies.1-4 NF-κB, as a family of related protein hetero- or
homodimers, promotes the downstream protein expression of anti-apoptosis (XIAP, Bcl-2 and Bcl-xL), proliferation (c-Myc) and
invasion (MMP-2 and MMP-9), and exhibits remarkable capabilities for regulating the transcription of hundreds of target genes.5-7
Increasing evidences indicated that the aberrant activation of NF-κB pathways allows cancer cells to escape apoptosis, invasion and
metastasis, which contributes to cancer drug resistance.6-10 Because activation of NF-κB is an essential feature of the survival of cancer
cells during treatment, which results in treatment resistance, considerable researchers have focused on targeting NF-κB for cancer
therapy. In facts, bortezomib (BTZ), one of the FDA approved anticancer drugs for treating multiple myeloma in the clinical, which
induces apoptosis by blocking NF-κB activation.11 This success greatly encouraged the development of NF-κB inhibitors to sensitize
cancer cells of solid tumors to anticancer drugs. Thus, designing NF-κB inhibitors is a good choice to develop new cancer therapeutics
and overcome drug resistance.
__________________
 Corresponding author at: School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China, State Key Laboratory for the
Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin
541004, PR China.
E-mail addresses: jiminseu@163.com, whengshan@163.com.
1 Co-first author: These authors contributed equally to this work.
In recent years, triterpenoids are highly multifunctional and have attracted many researchers interesting as potential anticancer agents
owing to their ability to interact with multiple biological targets.12-14 Glycyrrhetinic acid (GA), also known as enoxolone, is a
pentacyclic triterpenoid obtained from Glycyrrhiza glabra, served as one of the major effective elements of many traditional Chinese
medicines. 15,16 In recent years, GA and its derivatives were reported to exhibit moderate antitumor activity against several cancer cells
as well as induce cells apoptosis.17-19 Notably, these compounds were found as potent inhibitors of NF-κB.20 Liao and co-workers
reported that aniline-derived ursolic acid (pentacyclic triterpenoids, UA) derivatives as multidrug resistance reversers by blocking NF-
κB pathway.21 Previous work has also found that the modification in position C-3 and/or C-28 of GA or UA has properties of improving
the antitumor activity of anticancer drugs in various cells.22-24 Recently, Huang and co-workers reported that asiatic acid (AA), as a
pentacyclic triterpene acid isolated from Centella asiatica, containing ɑ-aminophosphonates moieties at C-28 position in AA showed
more potent antitumor activities against the tested human cancer cell lines compared to the parent compound AA.25 In facts, many
studies demonstrated that introduction of a phosphate ester moiety to chemotherapy drugs could significantly increase the solubility of
26
drugs and improve transport through cellular membrane.
It is generally acknowledged that most of natural or synthetic
aminophosphonate compounds exhibited a broad spectrum of biological activities such as antiviral, antimicrobial, antioxidant,

antitumor and anti-inflammatory, thus, application of these compounds is a good choice in the pharmacological and agrochemical
fields.27-30 In addition, some phosphate esters have a high affinity toward to calcium ions, thus they have been commonly applied to
design targeted drugs used for bone cancer treatment.31 Moreover, phosphonate esters can be hydrolyzed under physiological conditions,
therefore, introduction of aminophosphonate esters in drugs is a promising approach to develop targeted antitumor agents.32
In the light of above considerations, in this paper, a series of GA derivatives containing aminophosphonate ester species were
deliberately designed and obtained. These GA derivatives were expected to target tumor tissue and inhibit NF-κB, and they in vitro
cytotoxicity against a number of tumor cell lines and a human normal liver cell line were also evaluated. In addition, the mode of action
of a representative compound 12e that blocked the TNF-α-induced NF-κB pathway was investigated.
The general procedures for the synthesis of GA contained aminophosphonate ester derivatives are shown in Scheme 1. Compounds 1
were prepared by the treatment of 4-aminophenylacetic acid with benzaldehyde in methanol. Compounds 1 were followed by treatment
of diethyl phosphite to obtain Compounds 2. GA was acetylated in C-3 with acetic anhydride in pyridine to obtain ester 3, then 3
followed by its acylation with oxalyl chloride in the presence of dry dichloromethane to obtain the intermediate 4. Compound 5 and
compound 6 were achieved by the formation of amide bond between 4 and N-Boc-1, 3-propanediamine or N-Boc-1, 4-butanediamine.
Compound 5 and compound 6 were taken off protecting groups by TFA to afford compound 7 and 8. The target compounds 11a-11k
and 12a-12k were prepared by the treatment of compound 7 and 8 with EDCI, DMAP, and compounds 2, respectively, in DCM at room
temperature. Compounds 7 and 8 were hydrolyzed by NaOH aqueous solution to afford Compounds 9 and 10. The target compounds
13a-13k and 14a-14k were synthesized by the same method as compounds 11 and compounds 12. The structures of target compounds
11a-11k, 12a-12k, 13a-13k and 14a-14k were then confirmed by 1H NMR, 13C NMR and high resolution mass spectrometry (HR-MS).
Scheme 1. Synthetic route for the final compounds 11a-11g, 12a-12g, 13a-13g and 14a-14g from 18β-glycyrrhetinic acid. Reagents and conditions: (a) CH3OH,
reflux, overnight; (b) diethyl phosphite, 50 °C, 2 h; (c) acetic anhydride, pyridine, 0 °C, 24 h; (d) oxalyl chloride, CH2Cl2, 0 °C, 12 h; (e) TEA, CH2Cl2, 0 °C, 2 h;
(f) TFA, CH2Cl2, 0 °C, 1 h; (g) EDCI, DMAP, CH2Cl2, rt, 1 h; (h) NaOH aq., MeOH/THF, 40 °C, 5 h, 12 h; (i) EDCI, DMAP, CH2Cl2, rt, 1 h.

The in vitro anti-proliferative efficacy of the GA analogues 11a-11g, 12a-12g, 13a-13g and 14a-14g were evaluated by the MTT
assay against HepG-2 (hepatoma), NCI-H460 (lung), MGC-803 (gastric), A549 (breast), and MG-63 (osteosarcoma) human cancer cell
lines as well as human normal cell line HL-7702 (liver). GA and 10-hdroxycamptothecin (HCPT) were used as the reference
compounds. As shown in Table 1, most of newly synthesized GA derivatives 11-14 showed much higher inhibitory activity than GA
against the HepG-2, NCI-H460, MGC-803, A549 and MG-63 cell lines, indicating the introduction of aminophosphonate ester on GA
could markedly increase of antiproliferative activity. Moreover, 12e was the most potent compound in this series, with IC50 values
ranging from 6.25 to 9.11 μM in the cancer cell lines, and even more potent than HCPT as well as showed lower cytotoxicity toward to
human normal live cells HL-7702, respectively. Interestingly, compounds 11 (11b, 11d and 11f), 12 (12b, 12d and 12f), 13 (13b, 13d
and 13f) and 14 (14b, 14d and 14f) with a electron donor substituent (-OCH3) and halogen group (-F or -Br) at 3-position in benzene
ring showed a reduction in anti-proliferative activity compared with 11a, 12a, 13a and 14a on HepG-2 cells, but in contrast,
introduction of electron donor substituent (-OCH3) and halogen group (-F and -Br) at 4-position in benzene ring (11c, 11e, 11g, 12c, 12e,
12g, 13c, 13e, 13g, 14c, 14e and 14g) displayed a increasing of antitumor activity. The same results were also observed in NCI-H460,
MGC-803, A549 and MG-63. Furthermore, 12 or 14, with the increase of the length of the alkyl chain of the amide, showed a
significant increase in antiproliferative activity compared with 11 or 13, respectively. Interestingly, compounds 11 and 12, an acetyl
group was introduced at the 3-OH position, displayed better cytotoxicity than 13 and 14, indicating an acetyl group introduction of the
3-OH position caused a significant increase in the antitumor activity. In shorts, these cytotoxicity screening results implied that the
introduction of aminophosphonate moiety at C-28 position and an acetyl group at 3-OH in GA could obviously improve the antitumor
activity.
Table 1. Effects of GA derivatives 11a-11g, 12a-12g, 13a-13g and 14a-14g against different human cancer cell lines including
human normal liver cells HL-7702.
Compd.
IC50 (μM)a
HepG-2
31.63±2.21
38.19±2.03
19.31±2.16
37.74±1.09
15.31±1.13
>50
NCI-H460
23.25±1.91
31.65±1.88
18.77±2.04
29.47±1.08
14.88±1.23
>50
MGC-803
21.03±1.68
36.73±2.04
18.23±1.25
27.25±0.99
16.33±1.17
>50
A549
MG-63
25.29±2.29
39.51±1.92
19.13±2.08
>50
HL-7702
>50
30.28±2.25
39.33±2.96
19.28±2.12
33.96±1.07
15.01±1.02
>50
11a
11b
11c
11d
11e
11f
>50
>50
>50
13.72±1.32
>50
>50
>50
23.62±1.37
26.28±1.54
35.26±2.05
15.35±1.25
21.33±1.09
9.11±1.23
48.19±2.11
19.78±1.63
40.41±1.74
>50
17.08±1.37
13.23±1.37
27.37±1.61
14.12±1.15
28.18±2.31
8.28±1.07
47.60±2.18
10.18±1.07
46.08±1.43
>50
29.85±1.24
15.24±1.70
32.90±1.67
16.33±1.18
21.02±1.24
8.73±1.09
41.77±2.21
17.35±1.53
31.07±1.28
45.23±1.84
25.69±1.51
>50
20.47±1.35
23.08±1.29
27.25±2.03
15.25±1.07
26.41±1.52
6.25±0.95
39.63±2.16
17.27±1.22
36.01±2.03
>50
21.34±0.97
20.16±1.64
36.61±2.30
13.93±1.29
22.83±1.26
9.01±1.01
>50
>50
11g
12a
12b
12c
12d
12e
12f
>50
>50
>50
>50
>50
>50
19.21±1.29
31.04±1.91
46.55±2.13
23.61±1.43
>50
>50
12g
13a
13b
13c
13d
13e
13f
>50
>50
36.81±1.42
>50
29.44±1.39
>50
23.08±1.49
>50
>50
>50
29.11±1.50
>50
17.27±1.13
>50
20.52±1.36
>50
17.93±1.23
>50
16.48±1.37
>50
>50
>50
35.63±1.27
37.15±0.79
46.39±2.01
32.19±1.65
>50
20.27±1.82
30.48±1.34
39.25±1.73
24.04±1.09
>50
35.72±1.89
36.27±1.05
>50
31.93±1.67
33.21±1.86
48.31±1.79
25.18±1.41
>50
25.13±1.81
27.84±2.01
45.61±2.17
17.22±1.51
>50
>50
13g
14a
14b
14c
14d
14e
14f
>50
>50
22.21±2.08
>50
>50
>50
20.21±1.56
>50
19.37±1.39
>50
18.31±1.66
>50
19.76±1.33
>50
12.39±1.19
>50
>50
>50
29.23±1.81
44.51±2.41
10.21±1.06
25.16±1.34
37.65±2.12
10.08±1.05
30.93±1.93
>50
24.89±1.39
36.15±2.38
9.13 ± 1.09
24.63±1.72
>50
>50
14g
GA
HCPT
>50
9.71 ± 1.03
10.69 ± 1.15
10.23±1.19
a Each data represents mean ± S.D. from three different experiments performed in triplicate.
All synthesized compounds were further assayed for their inhibitory potency against the TNF-α-induced NF-κB transcriptional
activity, which considered as an important therapeutic target in hematological cancer and malignancies as well as in human non-small
cell lung cancer A549 cells.33, 34 The non-small cell lung cancer A549 cells transiently cotransfected by NF-κB-Luc was used to detected

the effects of GA derivates on tumor necrosis factor-alpha (TNF-α)-induced NF-κB activation. The target compounds 11, 12, 13 and 14
were exposed in a concentration-dependent manner to confirm 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 cells by tested title compounds are depicted in
Table 2. As shown in Table 2, most of title compounds showed significant inhibitory potency against NF-κB, with IC50 values mostly in
the micromolar range. Moreover, the tested compounds displayed a significant increase in inhibition of TNF-α-induced NF-κB
activation compared with corresponding parent compound GA, supporting the conclusion that introduction of aminophosphonate
moiety at C-28 position in GA result to improve the anticancer activity. Interestingly, compounds 11c (5.03±0.46 μM), 11e (3.01±0.51
μM), 12c (2.01±0.33 μM) and 12e (0.97±0.21 μM), introduction of electron donor substituent (-OCH3) or halogen group (-F) at 4-
position in benzene ring and acetyl group introduction of the 3-OH position in GA, exhibited a significant increase in inhibition of TNF-
α-induced NF-κB activation compared with 11a (22.05±0.83 μM) and 12a (13.23±0.72 μM), and the inhibitory activity was increased
4.4-fold, 7.3-fold, 6.6-fold and 13.6-fold compared with 11a and 13a, respectively. In contrast, introduction of electron donor
substituent (-OCH3) or halogen group (-F) at 3-position in benzene ring and deacetylation of the 3-OH position in GA, the inhibition
potency was markedly decreased, such as 11a-11g and 12a-12g exhibited more potent inhibition of TNF-α-induced NF-κB than 13a-
13g and 14a-14g, respectively. More importantly, compounds (such as 11a-11g and 12a-12g), different in the carbon chain length,
exhibited different inhibition of TNF-α-induced NF-κB activation that are enhanced with an increase of the carbon chain length,
consistent with the results for in vitro cytotoxicity assay.
Table 2. In vitro inhibition of TNF-α-induced NF-κB activation in A549 cells.
Compounds
IC50 (μM)
Compounds
IC50 (μM)
22.05±0.83
>30
28.25±0.53
N.I.
11a
11b
11c
11d
11e
11f
13a
13b
13c
5.03±0.46
>30
17.09±0.71
N.I.
13d
13e
13f
13g
14a
14b
14c
14d
14e
14f
14g
GA
3.01±0.51
>30
8.89±0.63
N.I.
28.39±0.91
13.23±0.72
23.41±0.68
2.01±0.33
27.36±0.82
0.97±0.21
27.05±0.62
11.09±0.30
>30
11g
12a
12b
12c
12d
12e
12f
12g
25.13±0.59
29.45±0.36
13.09±0.41
>30
6.09±0.35
>30
16.29±0.85
>30
In order to confirm the interactions between the most active compound 12e and the target of interest (NF-κB), we performed
molecular docking calculations on the active site of NF-κB (PDB ID: 1IKN) 35 using SYBYL-X 2.0 software and the score results are
summarized in Table S1. The results of the molecular docking study are depicted in Fig.1, and the surflex docking score is 9.41 for 12e,
where higher score indicate greater binding affinity. It is observed that the GA moiety of compound 12e goes deep into the pocket, and
surrounded by residues ILE298, HIS193, LEU207, PR290 and ARG295, indicating that 12e can indeed bind to the NF-κB (Fig.1).
Notably, the binding pocket of NF-κB interacted with the aminophosphonate moiety of compound 12e to form five hydrogen bonds
through the side chains of ARG253 and ILE212, respectively, which plays an important role in binding with NF-κB. Overall, the result
of the molecular docking study proves that compound 12e is a potential inhibitor of NF-κB.
Fig. 1 Binding modes of compound 12e in the active site of NF-κB (PDB ID: 1IKN). 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).
Because of the major role of the transcription factor NF-κB in tumorigenesis and survival 36, 37, it was supposed that compound 12e
might mediate its anticancer effects through modulating the NF-κB activation pathway. Thus, we next detected the effect of 12e on
constitutive NF-κB activation in A549 cells. As expected, results of ELISA-based DNA binding assay obviously indicated that 12e
clearly suppressed TNF-α-induced NF-κB activation after cells treated with 12e at the tested concentrations (5, 10 and 20 μM) (Fig. 2).

Interestingly, compound 12e decreased the NF-κB DNA binding ability by nearly 2.3-fold than that of TNF-α, and these results
suggested that 12e can significantly modulate constitutive NF-κB activation in A549 cells.
κ
α
The experiments were performed three times, and the results of the representative
experiments are shown.
In recent years, several studies have shown that pentacyclic triterpenoids effectively suppressed NF-κB translocation to the nucleus
by inhibition the activity of IκB kinase, which is known to be involved in the process of the activation of NF-κB.38, 39 Thus, in order to
determine the effect of the highly potent compound 12e on the NF-κB pathway, the relative levels of NF-κB, IκBα and phosphorylated
IKKβ were investigated by Western blotting assay. As shown in Fig. 3, consistent with expectations, phosphorylated IκBα was
significantly inhibited in a concentration-dependent manner and the IκBα protein was observed to be slightly suppressed after cells
treated with compound 12e at the indicated concentration. Meanwhile, as illustrated in Fig. 3, incubation of the cells with compound
12e for 24 h obviously suppressed the phosphorylation of IKKα/β. In summary, these results further suggested that compound 12e
suppressed NF-κB activation, mainly through inhibition of IκBα phosphorylation mediated by the IKKα/β phosphorylation.
Drug resistance has seriously limited the efficacy of cisplatin in clinical applications for treatment of solid tumors.31 In terms of the
strong inhibiting abilities on the tested cancer cell lines, compounds 11e and 12e were further evaluated against cisplatin-resistant SK-
OV-3/CDDP and cisplatin sensitive SK-OV-3 cancer cells. As shown in Table 3, the IC50 values of cisplatin against SK-OV-3/CDDP
was increased to 27.62±1.41 μM, whiles the cytotoxicity of title compounds 11e and 12e against SK-OV-3 and SGC-7901/CDDP cancer
cells were quite similar, indicating that GA derivates 11e and 12e may have the potential to overcome cisplatin resistance. Importantly,
compound 12e is very active in cisplatin-resistant SK-OV-3 cancer cells with a resistance factor value of 0.92.
Fig. 3. A549 cells were incubated with compound 12e at the tested concentrations (5, 10 and 20 μM) for 24h. The expressions of NF-κB, p-IκBα, IκBα and p-
IKKα/β were determined by western blotting assay. GAPDH was used as internal control. The experiments were performed three times, and the results of the
representative experiments are shown.
Table 3. In vitro growth inhibitory effect of 11e and 12e on cisplatin-resistant cancer cells.
a
b
Cisplatin, Each data represents mean ± S.D. from three different experiments
Compd.
IC50 (μM) b
SK-OV-3/CDDP
13.36±1.13
performed in triplicate, c The values express the ratio between IC50 determined in
resistant and nonresistant cell lines.
SK-OV-3
resistant factor c
1.32
In order to investigate whether the observed cytotoxicity of
compound 12e is owing to induction of apoptosis, therefore, an
annexin V-FITC/PI dual staining assay was measured after treatment
of A549 cells. Q1, Q2, Q3, and Q4 represented four different cell
states: necrotic cells, late apoptotic or necrotic cells, early apoptotic
cells and living cells, respectively. As depicted in Fig. 4, after 24 h
of treatment, significant apoptotic effects were observed for 12e in
A549 cells. The analysis of the apoptotic effects in Fig. 4 obviously
10.11±1.03
11e
12e
9.75±1.09
8.94±1.05
0.92
CDDP a
GA
4.81±0.31
27.62±1.41
49.36±2.35
5.74
1.08
45.62±0.38
showed a concentration-dependent manner, especially for early apoptosis. At the maximal tested concentration, 12e (20 μM) caused
early and late apoptosis in 19.5% and 39.7% of A549 cells, respectively. In shorts, these results clearly indicated that 12e significantly
induced apoptosis in A549 cancer cells.

Fig. 4. Compound 12e induced the apoptosis of A549 cells. Cells were treated with 12e at the tested concentrations (5, 10 and 20 μM) for 24. Then, the cells were
harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry, respectively. The experiments were performed three times, and the results of
the representative experiments are shown.
It is well-known that the cell cycle is divided into G1, G2, S, and M stages. In order to further investigate that the inhibition of cancer
cell growth by the GA derivatives was caused by cell cycle arrest, untreated cells were used as a negative control and the A549 cells
treated with compound 12e (5, 10 and 20 μM) for 24 h, respectively, and the cell cycle distribution was detected by flow cytometry
analysis after staining of DNA with propidium iodide (PI). As shown in Fig. 5, G1 period cells gradually decreased, but in contrast, S
phase cells gradually increased compared with the control cells, S population of 38.76% was observed in control cells, while 40.09% (5
μM), 43.60% (10 μM) and 54.32% (20 μM) were also observed in compound 12e group compared with the control cells, respectively.
In short, these results indicated that compound 12e arrested the cell cycle of A549 cells at the S stage.
Fig. 5. Cell cycle arrest effect of compound 12e. A549 cells treated with 12e at the tested concentrations (5, 10 and 20 μM) for 24 h. The cells were trypsinized,
harvested and washed three times with ice-PBS for PI-stained DNA content detected by flow cytometry. The experiments were performed three times, and the
results of the representative experiments are shown.
The ability of compound 12e to induce apoptosis was further investigated by analyzing the nuclear morphology of the exposed A549
cells using Hoechst 33258 staining. Nuclei of cells were treated with compound 12e for 24 h and then stained by membrane permeable
blue Hoechst 33258 to evaluate apoptosis morphologically. As shown in Fig. 6, in the control cells, nuclei of the A549 cancer cells
appeared as regular round contours, and the control group A549 cells with smaller nuclei and also condensed chromatin were rarely
observed, but in contrast, after cells treated with 5, 10 and 20 μM of compound 12e, nuclei morphology of the cells slightly changed,
and much more nuclei of the A549 cells appeared hyper condensed (brightly stained). In facts, the observation results further indicated
that
compound
12e
remarkably
induced
apoptosis
in
A549
cells,
respectively.
Fig. 6. Morphological changes in the nuclei (typical of apoptosis) of A549 cells treated with compound 12e at the tested concentrations (5, 10 and 20 μM) for 24
h, respectively. Then, the cells were harvested, and stained by Hoechst 33258. The experiments were performed three times, and the results of the representative
experiments are shown.
Increasing evidence has indicated that the metastatic cancer cells exhibited great capability of migration and invasion.40, 41 Migration
plays an important role in later period of cancer progression. Thus, transwell migration assays were conducted to evaluate if GA
derivative 12e could prevent the migration of human lung cancer cells. As shown in Fig. 7, the presence of 12e obviously attenuated the
migration of A549 cells in a concentration-dependent manner. In short, these results indicated that 12e markedly inhibited migration of
human lung cells in a concentration-dependent manner.

Fig. 7. Migration inhibition of A549 cells after incubation with the tested compound 12e at the tested concentrations (5, 10 and 20 μM) for 24 h, respectively by
transwell assay. The experiments were performed three times, and the results of the representative experiments are shown.
Changes in mitochondrial membrane potential (MMP) have been shown to play an important role in both extrinsic and intrinsic
apoptosis.6, 30 In order to further investigate whether apoptosis induced by compound 12e was related to mitochondrial dysfunction, we
therefore decided to examine the influence of 12e on the MMP of A549 cells using the fluorescent probe JC-1 in this study. As shown in
Fig. 8, compared with control group cells, treatment with A549 cells of compound 12e caused obvious loss of MMP as presented by the
increase of J-monomers (exhibiting green fluorescence and showing depolarized mitochondria) and concurrent decrease of Jaggregates
(exhibiting red fluorescence and showing hyperpolarized mitochondria).
Fig. 8. Compound 12e decreased the MMP of A549 cells. The A549 cells were treated with 12e at the tested concentrations (5, 10 and 20 μM) for 24 h followed
by incubation with the fluorescence probe JC-1 for 30 min. Then, the cells were visualized by fluorescence microscope. The experiments were performed three
times, and the results of the representative experiments are shown.
Many studies indicated that mitochondria are the main organelles that produce reactive oxygen species (ROS), and ROS has been
found to have a double sword role in the cytotoxicity in many solid tumors.6, 30 Thus, intracellular ROS production by compound 12e
was further measured by the fluorescent probe DCF-DA (2’, 7’-Dichlorofluorescein Diacetate). No significant green fluorescence was
observed in the control group cells, but in contrast, 12e significantly induced ROS production at the indicated concentrations as
reflected by the increase green fluorescence in cytoplasm (Fig. 9).
Fig. 9. Intracellular production of ROS by compound 12e following a 24 h incubation visualized by fluorescence microscope. The experiments were performed
three times, and the results of the representative experiments are shown.
In summary, a novel series of GA derivatives containing aminophosphonate ester species were rationally designed and synthesized
and exhibited potent in vitro antitumor activities. Many compounds showed better antitumor activities than that of parent compound GA,
and some target compounds displayed better inhibitory activity than the commercial anticancer drug HCPT. Interestingly, the selected
target compounds significantly reversed multidrug resistance in cisplatin-resistant cancer cells. It is noted that compound 12e (IC50
=
6.25 ± 0.95 μM) displayed the best antitumor activity against the A549 cells and showed more potent inhibitory activity than that of
HCPT (9.13 ± 1.09 μM), and indicated low cytotoxicity toward to human normal liver cells HL-7702, respectively. In addition, a
docking study of the most active compound 12e revealed key interactions between 12e and the active site of NF-κB in which the
aminophosphonate ester moiety at the C-28 position and ester group at the C-3 position was important for increasing the antitumor
activity. Further biological studies indicated that compound 12e significantly caused cell cycle arrest at S phase and induced apoptosis
in A549 cells by blockage of NF-κB signaling pathway. Importantly, compound 12e caused obvious loss of MMP and effectively
induced ROS production, and exhibited inhibition of cell migration against A549 cells in vitro. Consequently, GA derivatives containing
an aminophosphonate ester moiety at the C-28 position were able to effectively improve the anticancer activity and potentially provide
anticancer agents with bone-targeting ability. In short, this rational design offers a significant potential for the discovery of a new class
of NF-κB inhibitors with the ability to suppress migration and induce apoptosis in the A549 cells.
Acknowledgments

This research was financially supported by National Key Basic Research Program of China (grant No. 2013CB933904), by Natural
Science Foundation of China (Grant Nos. 81671745, 21431001 and 81760626), by Natural Science Foundation of Guangxi Province
(AB17292075), by Industry Project of Jiangsu Science-technology Support Plan (grant No. BE2013840), by Science and Technology
Development Program of Suzhou (grant No. ZXY201412).
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Graphical abstract
Glycyrrhetinic acid derivatives containing aminophosphonate ester species as multidrug resistance
reversers that block the NF-κB pathway and cell proliferation

a,1
b,1
b
a,

b
b
Le Jin , Bin Zhang , Shixian Hua , Min Ji , Xiaochao Huang , Rizhen Huang , and Hengshan
Wangc,

Highlights
 Representative compound 12e showed
κ .

κ
 12e
.
 12e effectively induced apoptosis and arrested cell cycle at S stage in A549 cells.