Novel hybrids of podophyllotoxin and formononetin inhibit the growth, migration and invasion of lung cancer cells

Article abstract of DOI:10.1016/j.bioorg.2019.02.019

In this study, three hybrids of podophyllotoxin and formononetin were synthesized and evaluated for anticancer efficacy. Some of the derivatives exhibited potent cytotoxicity against a panel of human and mouse cancer cell lines, with IC₅₀ value

Full text of DOI:10.1016/j.bioorg.2019.02.019

Accepted Manuscript
Novel hybrids of podophyllotoxin and formononetin inhibit the growth, migra-
tion and invasion of lung cancer cells
Chengli Yang, Qiongli Xie, Xian Zeng, Nengyin Tao, Yingshu Xu, Yongzheng
Chen, Jing Wang, Lei Zhang
PII:
S0045-2068(18)31107-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.019
YBIOO 2794
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 September 2018
22 January 2019
6 February 2019
Please cite this article as: C. Yang, Q. Xie, X. Zeng, N. Tao, Y. Xu, Y. Chen, J. Wang, L. Zhang, Novel hybrids of
podophyllotoxin and formononetin inhibit the growth, migration and invasion of lung cancer cells, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.019
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Contents lists available at ScienceDirect
Bioorganic Chemistry
Novel hybrids of podophyllotoxin and formononetin inhibit the growth,
migration and invasion of lung cancer cells
Chengli Yang, Qiongli Xie, Xian Zeng, Nengyin Tao, Yingshu Xu, Yongzheng Chen, Jing Wang*,
Lei Zhang
Generic Drug Research Center of Guizhou Province, Green Pharmaceuticals Engineering Research Center of Guizhou Province, School of Pharmacy, Zunyi
Medical University, Zunyi 563003, PR China
ARTICLE INFO
ABSTRACT
In this study, three hybrids of podophyllotoxin and formononetin were synthesized and evaluated for anticancer efficacy.
Some of the derivatives exhibited potent cytotoxicity against a panel of human and mouse cancer cell lines, with IC50
values in the low micromolar to submicromolar range. Evaluation against A549 lung tumor cell line identified that the
IC50 value of compound 10a was 0.753 μM, indicating that 10a was 2.568-fold more efficacious than parent
podophyllotoxin. Mechanistic studies revealed that 10a induced A549 cell apoptosis mainly via caspase pathway, as well
as disrupted the microtubule organization by occupying the colchicine binding site of the tubulin. Moreover, wound
healing assay and transwell invasion assay indicated that 10a displayed potent inhibitory effects on invasion and
migration in A549 cancer cells. In additiona, a decrease in vimentin immunostaining was also observed in A549 cells
after treatment with 10a. Overall, hybrid 10a might be a promising candidate for the potential treatment of human lung
carcinoma.
Keywords:
Podophyllotoxin
Formononetin
Hybridization
Antiproliferative activity
Migration
Invasion
———
E-mail addresses: wangjing@zmc.edu.cn (J. Wang); lzhang@zmc.edu.cn (L. Zhang).
1

1. Introduction
Cancer has been regarded as a leading cause of death in China, according to the National Central Cancer Registry of China [1].
Although considerable progresses in cancer therapy have been reported, the development of novel anticancer drugs is still a challenge in
medicinal chemistry.
Natural products are considered as “privileged structures” due to their diverse curative potential [2]. Among them, cyclolignans
represent a unique chemical class, such as podophyllotoxin (1, Fig. 1). Podophyllotoxin, derived from Podophyllum species, has
attracted much attention for its various biological activities, such as antirheumatic, antiviral and pesticidal properties [3]. More
importantly, podophyllotoxin shows interesting antiproliferative effect against many different types of cancers, such as breast cancer,
prostate cancer, colon cancer and osteosarcoma, by the inhibition of tubulin polymerization [4]. However, serious side effects like
nausea, diarrhea, vomiting have hindered its use as an efficient anticancer agent. To overcome these drawbacks, various structure
modification of podophyllotoxin have been performed worldwide [5]. In particularly, the two semisynthetic glucosidic derivatives,
etoposide (2, Fig. 1) and teniposide (3, Fig. 1), are widely used as anticancer drugs in the chemotherapy of lung cancer, lymphomas and
Kaposi's sarcoma [6]. Intriguingly, these two drugs are topoisomerase II inhibitors [7]. However, the two clinic drugs have been
reported to own some issues, such as poor bioavailability and drug resistance. Due to the toxic effects of podophyllotoxin and its
derivatives, there is an urgent need for the development of more potent and/or less toxic analogs of this natural product. Structure-
activity relationship and molecular docking experiments have unambiguously indicated that C4 of podophyllotoxin is the major site
tolerant to structural diversification [8,9]. At present, extensive efforts have been made [10]. For example, a series of N-
(aminosulfonyl)-4-podophyllotoxin carbamates were synthesized by Xu and coworkers. Among them, N-(morpholinosulfonyl)-4-
podophyllotoxin carbamate 4 (Fig. 1) exhibited potent anticancer activity against HeLa cells (IC50 = 0.5 μM). In addition, compound 4
could induce cell cycle arrest in the G2/M phase, and inhibit tubulin polymerization and microtubule assembly in HeLa cells [11].
Piperazine acetate podophyllotoxin ester derivative 5 (Fig. 1) was proved to manifest selectively prominent cytotoxicity towards MCF-7
cells in MTT assay, with an IC50 value of 2.78 μM, and displayed low cytotoxicity against human normal L02 cells. Moreover,
compound 5 could block cell cycle at G2 phase, and inhibit the microtubule network in MCF-7 cells as well [12]. More recently, our
group reported that the podophyllotoxin-pterostilbene fused conjugate 6 (Fig. 1) exhibited a significant IC50 value of 0.081 μM against
MUM-2B melanoma cells. Importantly, compound 6 showed antimigratory activity via up-regulating the level of E-cadherin, and
down-regulating the expression of VEGFR-2 and MMP-2 in MUM-2B cells [13].
Fig. 1. The structures of podophyllotoxin and its analogues.
Formononetin (7, Fig. 2) is an isoflavone found in Leguminosae family, such as Trifolium pretense and Glycyrrhiza. It has
previously been reported to have many potent biological activities, including antidiabetic, antioxidant, antiangiogenic,
antimycobacterial, neuroprotective, anti-inflammatory effects, and so on [14-17]. Recently, formononetin has attracted broad attention
for its potential anticancer activity against many cancer cell lines, such as A549, HCT116, A2780 and MDA-MB-231 and U266 cells
[18-22], and many formononetin derivatives were prepared as antiproliferative agents and explored their pharmacological mechanism
[23,24]. For example, Ren et. al reported that formononetin nitrogen mustard derivatives 8 (Fig. 2) showed potent antitumor activity
against HCT-116 cells with an IC50 value of 3.8 μM, and induced cell cycle arrest at G2/M phase and cell apoptosis [25]. In addition,
formononetin-dithiocarbamate hybrid 9 (Fig. 2) was proved to arrest cell cycle, and inhibit cell growth and migration via MAPK/Wnt
signaling pathways in PC-3 cells, with an IC50 value of 1.97 μM [26].
2

Fig. 2. The structures of formononetin and its analogues.
Molecular hybridization is an attractive structural modification strategy to design of new molecules based on the combination of two
or more bioactive compounds or pharmacophoric units [27]. Additionally, a number of lines of evidence have demonstrated that C4 of
podophyllotoxin was tolerant of structural diversification, and the C4 substituent was reported to insert into a hydrophobic cavity of a-
tubulin or the narrow gap in αβ-interface, which might provid more enhanced anticancer activity [28-31]. These above interesting
findings and our ongoing efforts to develop potent anticancer candidates, led to the molecular hybridization of podophyllotoxin and
formononetin to integrate them in one molecule to create new hybrid (10, Fig.3) with excellent antiproliferative activity. To verify the
hypothesis, herein, we reported the synthesis of novel hybrids of podophyllotoxin and formononetin using a molecular hybridization
approach, as well as the evaluation of their antitumor efficacy in vitro. In addition, the underlying mechanism of the representative
hybrid was also evaluated in the current study.
Fig. 3. Targeted hybrids of podophyllotoxin and formononetin.
2. Results and discussion
2.1. Chemistry
The general synthetic route for the hybrids of podophyllotoxin and formononetin was illustrated in Scheme 1. Initially,
podophyllotoxin was reacted with chloro acyl chloride in triethylamine and dichloromethane by esterification. The hybrids 10a-10c
were obtained via nucleophilic substitution reaction of above intermediate and formononetin in the presence of potassium iodide and
potassium carbonate. All compounds were fully characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry.
Scheme 1. Synthesis of hybrids of podophyllotoxin and formononetin.
2.2. Biology
2.2.1. MTT assay
To evaluate the antiproliferative effects of the hybrids of podophyllotoxin and formononetin, compounds 10a-10c were screened for
cytotoxic activity against a panel of seven different cancer cell lines [SKOV3 (human ovarian carcinoma), MCF-7 (human breast
carcinoma), HepG2 (human hepatocellular carcinoma), HeLa (human cervical carcinoma), A549 (human lung carcinoma), CT26
(mouse colon carcinoma), B16f10 (mouse melanoma carcinoma)] and a human endothelial cell line [HUVEC (human umbilical vein
endothelial cell)]. Podophyllotoxin (1) and formononetin (7) were used as positive compounds. As shown in Table 1, three hybrids not
only displayed significantly antiproliferative activity against human cancer cell lines with IC50 values varying from low micromolar to
3

submicromolar range, but also exhibited growth inhibitory effects on mouse cancer cell lines. Among them, hybrids displayed more
potent activity against SKOV3 and HepG2 cells. Moreover, the results showed that compound 10a, possessing two carbon chain,
exhibited more potent activity than the other two compounds with longer chains, 10b and 10c, against all cancer cell lines. For example,
the IC50 values of 10a against SKOV3 and HepG2 cells were 0.213 and 0.277 μM, respectively. However, no significant
antiproliferative activity was observed in cells exposed to positive compound formononetin. And as expected, natural product
podophyllotoxin exhibited excellent antiproliferative activity. Interestingly, among the test cancer lines, only one, A549 cell line, was
observed more sensitive to hybrid (10a) than parent podophyllotoxin, meaning the IC50 value of 10a was 2.568-fold more potent than
podophyllotoxin. Additionally, angiogenesis is an essential process during tumorigenesis, and podophyllotoxin was reported to inhibit
this process. For example, Guo et al. showed that podophyllotoxin had an obvious effect on antiangiogenisis of chick chorioallantoic
membrane [32]. As shown in Table 1, compound 10a was more active towards HUVEC cells among the hybrids of podophyllotoxin
and formononetin, indicating that 10a might reveal antiangiogenic activity and have potential ability to inhibit tumor growth, but the
effect was slightly weaker than parent 1.
2.2.2. MTT using Necrostatin-1 & Z-VAD-FMK
To investigate the mechanism of how hybrid 10a killed lung carcinoma, we further added necroptosis inhibitor or apoptosis inhibitor
prior to drug treatment. A549 cell viability was measured for 48 h by MTT assay. The cytotoxicity was descripted as cell viability
inhibition rate (%). Firstly, after treatment with 10a at the concentrations of 0.135, 1.215 and 3.645 μM, the cell viability inhibition rate
(%) was increased to 40.160, 44.257 and 55.622 (Fig. 4), respectively, indicating that 10a inhibited A549 cell viability in a dose-
dependent manner. However, above inhibition was attenuated by pretreatment with 10 μM Necrostatin-1 (a potent necroptosis inhibitor)
or 20 μM Z-VAD-FMK (a general caspase inhibitor). Similar tendency was observed in 1-treated group. The results indicated that
hybrid 10a might induce cell death in A549 cells mainly via caspase pathway.
2.2.3. Immunofluorescence of Caspase-8
Caspases play a crucial role in the apoptotic process [33]. And caspase-8 acts as the most upstream caspase in apoptotic signaling.
To investigate this further, we used an immunofluorescence approach to determine the distribution of caspase-8 in 10a-treated A549
cells. As shown in Fig. 5, in untreated A549 cells, caspase-8 was preferentially detected as green fluorescence in the perinuclear
cytoplasm. After the treatment of A549 ꢀcells with 10a at the concentrations of 0.5, 1 and 2 μM for 24ꢀh, the decreased green
fluorescence of caspase-8 was observed in a concentration-dependent manner, which suggested that the 10a could suppress the
expression of caspase-8 in A549 cells. Similar phenomenon was observed in 1-treated group.
2.2.4. Acridine orange/ethidium bromide (AO/EB) dual staining
To evaluate the effect of 10a on apoptosis in A549 cells, acridine orange (AO) and ethidium bromide (EB) were used as fluorescent
probes. The morphological analysis was depicted in Fig. 6. In the control group, A549ꢀcells were stained with uniform green
fluorescence and cell nucleus presented intact. After treatment with 10a for a period of 24 h, it was found that the cells which were dyed
to light orange fluorescence exhibited the representative apoptotic features, such as cell shrinkage and chromatin condensation. Similar
result was also observed in the cells incubated with compound 1. The results indicated that hybrid 10a could induce apoptosis in A549
lung cancer cells.
2.2.5. Immunofluorescence of microtubules
To investigate whether the hybrid 10a was a tubulin-targeting agent, we further examined the effect of 10a on the cellular
microtubule network by using immunofluorescence staining. As shown in Fig. 7, the untreated A549 cells exhibited a normal
arrangement and organization of microtubule. By contrast, after 24 ꢀh of exposure to 10a, the microtubule polymerization and spindle
formation became disorganized. Similar phenomenon was also observed in 1-treated group. These data revealed that compound 10a
induced depolymerization of microtubule network in A549 cells.
2.2.6. Molecular modeling
To further elucidate the binding mechanism of the hybrid 10a, molecular modeling of 10a was performed into the tubulin (PDB code:
1SA0) by using the GLIDE module in Schrödinger suite. As shown in Fig. 8, the trimethoxyphenyl ring in 10a occupied the colchicine
binding site of β-subunit as expected. The formononetin ring was located close to the αβ-interface, and formed a hydrogen bond with
LEU-248 residue (d = 3.1 Å) from the β-subunit. The docking results showed that hybrid 10a disrupted the microtubule network by
occupying the colchicine binding site of the tubulin.
2.2.7. Wound healing assay
The progression and metastasis of cancer are dependent on invasion and migration of malignant cells [34]. In the present study,
wound healing assay was performed to investigate the effect of hybrid 10a on the migration of A549 cells. A549 is a type of human
non-small cell lung cancer (NSCLC) that is prone to migration and invasion [35]. After the compounds treatment of 0ꢀ h and 24ꢀ h, we
captured the images of the injured area. The inhibition of the cell migration rate was quantified and compared with that of untreated
cells. As shown in Fig. 9, the wound area was almost completely healed in DMSO-treated control after 24 ꢀh. In contrast, compound
10a inhibited the A549 migration in a dose-dependent manner. What's more, similar inhibition of cell migration was observed in 1-
treated group. These results demonstrated that 10a had the ability to inhibit the migration capability of A549 ꢀcells.
2.2.8. Transwell invasion assay
In order to further determine the anti-invasion effect of compound 10a, in vitro transwell assay was performed on A549 cells. The
inhibition of the cell invasion rate was quantified and compared with that of untreated cells. The results in Fig. 10 revealed that
compound 10a markedly decreased A549 tumor cell invasion at various concentrations (0.05 and 0.5 μM). In addition, the inhibitory
4

effect on invasion of 10a at 0.5 μM was similar to that of compound 1 at 0.5 μM. The results indicated that 10a significantly inhibited
A549 cell invasion in a concentration-dependent manner.
2.2.9. Immunofluorescence of Vimentin
Vimentin, an intermediate filament protein, is associated with increased metastasis and poor prognosis for non-small cell lung cancer
patients [36]. Mounting evidence implicates vimentin as a key regulator of lung adenocarcinoma invasion and metastasis [37,38]. Thus,
we evaluated the effect of compound 10a on the vimentin filament distribution by mean of immunofluorescence staining assays in
A549 lung cancer cells. As shown in Fig. 11, A549 cells in control group exhibited a normal organization of vimentin filaments.
However, treatment with compound 10a induced a morphological rearrangement of vimentin filaments dose-dependently. Similar
phenomenon was found in 1-treated group. These results indicated that compound 10a might inhibit invasion and migration of A549
cells via degradation of vimentin filaments.
3. Conclusion
Podophyllotoxin, a tubulin polymerization inhibitor, was reported to bind at the colchicine site of tubulin. Meanwhile, high resolution
crystal structure showed that 3,4,5-trimethoxybenzoyl at ring-E of podophyllotoxin was located deeply inside the colchicine binding site
[39], and substituent at the C4 hydroxyl could insert into a hydrophobic cavity of a-tubulin or the narrow gap in αβ-interface, probably
resulting in an enhanced anticancer activity [12]. In present study, three hybrids of podophyllotoxin bearing natural product
formononetin fragment at C4 hydroxyl position were synthesized. Among them, molecule 10a with shortest linker showed the best
antineoplastic activity, and its A549 tumor growth inhibition activity was stronger than that of podophyllotoxin (Table 1). Moreover,
the formononetin fragment in 10a could insert into the αβ-interface, and formed a hydrogen bond (Fig. 8), leading to the destruction of
tubulin cytoskeleton (Fig. 7).
Migration and invasion have been reported to play important roles in the progression and metastasis of neoplasm [40]. The results of
wound healing assay, transwell invasion assay and immunofluorescence of vimentin demonstrated that compound 10a could
significantly inhibit the migration (Fig. 9) and invasion (Fig. 10) of A549 lung cancer cells.
Our results demonstrated that hybrid 10a might be a promising anticancer candidate. In the future, it will be interesting to evaluate its
metabolic stability and in vivo efficiency.
Table 1
In vitro antiproliferative activities of compounds.
IC50 (μM)a
Compound
SKOV3
MCF-7
HepG2
HeLa
A549
CT26
B16f10
HUVEC
10a
10b
10c
1
0.213±0.112
2.128±0.841
2.182±1.626
0.096±0.075
>50
2.384±0.408
20.775±7.688
74.556±11.198
1.059±0.549
>50
0.277±0.031
2.447±0.866
1.524±0.290
0.153±0.025
>50
0.763±0.244
3.559±0.338
2.752±1.039
0.375±0.155
>50
0.753±0.173
7.656±1.907
4.116±1.445
1.934±0.089
>50
0.679±0.109
3.240±0.338
5.739±1.070
0.411±0.290
>50
1.066±0.366
1.715±0.763
2.431±0.546
0.611±0.118
>50
0.568±0.130
20.409±6.234
28.356±9.347
0.236±0.098
46.389±14.346
7
a Cell lines were incubated with compounds for 72 h. Cell viability was measured by MTT assay as described in the Experimental Section. IC50 values were indicated as mean IC50 ± SD (μM).
Fig. 4. Cytotoxicity effects. A549 cells were treated with 10a and 1 for 48 h in the absence or presence of Necrostatin-1 (10 μM) or Z-VAD-FMK (20 μM). The
control cells were treated with DMSO. The cell viability was measured by MTT assay. Necrostatin-1 or Z-VAD-FMK was pretreated for 1 h. The cytotoxicity
*
was descripted as: cell viability inhibition rate (%) = (MTT value of control group - MTT value of treated group)/MTT value of control group. represents
pꢀ<ꢀ0.05, ** represents pꢀ<ꢀ0.01 and *** represents pꢀ<ꢀ0.001.
5

Fig. 5. The distribution of caspase-8 in A549 cells. A549 cells were incubated with vehicle, 10a and 1 for 24 h, respectively. Nucleuses were stained with DAPI
(blue), and caspase-8 were stained with anti-caspase-8 antibody (green). Magnification 20×.
Fig. 6. A549 cells were stained by AO/EB and detected under fluorescence microscopy. A549 cells were incubated with vehicle, 10a and 1 for 24 h, respectively.
The cells were stained with AO/EB. Magnification 20×.
6

Fig. 7. Effects of 10a on the microtubule network in A549 cells. A549 cells were incubated with vehicle, 10a and 1 for 24 h, respectively. Microtubules were
visualized with anti-α-tubulin antibody (green), and cell nuclei were stained with DAPI (blue). Magnification 20×.
Fig. 8. The binding mode of compound 10a (green color stick) in the colchicine site of tubulin. The docking pose was illustrated by two different visualizations:
A (ribbon) and B (protein surface) representations. Yellow dashed line represented hydrogen bond.
7

Fig. 9. Wound healing migration assay of A549 cells. (A) The images were captured before (0ꢀ h), and after 24 ꢀh of treatment with vehicle, 10a and 1.
Magnification 20×. (B) Relative inhibition of cell migration in (A). The percentage of inhibition was calculated relative to untreated cells (control).
Fig. 10. The effects of 10a on the invasion of A549 cells were assessed by transwell invasion assay. (A) Images of transwell invasion assay. A549 cells were
treated with vehicle, 10a and 1 at various concentrations. Magnification 20×. (B) Relative inhibition of cell invasion in (A). The percentage of inhibition was
calculated relative to untreated cells (control).
8

Fig. 11. Effects of 10a on vimentin in A549 cells. A549 cells were incubated with vehicle, 10a and 1 for 24 h, respectively. Vimentin and nuclei were stained
with anti-vimentin antibody (green) and DAPI (blue), respectively. Magnification 20×.
4. Experimental
4.2.1.
4α-(4-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl-
acetyl)-4-desoxypodophyllotoxin (10a)
4.1. General
1
White solid, yield 85%; mp: 133-35°C; H NMR (400 MHz,
CDCl3) δ 8.25 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 5.2 Hz, 1H), 7.49
(d, J = 8.4 Hz, 2H), 7.01 (d, J = 9.2 Hz, 1H), 6.97 (d, J = 8.4Hz,
2H), 6.85 (s, 1H), 6.56 (s, 1H), 6.52 (s, 1H), 6.35 (s, 2H), 6.01 (d,
J = 8.8 Hz, 1H), 5.94 (s, 2H), 4.86 (d, J = 5.2 Hz, 1H), 4.59 (d, J
= 3.2 Hz, 1H), 4.49-4.35 (m, 2H), 4.20 (t, J = 9.6 Hz, 1H), 3.82 (s,
3H), 3.79 (s, 3H), 3.74 (s, 6H), 2.95-2.86 (m, 2H); 13C NMR (100
MHz, CDCl3) δ 176.78, 175.66, 173.27, 168.56, 161.49, 159.62,
157.57, 153.17, 152.64, 152.19, 148.37, 147.61, 137.15, 134.51,
132.52, 130.06, 128.45, 127.06, 125.05, 123.85, 119.44, 114.11,
113.98, 109.79, 107.99, 106.69, 105.37, 101.72, 101.28, 75.20,
71.03, 65.26, 60.76, 56.31, 56.14, 55.33, 45.50, 43.59, 38.47;
HRMS-ESI (m/z): calcd for C40H35O13 [M + H]+ 723.2072, found
723.2063.
Melting points were determined on a SGWX-4 microscope
1
meltingpoint apparatus and given uncorrected. H and 13C NMR
analyses were performed using 400ꢀMHz Agilent spectrometer.
High resolution mass spectrometric data were determined on
Agilent Accurate-Mass-Q-TOF-MS 6520. All analytical grade
solvents and reagents were purchased from commercial sources
and used without further purification.
4.2. General preparation of compounds 10a-10c
A mixture of podophyllotoxin (0.1 mmol) and triethylamine
(0.5 mmol) in dichloromethane (5 mL) was stirred at 0 °C.
Chloro acyl chloride (0.25 mmol) dissolved in dichloromethane
(5 mL) was added slowly under stirring. The mixture was stirred
at room temperature for 1 h, quenched with saturated NH4Cl
solution, and then extracted with dichloromethane for three times.
And the organic layer was washed with brine, dried by Na2SO4,
filtered, and removed by evaporation in vacuo. The crude product
was used in the next reaction without further. A mixture of above
residue product, formononetin (0.105 mmol), potassium
carbonate (0.1 mmol) and catalytic amount KI was stirred in
anhydrous DMF (3 mL) for 2 h at 100 °C under argon. Then, the
mixture was quenched with brine and extracted with
dichloromethane. The organic layer was washed with water and
brine, dried with Na2SO4, filtered and removed by evaporation in
vacuo. The residue was purified by silica gel chromatography to
give pure compound.
4.2.2.
4α-(4-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl-
butyryl)-4-desoxypodophyllotoxin (10b)
1
White solid, yield 71%; mp: 126-128°C; H NMR (400 MHz,
CDCl3) δ 8.18 (d, J = 8.8 Hz, 1H), 7.90 (s, 1H), 7.49 (d, J = 8.4
Hz, 2H), 6.96 (d, J = 8.4 Hz, 3H), 6.82 (d, J = 2.0 Hz, 1H), 6.73
(s, 1H), 6.37 (s, 2H), 5.96 (d, J = 10.0 Hz, 2H), 5.72 (d, J = 4.8
Hz, 1H), 4.43-4.37 (m, 2H), 4.31-4.25 (m, 1H), 4.02 (t, J = 6.0
Hz, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 3.77 (s, 6H), 3.31-3.28 (m,
1H), 2.95 (s, 1H), 2.33-2.26 (m, 2H), 1.82-1.71 (m, 3H); 13C
NMR (100 MHz, CDCl3) δ 177.22, 172.81, 163.13, 159.52,
157.86, 153.17, 152.03, 148.42, 147.22, 138.80, 131.21, 130.09,
127.74, 126.08, 124.14, 114.72, 113.92, 109.90, 108.37, 105.29,
9

101.44, 100.56, 72.49, 70.76, 67.89, 60.84, 56.14, 55.32, 45.38,
44.16, 39.68, 33.78, 28.35, 21.41; HRMS-ESI (m/z): calcd for
C42H39O13 [M + H]+ 751.2385, found 751.2379.
concentrations of compounds as upper chambers. After 24 h
incubation at 37 °C, non-invasive cells were scraped with a
cotton swab, and the invaded cells were fixed with 4%
paraformaldehyde for 10 min and stained with crystal violet for
15 min. Finally, images were taken with an inverted microscope,
and further measured using Image-Pro Plus 6.0.
4.2.3. 4α-(4-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl-
valeryl)-4-desoxypodophyllotoxin (10c)
1
White solid, yield 64%; mp: 111-113°C; H NMR (400 MHz,
4.3.5 Immunofluorescence assay
CDCl3) δ 8.19 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 4.4 Hz, 1H), 7.49
(d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.0 Hz,
1H), 6.81 (d, J = 2.8 Hz, 1H), 6.73 (d, J = 4.8 Hz, 1H), 6.55 (d, J
= 4.0 Hz, 1H), 6.37 (s, 2H), 5.96-5.90 (m, 2H), 5.74-5.71 (m, 1H),
4.43 (d, J = 9.2 Hz, 1H), 4.39 (s, 1H), 4.31-4.25 (m, 1H), 4.07-
4.00 (m, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 3.77 (s, 6H), 3.30-3.27
(m, 1H), 2.97 (s, 1H), 2.46-2.39 (m, 1H), 2.31-2.24 (m, 1H),
2.11-2.04 (m, 1H), 1.85-1.69 (m, 3H); 13C NMR (100 MHz,
CDCl3) δ 177.18, 172.52, 162.89, 159.54, 153.19, 152.04, 148.44,
147.22, 138.77, 131.22, 130.09, 127.85, 127.74, 126.00, 124.10,
114.72, 114.56, 113.93, 109.88, 108.37, 108.29, 105.46, 105.35,
101.44, 101.29, 100.65, 100.56, 72.62, 70.72, 67.89, 67.10, 60.83,
56.19, 55.32, 45.38, 44.15, 39.68, 33.79, 30.59, 28.35, 24.11,
21.41; HRMS-ESI (m/z): calcd for C43H41O13 [M + H]+ 765.2542,
found 765.2531.
A549 cells were grown in 24-well plates and treated with
different concentrations of compounds. After 24 h treatment,
cells were fixed with 4% paraformaldehyde for 20 min and
further perforated with 0.5% Triton X-100 for 10 min. Then,
cells were blocked by goat serum at room temperature for 30 min
followed by incubation with α-tubulin/Vimentin/Caspase-8
primary antibody overnight at 4 °C. Next, cells were incubated
with the corresponding Alexa Fluor 488 labeled goat anti-rabbit
secondary antibody at room temperature for 1 h. Cell nuclei were
stained by DAPI at room temperature for 15 min. Images were
obtained under a fluorescence microscope.
4.3.6 Molecular modeling
Docking simulation was investigated using Schrödinger
Maestro. The crystal structure of tubulin (PDB code 1SA0) was
arranged using the protein preparation wizard. The synthesized
compound (10a) used in the docking simulation was optimized
using the LigPrep module, and further docked into colchicine
binding site using Glide. The docking results were further
analyzed using PyMOL.
4.3. Pharmacology
4.3.1
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay
Cells were from the lab of Professor Zhiyong Qian (State Key
Laboratory of Biotherapy/Collaborative Innovation Center of
Biotherapy, West China Hospital, Sichuan University, China).
Cells were seeded in 96-well plates and incubated for overnight
at 37°C in 5% CO2 atmosphere. Then various amounts of
compounds were added to the wells and then incubated further
for 72 ꢀh. After treatment, the medium was removed, 20ꢀμL of
5ꢀmgꢀL−1ꢀMTT was added to each well, and then incubated at
37ꢀ°C for another 4ꢀh. The resulting formazan precipitate was
dissolved with 150ꢀμL DMSO before measurement at 570ꢀnm
with an enzyme-linked immunosorbent assay reader. All these
tests were repeated in triplicate.
4.3.7 Statistical analysis
Statistical significance was analyzed by Student's t-test. P <
0.05 was considered statistically significant.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (81860622), Department of Science
and Technology of Guizhou Province ([2017]1219), Joint Fund
of the Department of Science and Technology of Zunyi City and
Zunyi Medical University ([2018]27), and National First-Rate
Construction Discipline of Guizhou Province (Pharmacy)
(YLXKJS-YX-04).
For treatment with inhibitors, A549 cells were pretreated with
inhibitor, Necrostatin-1 (10 μM) or Z-VAD-FMK (20 μM), for 1
h, and incubated with test compounds for 48 h. The cell survival
rate was measured by above MTT assay.
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11

Highlights
·Three novel hybrids of podophyllotoxin and
formononetin were synthesized.
· Compound 10a showed potent anticancer
activity against A549 cells, with an IC50 value
of 0.753 μM.
·Compound 10a induced A549 cell apoptosis
mainly via caspase pathway.
·Compound 10a displayed potent inhibitory
effects on the migration and invasion of A549
cells.
12

Graphical Abstract
13