Design, synthesis and evaluation of structurally diverse chrysin-chromene-spirooxindole hybrids as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2019.115109

A series of structurally diverse chrysin-chromene-spirooxindole hybrids were designed, synthesized via a Knoevenagel/Michael/cyclization of chrysin and isatylidene malononitrile derivatives through utilizing a hybrid pharmacophore approach. The newly synthesized compounds were evaluated for their in vitro anticancer activity, and most of the compounds showed stronger anti-proliferative activity than parent compound chrysin. In particular, compound 3e had the highest cytotoxicity towards A549 cells (IC₅₀ = 3.15 ± 0.51 μM), and had better selectivity in A549 cells and normal MRC-5 cells. Furthermore, compound 3e could significantly inhibit the proliferation and migration of A549 cells in a dose-dependent manner, as well as induce the apoptosis possibly through mitochondria-mediated caspase-3/8/9 activation and multi-target co-regulation of the p53 signaling pathway. Thus, our results provide in vitro evidence that compound 3e may be a potential candidate for the development of new anti-tumour drugs.

Full text of DOI:10.1016/j.bmc.2019.115109

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of structurally diverse chrysin-chromene-
spirooxindole hybrids as anticancer agents
⁎
⁎
Wen-Hui Zhanga, Shuang Chena, Xiong-Li Liua, , Ting-Ting Fengb, Wu-De Yangb, Ying Zhoub,
a Guizhou Medicine Edible Plant Resources Research and Development Center, Guizhou University, Guiyang 550025, China
b College of Pharmaceutical Sciences, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of structurally diverse chrysin-chromene-spirooxindole hybrids were designed, synthesized via a
Knoevenagel/Michael/cyclization of chrysin and isatylidene malononitrile derivatives through utilizing a hybrid
pharmacophore approach. The newly synthesized compounds were evaluated for their in vitro anticancer ac-
tivity, and most of the compounds showed stronger anti-proliferative activity than parent compound chrysin. In
Chrysin
Spirooxindole
Chromene
Hybrids
particular, compound 3e had the highest cytotoxicity towards A549 cells (IC50 = 3.15
0.51 μM), and had
Anticancer agents
better selectivity in A549 cells and normal MRC-5 cells. Furthermore, compound 3e could significantly inhibit
the proliferation and migration of A549 cells in a dose-dependent manner, as well as induce the apoptosis
possibly through mitochondria-mediated caspase-3/8/9 activation and multi-target co-regulation of the p53
signaling pathway. Thus, our results provide in vitro evidence that compound 3e may be a potential candidate for
the development of new anti-tumour drugs.
1. Introduction
bioactivity (Fig. 1).
Chromene, as one of the privileged motif, has widely appeared in
Molecular hybridization is a valuable useful approach that com-
prises the incorporation of two or more pharmacophoric moieties of
different bioactive substances into a single entity.1 This structural de-
rivatization strategy can result in hybrid molecules acting through the
same mechanism of action or different mechanisms of action.1,2 In the
last few years, molecular hybridization strategy has emerged as a prime
strategy for the discovery of innovative anticancer drugs that can po-
tentially overcome some drawbacks of the conventional anticancer
drugs.
natural products and drug molecules.18 Moreover, in recent years
chromene-derivated hybrids have played an ever-increasing role in the
field of medicinal chemistry.19–21 For example, in the case of cancer
therapy, the tumour antagonist HA14–1 is a new functionalized com-
pound that induce apoptosis or programmed cell death in follicular
lymphoma B cells and leukemia HL-60 cells.22 The 2-amino-3-cyano-
4H-chromene MX58151 is a promising class of proapoptotic small-
molecule agent with multiple action modes against the lung cancer cell
line H1299, the breast cancer cell line T47D, and the colorectal cancer
cell line DLD-1 (Fig. 1).23,24
Flavonoids, as the privileged molecular scaffolds in drug discovery,
which are one of the most representative classes of plant secondary
metabolites, have attracted the scientific interest because they display a
remarkable spectrum of biological activities, including important che-
mopreventive and chemotherapeutic effects on cancer.3–8 It is im-
portant to know that flavonoids are generally safe and without adverse
effects and that many flavonoids occur in our diet. As a natural flavone,
chrysin9–14 has also been reported to exhibit anticancer activities.
However, few chrysin derivatives and their biological activities have
been reported by the modification at C-7 of A-ring.15–17 Based on the
above considerations, we proposed that incorporating two or more
pharmacophoric groups into the chrysin structure might be an effective
strategy for discovering novel chrysin-derivated hybrids with potential
Spirooxindoles25–31 are an inportant class of scaffolds found in
many natural products and drug cancidate molecules (such as CFI-
400945, SAR405838, etc.). CFI-400945 is a polo-like kinase 4 inhibitor,
as a potential anticancer agent,30 and SAR405838 is an optimized in-
hibitor of MDM2-p53 interaction that induces complete and durable
tumour regression.31 This is especially true for the last few years which
have seen a phenomenal increase in the number of reports on the
construction of structurally diverse spirooxindoles, which may in part
be due to the increasing recognition of their biological significance.
However, to the best of our knowledge, there have been no reports for
the synthesis of spirooxindole derivatives containing flavonoid moiety
(Fig. 1).
⁎ Corresponding authors.
E-mail addresses: xlliu1@gzu.edu.cn (X.-L. Liu), yzhou@gzu.edu.cn (Y. Zhou).
https://doi.org/10.1016/j.bmc.2019.115109
Received 22 June 2019; Received in revised form 4 September 2019; Accepted 11 September 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Wen-HuiZhang,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.115109

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 1. Representative anticancer agents and our target molecule.
Scheme 1. Our strategy for construction of chrysin-chromene-spirooxindole hybrids.
Over the last decade, numerous studies pointed out the importance
of spirooxindole-based anticancer hybrids as promising chemother-
apeutic agents. Several research groups adopted molecular hybridiza-
tion approach for the design of spirooxindole-derivated hybrids as po-
tent anti-proliferative agents.32–35 Considering the wide occurrence of
chromenes and flavonoids in natural products and drugs, herein, with
the prime aim of developing potent anticancer agents, we hypothesized
that the structurally diverse chrysin-chromene-spirooxindole hybrids
could be constructed via a Knoevenagel/Michael/cyclization of chrysin,
isatins and malononitrile. To the best of our knowledge, if successful,
this is the first example of chrysin-fused spirooxindoles, and is also the
first example of flavonoid-fused spirooxindoles (Scheme 1).
electron-donating groups in the C-ring to give the desired products 3v-z
with very good yields (70–86%).
All the target hybrids 3 were deduced from spectroscopic studies
(1H NMR, 13C NMR and EI-HRMS), and were determined through
single-crystal X-ray analysis of compounds 3c (Fig. 2).36
The plausible mechanism for the formation of compounds (3a-z) is
shown in Scheme 3 which involved three steps. The initial step is as-
sumed to be the Knoevenagel condensation between the isatin 2 and
malononitrile to form isatylidene malononitrile intermediate in situ. In
the next step, the intermediate undergoes Michael-type addition with
chrysin 1 followed by an intramolecular cyclization to give target
chrysin-chromene-spirooxindole hybrids 3.
2. Results and discussion
2.2. In vitro biological studies
2.1. Chemistry
2.2.1. MTT assay
Compounds 3a-z were evaluated for in vitro anticancer activities
towards human lung cancer cell lines (A549 and H1299), human leu-
kemic cell line (K562) and prostate cancer cell line (PC-3) by the MTT
assay with the parent compound chrysin as a positive control. Besides,
commercially available broad-spectrum anticancer drug cisplatin also
was used as the reference drug.37 As shown in Table 1, the newly
synthesized compounds were evaluated for their in vitro anticancer
activity, and some of the compounds showed stronger anti-proliferative
activity against A549, H1299, K562 and PC-3 than parent compound
chrysin, and demonstrated equipotent potency compared with the re-
ference drug of cisplatin.
Preparation of the chrysin-chromene-spirooxindole hybrids (3a-z)
was achieved via a Knoevenagel/Michael/cyclization of chrysins 1,
isatins 2 and malononitrile in methanol in the presence of 1.5 eq of Ca
(OH)2 in 70–87% yields (for details, reaction optimization experiments
see the SI). It is important to mention that the electronic nature of the
substituents on the oxindole core of isatins 2 had some impact on the
yields of the products. As indicated in Scheme 2, isatins 2 containing
electronwithdrawing groups provided higher yields than their electron-
donating counterparts. The reactions were also shown to work well
with a range of chrysins 1 bearing either electronwithdrawing or
2

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Scheme 2. Substrate scope to chrysin-chromene-spirooxindole hybrids. Reagents and conditions: a mixture of isatin 2 (0.8 mmol), malononitrile (1.0 mmol) and Ca
(OH)2 (0.8 mmol, 59.2 mg) in 5.0 mL of methanol was stirred at room temperature for 10 min followed by the addition of chrysin 1 (0.5 mmol), which was stirred
under reflux for 8 h.
3

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 2. X-ray crystallographic structure of 3c.
The MTT assay demonstrated that compounds 3v-x were equipotent
with cisplatin towards K562 (IC50 < 20.00 μM). Compounds 3b, 3d,
3e, 3t and 3u exhibited the highest anti-proliferative activity against
both A549 and K562 (IC50 < 10.00 μM). Due to the low solubility of
some of these compounds in RPMI-1640 medium, a general structure-
activity relationship to anticancer effect could not be summarized from
only 26 componds, the following points were noteworthy: (1) a chro-
mene-spirooxindole moiety located in the chrysin skeleton is beneficial
for the activity, and (2) a halogen atom substituent in the isatin im-
proves the potency, especially for K562.
These results indicate that compound 3e significantly inhibited the
anchorage-independent growth of A549 cells in vitro.
2.2.3. Compound 3e treatment induces apoptosis in A549 cells
To investigate whether compound 3e could induce apoptosis of
A549 cells, we first observed the morphological changes of A549 cells
after compound 3e treatment for 24 h. As shown in Fig. 4A, as the
concentration of compound 3e increased, the cells began to round, and
the adhesion became poor. Second, AO-EB double staining (Fig. 4B)
showed chromatin shrinkage, and apoptotic bodies occurred at espe-
cially high concentrations. Finally, PI single staining (Fig. 4C) was used
to confirm the morphological changes associated with apoptosis in
A549 cells, including cell membrane boundaries becoming compro-
mised and presenting fragments of varying sizes. Thus, compound 3e
treatment altered the morphology of A549 cells.
Gratifyingly, the results in Table 1 demonstrated that 5-brom-sub-
stituted derivative 3e showed the strongest cytotoxicity against A549
cells. Therefore, we tested the cytotoxicity of compound 3e against
normal lung fibroblast MRC-5. As shown in Table 2, it showed only
slight cytotoxicity towards normal lung fibroblast MRC-5. The results
demonstrate that compound 3e has good selectivity between A549 cells
and normal lung fibroblast MRC-5. Thus, compound 3e was taken
forward to further explore its possible cytotoxicity mechanism in A549
cells.
Based on the above results, the most important feature of apoptosis
is DNA damage.41 Therefore, we examined apoptosis in compound 3e-
treated A549 cells via the formation of a DNA ladder. As shown in
Fig. 4D, the DNA ladder became increasingly apparent as the compound
concentration increased. Next, the effect of compound 3e treatment on
apoptosis in A549 cells was confirmed by flow cytometry. The results
are shown in Fig. 4E. The percentage of apoptosis in A549 cells gra-
dually increased, especially at high concentrations. Thus, these results
suggest that compound 3e treatment induces apoptosis of A549 cells in
a dose-dependent manner.
2.2.2. Anti-proliferative effects of compound 3e on colony formation in
A549 cells
Anchorage-independent cell growth, which is a key aspect of the
tumour phenotype, has been connected with tumour cell aggressiveness
in vivo.38 Therefore, clone and soft agar assays were used to confirm the
effect of compound 3e on the colony formation ability of A549
cells.39,40 As shown in Fig. 3, compared with the control, as the con-
centration of compound 3e increased, the size of the colonies formed by
the A549 cells decreased, and the number of colonies became fewer.
2.2.4. Effect of compound 3e on ROS production in A549 cells
Studies early have demonstrated that overproduction of in-
tracellular ROS tended to attack DNA and cause DNA strand break
Scheme 3. A plausible reaction mechanism.
4

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Table 1
treated with 100 μM of compound 3e, the activity of caspase-3 in-
In vitro cytotoxic activity of the twenty compounds 3a-t towards K562, A549,
creased by 5.24
0.36 (P < .01) fold, caspase-8 increased by the
H1299 and PC-3 cells.
Compound
2.98
0.48 (P < .01), and caspase-9 increased by the 1.81
0.41
IC50 (μM)a
(P < .01) compared with the control group. These results demonstrate
that compound 3e could increase the levels of activated caspase in a
dose-dependent manner and induce A549 cell death by mitochondrial
apoptotic pathway. Besides, compound 3e may be considered as pro-
mising apoptotic inducer in A549 cells for future studies.
A549
K562
H1299
PC-3
3a
> 50.00
> 50.00
> 50.00
10.18
> 50.00
> 50.00
> 50.00
21.43
3b
3c
7.06
0.82
5.87
41.40
4.34
4.10
0.94
1.31
0.62
1.24
1.11
0.92
> 50.00
> 50.00
11.18
3d
3e
4.72
3.15
0.35
0.51
0.98
1.45
2.2.6. Expression of apoptosis-related protein treatment in A549 cells after
compound 3e treatment
7.61
0.63
20.28
3f
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
37.62
> 50.00
> 50.00
> 50.00
> 50.00
36.32
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
16.61
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
25.69
Apoptosis is the result of a series of gene activation, expression and
regulation changes. Therefore, we examined the expression levels of the
apoptosis-related proteins Bcl-2, Bax, Cyt c, Akt, 5-Lox, p53, p21 and
MDM2 to explore the mechanism by which compound 3e treatment
induces apoptosis in A549 cells. As shown in Fig. 7, compound 3e
treatment significantly downregulated Bcl-2, Akt, 5-Lox and MDM2 and
upregulated the expression of Bax, Cyt c, p53 and p21 proteins in a
dose- and time-dependent manner. All of these results demonstrate that
compound 3e may induce apoptosis in A549 cells via the p53 signaling
pathway.
3g
3h
3i
3j
1.50
1.22
1.35
3k
3l
36.40
47.84
3m
3n
3o
3p
3q
3r
> 50.00
> 50.00
> 50.00
> 50.00
36.41
> 50.00
> 50.00
8.47
> 50.00
37.47
3s
3t
9.25
2.2.7. Molecular docking of compound 3e with related proteins
3u
3v
3w
3x
9.34
8.70
14.38
24.77
Based on the in vitro inhibition results, compound 3e was selected as
a ligand, and molecular docking studies were further performed to
explore the binding modes of compound 3e with related proteins Akt,
5-Lox and MDM2 using AutoDock Vina. The generated docked com-
plexes were examined on the basis of minimum energy values (kcal/
mol) and bonding interaction pattern such as hydrophobic and hy-
drogen bonds, respectively. Hydrophobic and hydrogen bonds play an
important role in molecular docking because they help to stabilize and
strengthen the docked complexes.
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
17.58
12.15
1.07
0.81
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
15.24
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
> 50.00
19.15
8.45
0.75
16.12
3y
3z
> 50.00
> 50.00
> 50.00
12.17
Chrysinb
DDPc
1.12
0.84
1.04
1.28
a
IC50: The concentration which results in 50% of tumour cell proliferation
inhibition after 48 h of compounds treatment. Data were represented as
means
SD obtained in at least three independent experiments.
The theoretical binding mode between compound 3e and binding
site were shown in Fig. 8A–C, Table 3. Docking of compound 3e in Akt
(Fig. 8A) showed hydrogen bonding interaction benzene ring with
amino acids Arg4 (d = 3.5 Å), Lys179 (d = 3.4 Å), Asp292 (d = 2.5 Å),
and Gly159 (d = 3.0 Å), and showed hydrophobic interaction with
Phe438 and Met281. As shown in Fig. 8B, the bromobenzene ring of
compound 3e exhibited hydrophobic interaction with Pro569, Val604,
and Phe359, and exhibited hydrogen bonding with amino acid Asn180
(d = 3.1 Å). Similarly, compound 3e also exhibited hydrogen bonding
interaction with amino acids His96 (d = 3.2 Å) and Gln18 (d = 3.0 Å)
of MDM2 was depicted in Fig. 8C.
b
Parent compound chrysin used as a positive control.
DDP (cisplatin) used as the reference drug.
c
Table 2
In vitro cytotoxic activity of the compound 3e towards normal cells MRC-5.
Compound
IC50 (μM)a
A549
MRC-5
3e
3.15
0.51
> 100.00
> 100.00
> 100.00
Chrysinb
DDPc
> 50.00
16.58
1.12
2.2.8. Compound 3e inhibits the migration and invasion of A549 cells
The migration of tumour cells plays an important role in the late
stage of cancer.43 In early studies, we found that compound 3e could
induce the apoptosis of A549 cells. Thus, we adopted the wound-
healing assay to evaluate the lateral migration ability of A549 cells after
treatment with compound 3e. As shown in Fig. 9, compound 3e
treatment significantly inhibited wound healing in a dose-dependent
manner. Furthermore, we evaluated the ability of compound 3e to in-
hibit longitudinal migration using transwell and Matrigel transwell
assays (Fig. 10). The results also demonstrated that compound 3e could
inhibit the migration and invasion of A549 cells in a dose-dependent
manner.
a
IC50: The concentration which results in 50% of tumour cell proliferation
inhibition after 48 h of compounds treatment. Data were represented as
means SD obtained in at least three independent experiments.
b
c
Parent compound chrysin used as a positive control.
DDP (cisplatin) used as the reference drug.
leading to cell apoptosis.42 Therefore, we examined the level of in-
tracellular ROS in A549 cells treated with compound 3e at different
concentrations for 24 h by using a fluorescent probe DCFH-DA. As
shown in Fig. 5, there was no bright fluorescence image in the control
group, while brighter fluorescent images were detected as the con-
centration was changed, especially at high concentrations. These results
demonstrate that compound 3e could increase the production of in-
tracellular ROS in a dose-dependent manner.
3. Conclusions
In conclusion, based on molecular hybridization, we designed and
synthesized a promising class of novel structurally diverse chrysin-
chromene-spirooxindole hybrids. The newly synthesized compounds
were evaluated for their in vitro anticancer activity, and some of the
compounds showed stronger anti-proliferative activity against A549,
H1299, K562 and PC-3 than parent compound chrysin. In particular,
compound 3e had the highest cytotoxicity towards A549 cells, and had
2.2.5. Activation of caspase in A549 cells after compound 3e treatment
The caspase family plays an important role in the process of apop-
tosis.36 Therefore, we examined the activities of caspase-9, caspase-8
and caspase-3 via measurement of their active forms, respectively. As
shown in Fig. 6, compound 3e treatment significantly increased the
activity of caspase-9, caspase-8 and caspase-3. When A549 cells were
5

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 3. (A) The plate colony formation of A549 cells were treated with compound 3e at different concentrations for 10 days; (B) The soft agar colony formation of
A549 cells were treated with compound 3e at different concentrations for 20 days; (C) Data were represented as means SD obtained in at least three independent
experiment. ⁎P < .05; ⁎⁎P < .01 compared with the control group.
better selectivity in A549 cells and normal MRC-5 cells.
standard. Chemical shifts were reported as δ values (ppm). High-re-
solution mass spectra (HRMS-ESI) were obtained on a Micro™ Q-TOF
Mass Spectrometer. Melting points were uncorrected and recorded on
an Electrothermal 9100 digital melting point apparatus.
Preliminary mechanism studies demonstrated that compound 3e
could significantly inhibit A549 colony formation and alter cell mor-
phology to induce cell apoptosis, which may be dependent on mi-
tochondria-mediated caspase activation. It appears that compound 3e
treatment increased Bax and decreased Bcl-2 protein levels, thus in-
creasing the ratio of Bax/Bcl-2, releasing cytochrome c and activating
caspase to cause apoptosis. Simultaneously, compound 3e treatment
regulated the expression of MDM2, p21, Akt and 5-lox proteins asso-
ciated with the tumour suppressor p53, thereby inducing cell death.
Our results demonstrate that compound 3e could significantly induce
apoptosis and inhibit the migration and invasion of A549 cells.
Apoptosis may be the result of mitochondria- mediated caspase-3/8/9
activation and multi-target co-regulation of the p53 signaling pathway.
In addition, the molecular docking of active compound 3e was also
investigated. The 3e interact with the receptor through hydrophobic
hydrophobic interaction and hydrogen bonding with essential amino
acid. Thus, our results provide evidence that the most active compound
of this series was 3e, which represents a good lead for drug develop-
ment.
Reagents were purchased from commercial sources and were used
as received unless stated otherwise. Reactions were monitored by thin
layer chromatography using silica gel GF254 plates. Column chroma-
tography was performed on silica gel (300–400 mesh).
4.2. General procedure for synthesis of compounds 3a-z
A mixture of isatin 2 (0.8 mmol), malononitrile (1.0 mmol) and Ca
(OH)2 (0.8 mmol, 59.2 mg) in 5.0 mL of methanol was stirred at room
temperature for 10 min followed by the addition of chrysin
1
(0.5 mmol), which was stirred under reflux for 8 h. After completion of
the reaction, as indicated by TLC, the mixture was cooled to room
temperature and then purified by flash chromatography to afford the
corresponding product 3.
4.2.1. 2′-Amino-5′-hydroxy-1-methyl-2,6′-dioxo-8′-phenyl-6′H-spiro
[indoline-3,4′-pyrano[3,2- g] chromene]-3′‑carbonitrile (3a)
4. Experimental section
4.1. General
The 1H NMR spectra were recorded on Bruker Avance DMX 400 or
500 MHz NMR spectrometers in DMSO‑d6 using TMS as an internal
standard. The 13C NMR spectra were recorded on Bruker Avance DMX
100 MHz NMR spectrometers in DMSO‑d6 using TMS as an internal
6

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 4. Compound 3e induced apoptosis of cells. A549 cells were treated with various concentrations of compound 3e for 24 h. (A) Morphological observation; (B)
AO-EB double staining assay; (C) PI staining assay; (D) The compound 3e-induced apoptosis was examined by observation of the formation of DNA ladder; (E) The
compound 3e-induced apoptosis was evidenced by flow cytometry and Annexin V-FITC/PI double staining.
Yellow solid; m.p. > 300 °C; yield 72%; 1H NMR (DMSO‑d6,
500 MHz) δ: 3.22 (s, 3H), 6.98–7.00 (m, 1H), 7.03–7.04 (m, 2H),
7.06–7.09 (m, 2H), 7.28–7.31 (m, 1H), 7.39 (s, 2H), 7.57–7.60 (m, 2H),
7.62–7.64 (m, 1H), 8.12 (d, J = 7.5 Hz, 2H), 13.32 (br s, 1H); 13C NMR
(DMSO‑d6, 125 MHz) δ: 30.7, 46.9, 55.8, 103.9, 105.23, 106.7, 108.4,
117.5, 122.8, 123.4, 126.7, 128.7, 129.2, 130.3, 132.5, 133.2, 143.6,
153.8, 155.7, 159.5, 176.1, 182.4; HRMS (ESI-TOF) m/z: Calcd. for
4.2.2. 2′-Amino-5′-hydroxy-1,5-dimethyl-2,6′-dioxo-8′-phenyl-6′H-spiro
[indoline-3,4′-pyrano [3,2-g]chromene]-3′-carbonitrile (3b)
C
27H18N3O5 [M + H]+: 464.1241; Found: 464.1242.
7

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 5. Effect of compound 3e on the production of ROS in A549 cells. Cells were treated with various concentrations of compound 3e for 24 h and detected by
examining the fluorescence of DCF in cells by the fluorescence microscope.
7.50–7.60 (m, 3H), 8.06 (d, J = 7.2 Hz, 3H), 8.05 (br s, 2H), 13.38 (br
s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 30.2, 47.4, 55.8, 95.6, 107.1,
114.3, 114.5, 117.1, 117.8, 123.3, 124.6, 127.0, 129.5, 130.6, 131.1,
132.9, 136.7, 139.6, 154.0, 156.2, 158.6, 159.0, 159.9, 164.8, 177.1,
182.7; HRMS (ESI-TOF) m/z: Calcd. for C27H17ClN3O5 [M + H]+
:
498.0851; Found: 498.0855.
4.2.4. 2′-Amino-5-chloro-5′-hydroxy-1-methyl-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3d)
Yellow solid; m.p. > 300 °C; yield 84%; 1H NMR (DMSO‑d6,
400 MHz) δ: 3.21 (s, 3H), 6.76 (br s, 2H), 7.04 (d, J = 14.8 Hz, 2H),
7.10 (d, J = 8.4 Hz, 1H), 7.21 (s, 1H), 7.34–7.36 (m, 1H), 7.47 (s, 1H),
7.53–7.62 (m, 3H), 8.09 (d, J = 7.2 Hz, 1H), 13.35 (br s, 1H); 13C NMR
(DMSO‑d6, 100 MHz) δ: 26.8, 47.4, 55.3, 95.4, 103.3, 105.4, 106.8,
110.0, 114.4, 116.7, 117.6, 123.9, 126.8, 128.8, 129.3, 130.4, 132.7,
135.3, 142.7, 154.0, 155.9, 158.5, 158.8, 159.8, 164.6, 176.1, 182.5;
Fig. 6. A549 cells were treated with various concentrations of compound 3e for
24 h, the effects of compound 3e on activation of caspase-9, caspase-8 and
caspase-3, respectively. Data were represented as means SD obtained in at
least three independent experiment. ⁎P < .05; ⁎⁎P < .01 compared with the
control group.
HRMS (ESI-TOF) m/z: Calcd. for
C
27H16ClN3NaO5 [M + Na]+
:
Yellow solid; m.p. > 300 °C; yield 71%; 1H NMR (DMSO‑d6,
400 MHz) δ: 2.20 (s, 3H), 3.19 (s, 3H), 6.86 (s, 1H), 6.95 (d, J = 7.9 Hz,
1H), 7.04 (s, 1H), 7.10 (d, J = 8.1 Hz, 2H), 7.37 (s, 2H), 7.56–7.65 (m,
3H), 8.12 (d, J = 7.3 Hz, 2H), 13.32 (br s, 1H); 13C NMR (DMSO‑d6,
100 MHz) δ: 20.6, 26.6, 47.1, 56.0, 95.1, 104.0, 105.3, 106.7, 108.2,
117.6, 124.0, 126.8, 129.0, 129.2, 130.3, 131.8, 132.6, 133.3, 141.3,
153.8, 155.6, 158.2, 159.5, 164.4, 176.0, 182.5; HRMS (ESI-TOF) m/z:
Calcd. for C28H20N3O5 [M + Na]+: 478.1397; Found: 478.1390.
520.0671; Found: 520.0677.
4.2.5. 2′-Amino-5-bromo-5′-hydroxy-1-methyl-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3e)
4.2.3. 2′-Amino-7-chloro-5′-hydroxy-1-methyl-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3c)
Yellow solid; m.p. > 300 °C; Yield 87%; 1H NMR (DMSO‑d6,
500 MHz) δ: 3.22 (s, 3H), 7.00 (s, 1H), 7.05–7.08 (m, 2H), 7.33 (s, 1H),
7.49 (d, J = 6.9 Hz, 3H), 7.52–7.55 (m, 2H), 7.58–7.61 (m, 1H), 8.07
(d, J = 7.6 Hz, 2H), 13.34 (br s, 1H); 13C NMR (DMSO‑d6, 125 MHz) δ:
31.2, 47.7, 55.6, 95.7, 103.6, 105.7, 107.2, 111.0, 115.1, 118.0, 126.9,
127.2, 129.7, 130.7, 132.0, 133.1, 136.0, 143.5, 154.3, 156.3, 158.7,
160.2, 164.9, 176.3, 182.9, 207.1; HRMS (ESI-TOF) m/z: Calcd. for
Yellow solid; m.p. > 300 °C; yield 86%; 1H NMR (DMSO‑d6,
400 MHz) δ: 3.54 (s, 3H), 6.97–7.06 (m, 3H), 7.28–7.30 (m, 1H),
C
27H17BrN3O5 [M + H]+: 542.0346; Found: 542.0344.
8

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 7. Expression level of apoptosis-related protein, Bax, Bcl-2, Cyt c, p53, p21, MDM2, Akt and 5-Lox. (A-D) A549 cells were treated with compound 3e at different
concentrations for 24 h; (E-H) A549 cells were treated with 10 μM of compound 3e for different time. Data were represented as means SD obtained in at least three
independent experiments. ⁎P < .05; ⁎⁎P < .01 verse the control group. The grayscale assay was quantified using Image Lab software.
9

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 8. (A–B) The theoretical binding models of compound 3e with Akt, 5-Lox and MDM2. (A) Molecular docked model of compound 3e with Akt (PDB ID: 3MV5);
(B) Molecular docked model of compound 3e with 5-Lox (PDB ID: 3V99); (C) Hydrogen bonding interactions between compound 3e and key amino acid residues of
MDM2 (PDB ID: 4ZYF).
136.3, 140.5, 153.9, 155.7, 158.5, 159.7, 164.5, 176.4, 182.5; HRMS
(ESI-TOF) m/z: Calcd. for C34H24N3O5 [M + H]+: 554.1710; Found:
554.1713.
Table 3
Binding affinities of compound 3e to related proteins by molecular
docking.
Docking model
Docking score (kcal/mol)
Akt
−5.6
−6.4
−5.4
4.2.7. 2′-Amino-1-benzyl-5′-hydroxy-7-methyl-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3g)
5-Lox
MDM2
4.2.6. 2′-Amino-1-benzyl-5′-hydroxy-5-methyl-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3f)
Yellow solid; m.p. > 300 °C; yield 75%; 1H NMR (DMSO‑d6,
500 MHz) δ: 2.23 (s, 3H), 5.14 (d, J = 17.4 Hz, 1H), 5.26 (d,
J = 17.2 Hz, 1H), 6.88–6.93 (m, 2H), 6.98 (d, J = 6.9 Hz, 1H), 7.05 (d,
J = 14.2 Hz, 2H), 7.25–7.28 (m, 1H), 7.32–7.35 (m, 2H), 7.42 (d,
J = 7.5 Hz, 2H), 7.53–7.56 (m, 2H), 7.59–7.61 (m, 1H), 8.09 (d,
J = 7.6 Hz, 2H), 9.73 (br s, 2H), 13.54 (br s, 1H); 13C NMR (DMSO‑d6,
125 MHz) δ: 18.1, 45.3, 46.8, 56.5, 95.2, 104.5, 105.3, 106.8, 114.0,
116.3, 118.1, 119.2, 122.0, 123.1, 126.0, 126.8, 127.1, 128.6, 129.2,
130.3, 132.5, 134.3, 138.4, 141.0, 153.9, 155.8, 158.0, 158.3, 158.5,
158.6, 159.6, 164.6, 177.6, 182.6; HRMS (ESI-TOF) m/z: Calcd. for
Yellow solid; m.p. > 300 °C; yield 78%; 1H NMR (DMSO‑d6,
500 MHz) δ: 2.16 (s, 3H), 4.91 (d, J = 15.8 Hz, 1H), 5.03 (d,
J = 15.9 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.90 (s, 1H), 6.98 (d,
J = 7.9 Hz, 3H), 7.25–7.28 (m, 1H), 7.30–7.33 (m, 2H), 7.44–7.55 (m,
7H), 8.05 (br s, 2H), 13.35 (br s, 1H); 13C NMR (DMSO‑d6, 125 MHz) δ:
20.6, 43.7, 47.3, 56.2, 95.2, 10.3.9, 105.2, 106.7, 108.9, 117.9, 124.3,
126.7, 127.4, 127.6, 128.4, 128.9, 129.2, 130.2, 132.1, 132.6, 133.4,
C
34H24N3O5 [M + H]+: 554.1710; Found: 554.1712.
Fig. 9. With different concentrations of compound 3e inhibits migration abilities of A549 cells by using wound healing assay. Data were represented as means SD
obtained in at least three independent experiments. ⁎P < .05; ⁎⁎P < .01 verse the control group.
10

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 10. With different concentrations of compound 3e inhibits migration and invasion abilities of A549 cells by using transwell and matrigel transwell assay. Data
were represented as means SD obtained in at least three independent experiments. ⁎P < .05; ⁎⁎P < .01 verse the control group.
4.2.8. 2′-Amino-1-benzyl-5′-hydroxy-5-methoxy-2,6′-dioxo-8′-phenyl-
6′H-spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3h)
4.2.10. 2′-Amino-1-benzyl-5-chloro-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3j)
Yellow solid; m.p. > 300 °C; yield 71%; 1H NMR (DMSO‑d6,
400 MHz) δ:; 13C NMR (DMSO‑d6, 100 MHz) δ: 44.1, 48.4, 54.6, 57.9,
106.9, 107.5, 107.7, 108.5, 111.9, 119.4, 126.5, 127.3, 128.7, 129.6,
131.3, 132.1, 136.1, 136.5, 137.4, 154.4, 154.9, 159.1, 160.3, 161.2,
168.9, 178.1, 180.1; HRMS (ESI-TOF) m/z: Calcd. for C34H24N3O6
[M + H]+: 570.1660; Found: 570.1660.
Yellow solid; m.p. > 300 °C; yield 86%; 1H NMR (DMSO‑d6,
500 MHz) δ: 4.93 (d, J = 16.0 Hz, 1H), 5.04 (d, J = 15.9 Hz, 1H),
6.88–6.90 (m, 1H), 7.06 (d, J = 10.5 Hz, 2H), 7.26–7.34 (m, 5H), 7.45
(d, J = 7.1 Hz, 2H), 7.53–7.62 (m, 5H), 8.10–8.11 (m, 2H), 13.41 (br s,
1H); 13C NMR (DMSO‑d6, 125 MHz) δ: 43.7, 47.4, 55.1, 95.3, 103.1,
105.3, 106.7, 110.5, 117.7, 124.1, 126.8, 127.1, 127.5, 128.5, 128.6,
129.2, 130.3, 132.6, 135.3, 135.8, 141.7, 153.9, 155.9, 158.1, 158.3,
159.8, 164.5, 176.2, 182.5; HRMS (ESI-TOF) m/z: Calcd. for
4.2.9. 2′-Amino-1-benzyl-5-fluoro-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3i)
C
33H21ClN3O5 [M + H]+: 574.1164; Found: 574.1169.
4.2.11. 2′-Amino-1-benzyl-5-bromo-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3k)
Yellow solid; m.p. > 300 °C; yield 85%; 1H NMR (DMSO‑d6,
500 MHz) δ: 4.93 (d, J = 15.9 Hz, 1H), 5.04 (d, J = 15.9 Hz, 1H), 6.19
(br s, 2H), 6.85–6.88 (m, 1H), 7.03–7.06 (m, 3H), 7.10–7.12 (m, 1H),
7.27–7.29 (m, 1H), 7.31–7.34 (m, 1H), 7.46–7.62 (m, 6H), 8.10 (d,
J = 7.4 Hz, 2H), 13.41 (br s, 1H); 13C NMR (DMSO‑d6, 125 MHz) δ:
43.7, 47.6, 55.3, 95.2, 103.2, 105.3, 106.7, 109.9, 111.7, 111.9, 114.8,
115.0, 117.7, 126.7, 127.5, 128.4, 129.2, 130.2, 132.6, 135.0, 135.9,
139.0, 153.9, 155.8, 158.0, 158.3, 158.4, 158.7, 159.8, 159.9, 164.5,
Yellow solid; m.p. > 300 °C; yield 84%; 1H NMR (DMSO‑d6,
400 MHz) δ: 4.90 (d, J = 15.9 Hz, 1H), 5.01 (d, J = 15.9 Hz, 1H), 6.82
(d, J = 8.4 Hz, 1H), 7.02 (s, 1H), 7.06 (s, 1H), 7.25–7.43 (m, 7H), 7.49
(s, 2H), 7.52–7.61 (m, 3H), 8.09 (d, J = 7.3 Hz, 2H), 13.39 (br s, 1H);
13C NMR (DMSO‑d6, 100 MHz) δ: 44.1, 47.8, 55.6, 95.8, 103.5, 105.8,
176.4, 182.5; HRMS (ESI-TOF) m/z: Calcd. for C33H21FN3O5 [M + H]+
:
558.1460; Found: 558.1458.
11

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
107.1, 111.5, 115.3, 118.2, 127.2, 128.0, 128.9, 129.7, 130.7, 131.9,
133.1, 136.0, 136.2, 142.6, 154.4, 156.3, 158.8, 160.3, 164.9, 176.6,
Calcd. for C29H22N3O5 [M + H]+: 492.1554; Found: 492.1551.
183.0; HRMS (ESI-TOF) m/z: Calcd. for C33H21BrN3O5 [M + H]+
:
4.2.15. 2′-Amino-1-ethyl-5′-hydroxy-5-methyl-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3o)
618.0659; Found: 618.0653.
4.2.12. 2′-Amino-1-benzyl-7-chloro-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3l)
Yellow solid; m.p. > 300 °C;, yield 75%; 1H NMR (DMSO‑d6,
500 MHz) δ: 1.18–1.21 (m, 3H), 2.19 (s, 3H), 3.76–3.78 (m, 2H), 6.87
(s, 1H), 6.98–7.02 (m, 2H), 7.08 (d, J = 7.5 Hz, 1H), 7.37 (br s, 2H),
7.50–7.59 (m, 3H), 8.05–8.06 (m, 2H), 13.30 (br s, 1H); 13C NMR
(DMSO‑d6, 125 MHz) δ: 12.2, 20.6, 34.5, 47.0, 56.3, 95.0, 104.0, 105.3,
106.7, 108.2, 117.5, 124.2, 126.0, 126.7, 129.2, 130.3, 131.6, 132.5,
133.6, 140.1, 153.9155.6, 158.5, 159.5, 164.3175.5, 182.4; HRMS
Yellow solid; m.p. > 300 °C; yield 85%; 1H NMR (DMSO‑d6,
500 MHz) δ: 5.27 (d, J = 16.8 Hz, 1H), 5.33 (d, J = 16.7 Hz, 1H),
6.99–7.02 (m, 1H), 7.03 (d, J = 1.6 Hz, 2H), 7.11 (d, J = 7.3 Hz, 1H),
7.22–7.24 (m, 2H), 7.28–7.31 (m, 2H), 7.39 (d, J = 7.4 Hz, 2H),
7.52–7.55 (m, 2H), 7.57–7.60 (m, 1H), 8.07 (d, J = 7.3 Hz, 2H), 13.26
(br s, 3H); 13C NMR (DMSO‑d6, 125 MHz) δ: 45.5, 47.4, 55.8, 95.7,
103.5, 107.1, 111.9, 114.2, 116.5, 118.0, 118.8, 123.4, 124.7, 126.6,
127.0, 128.6, 129.4, 130.6, 131.2, 132.8, 136.8, 138.2, 139.1, 154.0,
156.3, 158.2, 158.5, 158.8, 159.1, 160.0, 164.9, 169.4, 177.6, 182.7;
HRMS (ESI-TOF) m/z: Calcd. for C33H21ClN3O5 [M + H]+: 574.1164;
Found: 574.1159.
(ESI-TOF) m/z: Calcd. for
C
29H21N3NaO5 [M + Na]+
: 514.1373;
Found: 514.1379.
4.2.16. tert-Butyl 2-(2′-amino-3′-cyano-5′-hydroxy-2,6′-dioxo-8′-phenyl-
6′H-spiro[indoline- 3,4′-pyrano [3,2-g]chromene]-1-yl)acetate (3p)
4.2.13. 2′-Amino-5′-hydroxy-2,6′-dioxo-1,8′-diphenyl-6′H-spiro[indoline-
3,4′-pyrano[3,2-g] chromene]-3′‑carbonitrile (3 m)
Yellow solid; m.p. > 300 °C; yield 77%; 1H NMR (DMSO‑d6,
500 MHz) δ: 1.40 (s, 9H), 4.41 (d, J = 17.0 Hz, 1H), 4.48 (d,
J = 17.0 Hz, 1H), 6.97–7.01 (m, 4H), 7.06 (d, J = 6.7 Hz, 1H),
7.24–7.28 (m, 1H), 7.49–7.52 (m, 2H), 7.54–7.57 (m, 1H), 8.03 (d,
J = 7.3 Hz, 2H), 11.24 (br s, 2H), 13.23 (br s, 1H); 13C NMR (DMSO‑d6,
125 MHz) δ: 27.9, 43.2, 47.4, 56.2, 82.0, 95.5, 107.0, 109.3, 112.0,
114.3, 116.6, 117.6, 118.9, 123.3, 123.9, 127.0, 128.8, 129.5, 130.7,
132.8, 133.3, 143.1, 156.2, 158.4, 158.7, 159.0, 159.3, 160.1, 166.9,
Yellow solid; m.p. > 300 °C; yield 81%; 1H NMR (DMSO‑d6,
400 MHz) δ: 5.95 (br s, 2H), 6.72 (d, J = 7.9 Hz, 1H), 7.03–7.09 (m,
2H), 7.14 (d, J = 7.2 Hz, 1H), 7.21–7.25 (m, 1H), 7.44–7.64 (m, 9H),
8.11 (d, J = 7.3 Hz, 2H), 13.46 (br s, 1H); 13C NMR (DMSO‑d6,
100 MHz) δ: 47.3, 56.1, 95.3, 103.8, 105.3, 106.9, 108.8, 114.3, 117.2,
117.6, 123.5, 124.1, 126.8, 128.4, 128.8, 129.2, 129.9, 130.1, 132.6,
133.0, 134.6, 143.3, 153.8, 155.8, 158.2, 158.3, 158.6, 159.5, 164.5,
176.4, 182.7; HRMS (ESI-TOF) m/z: Calcd. for
C32H25N3NaO7
[M + Na]+: 586.1585; Found: 586.1579.
175.8, 182.5; HRMS (ESI-TOF) m/z: Calcd. for C32H20N3O5 [M + H]+
:
526.1397; Found: 526.1402.
4.2.17. 2′-Amino-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-spiro[indoline-
3,4′-pyrano[3,2-g] chromene]-3′‑carbonitrile (3q)
4.2.14. 2′-Amino-1-ethyl-5′-hydroxy-7-methyl-2,6′-dioxo-8′-phenyl-6′H-
spiro[indoline-3,4′- pyrano[3,2-g]chromene]-3′‑carbonitrile (3n)
Yellow solid; m.p. > 300 °C; yield 74%; 1H NMR (DMSO‑d6,
500 MHz) δ: 6.88 (d, J = 10.0 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.99
(d, J = 8.5 Hz, 1H), 7.03 (s, 1H), 7.08 (s, 1H), 7.19–7.22 (m, 1H), 7.31
(s, 2H), 7.55–7.64 (m, 3H), 8.12 (d, J = 9.0 Hz, 2H), 10.64 (br s, 1H),
13.4 (br s, 1H); 13C NMR (DMSO‑d6, 125 MHz) δ: 47.9, 56.9, 95.4,
104.5, 105.7, 107.1, 109.8, 122.5, 124.1, 127.2, 129.0, 129.6, 130.8,
134.5, 142.6, 154.4, 156.1, 158.9, 159.9, 164.9, 178.0, 182.9; HRMS
Yellow solid; m.p. > 300 °C; yield 73%; 1H NMR (DMSO‑d6,
400 MHz) δ: 1.20–1.29 (m, 3H), 2.57 (s, 3H), 3.97–4.02 (m, 2H),
6.85–6.91 (m, 2H), 7.01–7.13 (m, 3H), 7.34–7.41 (m, 2H), 7.56–7.60
(m, 2H), 7.62–7.66 (m, 1H), 8.08–8.17 (m, 2H); 13C NMR (DMSO‑d6,
100 MHz) δ: 14.3, 18.4, 36.5, 46.4, 56.5, 95.0, 104.2, 105.2, 106.6,
117.5, 118.7, 121.8, 122.6, 126.7, 129.1, 130.2, 132.5, 134.5, 140.4,
153.8, 155.6, 158.4, 159.4, 164.3, 176.6, 182.4; HRMS (ESI-TOF) m/z:
(ESI-TOF) m/z: Calcd. for
C
26H15N3NaO5 [M + Na]+
: 472.0904;
Found: 472.0891.
12

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
4.2.18. 2′-Amino-5′-hydroxy-5-methyl-2,6′-dioxo-8′-phenyl-6′H-spiro
[indoline-3,4′-pyrano [3,2-g]chromene]-3′‑carbonitrile (3r)
107.1, 112.6, 117.9, 121.5, 125.2, 126.1, 127.2, 129.6, 130.7, 133.0,
133.8, 144.2, 154.3, 156.2, 158.8, 159.9, 164.9, 178.0, 182.9; HRMS
(ESI-TOF) m/z: Calcd. for C26H14BrN3NaO5 [M + Na]+: 550.0009;
Found: 550.0009.
4.2.22. 2′-Amino-8′-(2,4-dimethoxyphenyl)-5′-hydroxy-1,5-dimethyl-2,6′-
dioxo-6′H-spiro [indoline-3,4′-pyrano[3,2-g]chromene]-3′‑carbonitrile (3v)
Yellow solid; m.p. > 300 °C; yield 70%; 1H NMR (DMSO‑d6,
500 MHz) δ: 1.66 (s, 3H), 6.28 (s, 1H), 6.35 (s, 1H), 6.41 (s, 1H),
6.93–6.96 (m, 3H), 7.54–7.56 (m, 4H), 8.01–8.03 (m, 2H); 13C NMR
(DMSO‑d6, 125 MHz) δ: 20.8, 48.9, 57.5, 106.6, 106.8, 109.7, 112.4,
118.9, 123.1, 126.2, 126.8, 129.5, 129.8, 131.7, 135.8, 140.2, 154.2,
159.1, 160.4, 160.5, 169.3, 179.1, 182.1; HRMS (ESI-TOF) m/z: Calcd.
for C27H17N3NaO5 [M + Na]+: 486.1060; Found: 486.1049.
Yellow solid; m.p. > 300 °C; yield 70%; 1H NMR (DMSO‑d6,
500 MHz) δ: 2.20 (s, 3H), 3.20 (s, 3H), 3.83 (d, J = 1.7 Hz, 6H),
6.62–6.67 (m, 2H), 6.84–6.88 (m, 3H), 6.95 (d, J = 8.0 Hz, 1H), 7.09
(d, J = 7.4 Hz, 1H), 7.34 (br s, 2H), 7.89 (d, J = 8.8 Hz, 1H), 13.46 (br
s, 1H); 13C NMR (DMSO‑d6, 125 MHz) δ: 20.6, 26.6, 47.1, 55.7, 56.0,
94.7, 98.7, 103.6, 106.2, 106.4, 107.6, 108.1, 110.9, 117.7, 124.0,
128.9, 130.5, 131.8, 133.4, 141.3, 153.6, 155.4, 158.1, 159.6, 160.0,
162.0, 163.9, 176.1, 182.2; HRMS (ESI-TOF) m/z: Calcd. for
4.2.19. 2′-Amino-7-chloro-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-spiro
[indoline-3,4′-pyrano [3,2-g]chromene]-3′‑carbonitrile (3s)
C
30H24N3O7 [M + Na]+: 538.1609; Found: 538.1608.
4.2.23. 2′-Amino-5-chloro-8′-(2,4-dimethoxyphenyl)-1-ethyl-5′-hydroxy-
2,6′-dioxo-6′H-spiro [indoline-3,4′-pyrano[3,2-g]chromene]-3′‑carbonitrile
(3w)
Yellow solid; m.p. > 300 °C; yield 77%; 1H NMR (DMSO‑d6,
500 MHz) δ: 6.21 (s, 1H), 6.68 (s, 1H), 6.71–6.74 (m, 2H), 6.97–7.00
(m, 3H), 7.58–7.59 (m, 3H), 8.01–8.03 (m, 2H), 10.20 (br s, 1H); 13C
NMR (DMSO‑d6, 125 MHz) δ: 49.3, 57.8, 106.3, 107.3, 111.5, 113.7,
118.7, 121.8, 122.7, 126.4, 129.6, 131.5, 131.9, 138.0, 154.5, 159.1,
160.3, 169.1, 179.6; HRMS (ESI-TOF) m/z: Calcd. for C26H14ClN3NaO5
[M + Na]+: 506.0514; Found: 506.0504.
4.2.20. 2′-Amino-6-chloro-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-spiro
[indoline-3,4′-pyrano [3,2-g]chromene]-3′‑carbonitrile (3 t)
Yellow solid; m.p. > 300 °C; yield 85%; 1H NMR (DMSO‑d6,
500 MHz) δ: 1.17–1.19 (m 3H), 3.74–3.82 (m, 2H), 3.84 (s, 3H), 3.86 (s,
3H), 6.66–6.69 (m, 2H), 6.89 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 8.4 Hz,
1H), 7.20 (d, J = 2.0 Hz, 1H), 7.32–7.34 (m, 1H), 7.41 (br s, 2H), 7.94
(d, J = 8.8 Hz, 1H), 13.50 (br s, 1H); 13C NMR (DMSO‑d6, 125 MHz) δ:
12.2, 34.9, 47.3, 55.4, 55.8, 56.1, 95.1, 99.0, 106.4, 111.1, 111.2,
111.8, 114.1, 116.4, 117.6, 118.7, 124.1, 126.9, 128.7, 130.7, 135.7,
141.6, 155.8, 158.1, 158.4, 158.8, 159.8, 160.2, 164.1, 175.7, 182.4;
HRMS (ESI-TOF) m/z: Calcd. for C30H23ClN3O7 [M + Na]+: 572.1219;
Found: 572.1225.
Yellow solid; m.p. > 300 °C; yield 76%; 1H NMR (DMSO‑d6,
400 MHz) δ: 6.91 (s, 1H), 6.96–7.00 (m, 2H), 7.05–7.07 (m, 2H), 7.43
(br s, 2H), 7.53–7.62 (m, 3H), 8.07–8.10 (m, 2H), 10.86 (br s, 1H),
13.45 (br s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 47.6, 56.1, 95.5,
103.8, 105.7, 107.1, 109.9, 118.0, 122.3, 125.7, 127.1, 129.6, 130.6,
133.2, 133.3, 144.1, 154.3, 156.1, 158.8, 159.9, 164.8, 178.1, 182.8;
4.2.24. 2′-Amino-5-bromo-8′-(2,4-dimethoxyphenyl)-1-ethyl-5′-hydroxy-
2,6′-dioxo-6′H-spiro [indoline-3,4′-pyrano[3,2-g]chromene]-3′‑carbonitrile
(3x)
HRMS (ESI-TOF) m/z: Calcd. for
C
26H14ClN3NaO5 [M + Na]+
:
506.0514; Found: 506.0502.
4.2.21. 2′-Amino-6-bromo-5′-hydroxy-2,6′-dioxo-8′-phenyl-6′H-spiro
[indoline-3,4′-pyrano [3,2-g]chromene]-3′‑carbonitrile (3u)
Yellow solid; m.p. > 300 °C; yield 86%; 1H NMR (DMSO‑d6,
400 MHz) δ: 1.13–1.17 (m, 3H), 3.71–3.82 (m, 8H), 6.54 (s, 1H),
6.58–6.61 (m, 1H), 6.74 (s, 1H), 6.80 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H),
7.30 (s, 1H), 7.42–7.44 (m, 1H), 7.83 (d, J = 8.9 Hz, 1H), 10.43 (br s,
2H), 13.47 (br s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 12.4, 35.2,
47.5, 55.7, 56.0, 56.2, 99.1, 103.1, 106.6, 111.3, 114.1, 114.8, 116.9,
119.8, 127.1, 130.8, 131.9, 136.3, 142.3, 154.0, 156.0, 158.3, 158.7,
159.1, 159.5, 160.1, 160.4, 164.4, 175.9, 182.6; HRMS (ESI-TOF) m/z:
Calcd. for C30H23BrN3O7 [M + H]+: 616.0714; Found: 616.0722.
Yellow solid; m.p. > 300 °C; yield 75%; 1H NMR (DMSO‑d6,
400 MHz) δ: 6.99–7.05 (m, 3H), 7.10–7.13 (m, 2H), 7.43 (br s, 2H),
7.59–7.65 (m, 3H), 8.13 (d, J = 7.2 Hz, 2H), 10.83 (br s, 1H), 13.47 (br
s, 1H); 13C NMR (DMSO‑d6, 100 MHz) δ: 47.7, 56.0, 95.6, 103.8, 105.7,
13

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
4.2.25. 2′-Amino-1-benzyl-8′-(2,4-dimethoxyphenyl)-5′-hydroxy-2,6′-
a microplate reader. The concentration of compounds when 50% of
cells were inhibited (IC50) was calculated with IBM SPSS Statistics
(version 19). The IC50 of each compound was obtained in at least three
independent experiments.
dioxo-6′H-spiro
(3y)
[indoline-3,4′-pyrano[3,2-g]chromene]-3′‑carbonitrile
4.5. Colony formation assay
Plate colony and soft agar colony formation assays were used to
assess the colony forming ability of cancer cells. Both experiments were
slightly modified from previously reported methods.41,44
Yellow solid; m.p. > 300 °C; yield 73%; 1H NMR (DMSO‑d6,
500 MHz) δ: 3.82 (d, J = 4.5 Hz, 6H), 4.91 (d, J = 15.9 Hz, 1H), 5.05
(d, J = 15.9 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.64–6.67 (m, 1H),
6.84–6.87 (m, 3H), 6.95–6.98 (m, 1H), 7.08 (d, J = 7.1 Hz, 1H),
7.16–7.19 (m, 1H), 7.24–7.28 (m, 1H), 7.30–7.33 (m, 2H), 7.47 (d,
J = 7.3 Hz, 2H), 7.90 (d, J = 8.9 Hz, 1H), 8.88 (br s, 2H), 13.53 (br s,
1H); 13C NMR (DMSO‑d6, 125 MHz) δ: 43.8, 47.4, 55.8, 56.1, 95.0,
98.9, 103.6, 106.4, 106.6, 109.2, 111.1, 111.9, 114.2, 116.5, 118.1,
118.7, 123.1, 123.9, 127.7, 128.6, 130.7, 133.6, 136.4, 143.0, 153.8,
155.7, 158.2, 158.5, 158.8, 159.1, 159.9, 160.2, 162.2, 164.2, 176.7,
For the plate colony formation assay, log-phase A549 cells were
seeded in each 6-well plate (Nest Biotechnology, China) at 1 × 103
cells/well. After incubation for 24 h, the medium was replaced with
fresh medium containing different concentrations of compound 3e and
incubated for another 10 days. Then, the cells were washed with PBS
three times, fixed with 4% paraformaldehyde for 30 min and then
stained with 0.1% Crystal violet stain. The plates were washed gently
and then observed under
a fluorescence microscope (Leica Inc.,
Germany). The percentage of colonies = number of treatment colonies/
number of control colonies×100%.
182.4; HRMS (ESI-TOF) m/z: Calcd. for
600.1765; Found: 600.1761.
C
35H26N3O7 [M + H]+
:
For the soft agar colony formation assay, A549 cells (1 × 105 cells/
mL) were treated with different concentrations of compound 3e in 1 mL
of 0.3% RPMI-1640 medium-agar. The suspension was added on top of
2 mL of 0.6% RPMI-1640 medium-agar. The cultures were maintained
at 37 °C and 5% CO2 for 14 days, and the cell colonies were stained with
1.5 mg/mL NBT (nitrotetrazolium blue chloride). The next processing
method was the same as that for the plate colony assay.
4.2.26. 2′-Amino-1-benzyl-8′-(2-fluorophenyl)-5′-hydroxy-5-methyl-2,6′-
dioxo-6′H-spiro
(3z)
[indoline-3,4′-pyrano[3,2-g]chromene]-3′‑carbonitrile
4.6. AO-EB and PI staining assay
A549 cells (3 × 105 cells/well) were seeded into 6-well plates in
RPMI-1640 medium (Nest Biotechnology, China) and incubated for
12 h. Then, the medium was replaced with fresh medium containing
different concentrations of compound 3e and incubated for another
24 h.
Yellow solid; m.p. > 300 °C; yield 71%; 1H NMR (DMSO‑d6,
500 MHz) δ: 2.17 (s, 3H), 4.89 (d, J = 15.8 Hz, 1H), 5.01 (d,
J = 15.8 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 6.79 (s, 1H), 6.89 (s, 1H),
6.99–7.01 (m, 2H), 7.26–7.33 (m, 3H), 7.43–7.48 (m, 6H), 7.68–7.69
(m, 1H), 8.04–8.07 (m, 1H), 13.20 (br s, 1H); 13C NMR (DMSO‑d6,
125 MHz) δ: 20.6, 43.6, 47.2, 56.1, 95.1, 104.0, 106.5, 108.9, 109.8,
117.0 (d, JCF = 21.3 Hz), 117.8, 118.7 (d, JCF = 8.8 Hz), 124.2, 125.3,
127.5, 128.3, 128.9, 129.7, 132.0, 133.3, 134.4, 136.2, 140.4, 154.0,
155.7, 158.3, 159.6, 159.9 (d, JCF = 253.8 Hz), 160.3, 176.2182.2;
HRMS (ESI-TOF) m/z: Calcd. for C34H23FN3O5 [M + H]+: 572.1616;
Found: 572.1618.
For the AO-EB double staining assay, cells were washed twice with
ice-cold PBS and stained with 100 μL of AO-EB stain (containing 1 mg/
mL AO and 1 mg/mL EB in PBS) at room temperature for 10 min in the
dark. Fluorescence was examined under a fluorescence microscope.
For the PI staining assay, A549 cells were harvested, centrifuged
and washed twice with ice-cold PBS. The cells were stained with PI
stain (50 mg/mL) at room temperature for 15 min in the dark. After
centrifugation, the cells were washed twice with ice-cold PBS. The
specimens were examined under a fluorescence microscope.
4.3. Cell culture
4.7. DNA fragmentation assay
All cell lines were purchased from the Chinese Academy of Sciences,
Kunming Cell Bank. These cells were cultured in RPMI-1640 medium
(Gibco, USA) supplemented with 10% foetal bovine serum (HyClone,
USA) 100 U/mL penicillin and 100 mg/mL streptomycin in a humidi-
fied atmosphere with 5% CO2 at 37 °C. The concentration of DMSO
was < 0.1%, and did not have any substantial effect on cellular func-
tion. Therefore, the DMSO concentration of the mixture was controlled
within this range.
The DNA cleavage pattern of A549 cells treated with compound 3e
was evaluated by observation of the formation of a DNA ladder45 and
according to the corresponding instructions of the genomic DNA iso-
lation kit (TIANGEN, China). Briefly, A549 cells were treated with
different concentrations of compound 3e for 24 h, harvested and wa-
shed with ice-cold PBS. Then, the cells were lysed with DNA lysis
buffer, treated with RNase A and digested with proteinase K. The DNA
was precipitated using absolute ethanol, collected by centrifugation and
subjected to 2% agarose gel electrophoresis for DNA fragmentation
analysis.
4.4. MTT assay
A total of 5 × 103 log-phase cells were inoculated into each well of a
96-well plate in RPMI-1640 medium (Nest Biotechnology, China) and
incubated for 24 h. The different concentrations of compounds were
added to each well and incubated for another 48 h. Next, 10 μL of MTT
reagent (5 mg/mL) was added to each well to react with the mi-
tochondria of living cells for approximately 4 h. Finally, the medium
was discarded, and the blue crystals were completely dissolved in
150 μL DMSO. Absorbance at the wavelength of 490 nm was read using
4.8. Annexin V-FITC/PI double staining assay
A549 cells (3 × 105 cells/well) were seeded into 6-well plates (Nest
Biotechnology, China) and incubated for 12 h. Then, the cells were
treated with different concentrations of compound 3e and incubated for
another 24 h, harvested with trypsin and washed with ice-cold PBS.
Finally, the collected cells were stained with Annexin V-FITC and PI
according to the instructions of the Annexin V-FITC/PI apoptosis kit
14

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
(CoWin Biosciences, China). The samples were analysed by flow cyto-
metry (BD Bioscience, USA).
area of the cells was calculated using the software provided with the
microscope, and the percentage of migration in the control cells was
taken as 100%.
4.9. Detection of reactive oxygen species (ROS) production
4.13. Transwell assay
A549 cells (3 × 105 cells/well) were seeded into 6-well plates (Nest
Biotechnology, China) and incubated for 12 h. Then, the cells were
treated with different concentrations of compound 3e and incubated for
another 24 h, the medium was discarded and cells were washed with
serum-free medium three times. Next, cells were stained with DCFH-DA
(10 μM) (Beyotime Institute of Biotechnology, China) for 20 min at
37 °C in the dark. Finally, the fluorescence of the cells from each well
was imaged with fluorescence microscope.
The cell migration and invasion assays were used to evaluate the
colony migration ability of the compound 3e-treated cells. Both ex-
periments were slightly modified from previously reported
methods.46,47
For the migration assay, A549 cells (1 × 104/mL) were suspended
in RPMI-1640 medium containing 1% foetal bovine serum, and 100 μL
of cell suspension was added to the upper chamber of each well of a 24-
well transwell plate (Corning, USA). A total of 600 μL of RPMI-1640
medium containing 10% foetal bovine serum was added to the lower
chamber. Then, the cells were treated with different concentrations of
compound 3e and incubated for 36 h. Each chamber was washed with
PBS, fixed with methanol for 30 min and stained with 0.1% crystal
violet for 20 min. A549 cells on the upper side of the membrane were
removed with cotton swabs and photographed under a microscope. The
cells that had migrated to the lower side of the membrane were
counted, and the percentage of migration in the control cells was taken
as 100%.
4.10. Caspase-3, -8 and -9 enzymatic activity assay
Caspase-3, -8 and -9 enzymatic activities were evaluated according
to the corresponding instructions of the caspase colorimetric assay kits
(KeyGEN BioTECH, USA). Briefly, 3 × 105 logarithmically growing
A549 cells were seeded into each 6-well plate (Nest Biotechnology,
China) and incubated for 12 h. The cells were treated with various
concentrations of compound 3e for 24 h, harvested with trypsin and
washed with ice-cold PBS. Then, the collected cells were lysed on ice for
60 min. After centrifugation, the total protein concentration was mea-
sured using the BCA protein assay kit (Beyotime, China). Next, 5 μL of
the corresponding caspase substrate was added to 50 μL of lysate con-
taining 200 μg of protein and incubated at 37 °Cfor 4 h in the dark. The
samples were measured at a wavelength of 405 nm using a microplate
reader. According to the instructions of the caspase colorimetric assay
kits, 50 μL PBS + 50 μL 2 × reaction was used as a blank control, and
the corresponding degree of caspase activation = (induced group
OD405 − blank group OD405) / (control group OD405 − blank group
OD405).
For the Matrigel-coated transwell assay, A549 cells (1 × 104/mL)
were suspended in RPMI-1640 medium containing 1% foetal bovine
serum, and 100 μL of cell suspension was seeded with Matrigel
(Changsheng, Beijing, China) in each well of a 24-well transwell plate
(Corning, USA). The remaining operations were the same as those for
the migration assay.
4.14. Molecular docking studies
For preparation before docking, the structure of the target com-
pound 3e was obtained using ChemBioDraw Ultra 14.0 software and
converted to 3D structure by ChemBio3D Ultra 14.0 software programs.
The 3D structure of Akt (PDB ID: 3MV5), 5-Lox (PDB ID: 3V99) and
MDM2 (PDB ID: 4ZYF) were downloaded from the Protein Data Bank,
respectively, and then subjected to removal of water molecules and
inhibitor treatment. Using Open Babel tool, the structure of the ligand
and acceptor proteins were converted from a pdb file to a pdbqt file.
After preparation, docking simulations were carried out using Autodock
Vina.48 The best-scoring poses as judged by the Vina docking score were
selected, and used PyMOL software for visual analysis.
4.11. Western blot assay
The A549 cells were treated with various concentrations of com-
pound 3e for 24 h, harvested and washed with cold PBS. Then, the
collected cells were lysed in RIPA lysis buffer (Beyotime, China) con-
taining 1 mM PMSF (Beyotime, China) on ice for 15 min. After cen-
trifugation, the total protein concentration was measured using the BCA
protein assay kit (Beyotime, China) and boiled with 5 × loading buffer
for 5 min at 100 °C. Then, 20 μg of protein was separated on 8–12%
SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) mem-
branes (Millipore, USA). The membranes were blocked with 5% skim
milk at room temperature for 1 h and then incubated with primary
antibodies overnight at 4 °C. The membranes were washed with TBST
and incubated with anti-rabbit secondary antibodies for 1 h at room
temperature. The immunoblots were observed with Clarity™ Western
ECL Substrate (BIO-RAD, USA). Reagents and imaged using ChemiDoc™
Touch Imaging System (BIO-RAD, USA). Antibodies used were against
MDM2 (#86934, Cell Signaling, USA), Cytochrome c (#11940, Cell
Signaling, USA), Bcl-2 (#3498, Cell Signaling, USA), Bax (#2772, Cell
Signaling, USA), Akt (#4691, Cell Signaling, USA), p21 (#2947, Cell
Signaling, USA), β-actin (#4970, Cell Signaling, USA), HRP-linked anti-
rabbit IgG (#7074, Cell Signaling, USA), 5-Lipoxygenase (ab39347,
Abcam, USA) and p53 (ab131442, Abcam, USA).
Acknowledgements
We are grateful for the financial support from the National Key R&D
Program of China (2018YFC1708100), NSFC (81760625, 81660576
and 81560563), and Guizhou Projects (GNYL[2017]008 and
QKHRC[2015]4032).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.115109.
References
4.12. Wound-healing assay
1. Viegas-Junior C, Danuello A, Bolzani VDS, Barreiro EJ, Fraga CAM. Curr Med Chem.
2007;14:1829–1852.
2. Fortin S, Bérubé G. Expert Opin Drug Discov. 2013;8:1029–1047.
3. Voskresensky ON, Levitsky AP. Curr Med Chem. 2002;2:691–714.
4. Li Y, Fang H, Xu W. Mini Rev Med Chem. 2007;7:663–678.
5. Wang HK. Expert Opin Investig Drugs. 2000;9:2103–2109.
6. Ren W, Qiao Z, Wang H, Zhang L. Med Res Rev. 2003;23:519–534.
7. Kelland LR. Expert Opin Investig Drugs. 2000;9:2903–2911.
8. Senderowicz AM, Sausville EA. J Natl Cancer Inst. 2000;92:376–387.
9. Fishkin RJ, Winslow JT. Psychopharmacology. 1997;132:335–341.
A549 cells were cultured in a 6-well plate (Nest Biotechnology,
China) at 1 × 105 cells/per well until confluent monolayers formed.
The monolayer of cells was scratched with a 10 μL sterile pipette tip.
Then, the medium was replaced with fresh medium containing various
concentrations of compound 3e and incubated for another 36 h. The
movement of cells was observed under the microscope. The migration
15

W.-H. Zhang, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
10. Habtemariam S. J Nat Prod. 1997;60:775–778.
M. M.; Ghabbour, H. A. 2017, (US9822128B).
11. Kubo I, Kinst Hori I, Choudhuri SK, Kubo Y, Sanchez Y, Ogura T. Bioorg Med Chem.
2000;8:1749–1755.
30. Mason JM, Lin DCC, Wei X, et al. Cancer Cell. 2014;26:163–176.
31. Wang S, Sun W, Zhao Y, et al. Cancer Res. 2014;74:5855–5865.
32. Yu B, Zheng YC, Shi XJ, Qi PP, Liu HM. Anti Cancer Agents Med Chem.
2016;16:1315–1324.
12. Liu YL, Ho DK, Cassady JM. J Nat Prod. 1992;55:357–363.
13. Pearce FL, Befs AD, Bienenstock J. Allergy J Clin Immunol. 1984;73:819–823.
14. Wolfman C, Viola H, Paladini A, Dajas F, Medina JH. Pharmacol Biochem Behav.
1994;47:1–4.
33. Han WY, Zhao JQ, Zuo J, Xu XY, Zhang XM, Yuan WC. Adv Synth Catal.
2015;357:3007–3031.
15. Peng CLV, Wang KR, Li QS, Chen J, Sun J, Zhu HL. Bioorg Med Chem.
2010;18:1117–1123.
34. Mei G, Shi F. Chem Commun. 2018;54:6607–6621.
35. Cao ZY, Zhou F, Zhou J. Acc Chem Res. 2018;51:1443–1454.
36. CCDC1890559 contains the supplementary crystallographic datas for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
16. Suresh Babu K, Hari Babu T, Srinivas PV, et al. Bioorg Med Chem Lett.
2006;16:221–224.
17. Xiao QZ, Sheng MP, Chang PH, Li FT, Han WD, Yuan JL. Bioorg Med Chem Lett.
2011;21:1222–1226.
37. Zhang Y, Chen S, Wei C, Pankin GO, Ye X, Chen YC. Eur J Med Chem.
2018;147:218–226.
18. Parmar VS, Jain SC, Bisht KS, et al. Phytochemistry. 1997;46:597–673.
19. Sun W, Cama LJ, Birzin ET, et al. Bioorg Med Chem Lett. 2006;16:1468–1472.
20. Stachulski AV, Berry NG, Low ACL, et al. J Med Chem. 2006;49:1450–1454.
21. Garino C, Bihel F, Pietrancosta N, et al. Bioorg Med Chem Lett. 2005;15:135–138.
22. Wang JL, Liu D, Zhang Z, et al. Proc Natl Acad Sci U S A. 2000;97:7124–7129.
23. Kemnitzer W, Kasibhatla S, Jiang S, et al. Bioorg Med Chem Lett. 2005;15:4745–4751.
24. Kemnitzer W, Drewe J, Jiang S, et al. J Med Chem. 2004;47:6299–6310.
25. Lotfy G, Said MM, El Ashry ESH, et al. Bioorg Med Chem. 2017;25:1514–1523.
26. Lotfy G, El Ashry ESH, Said MM, et al. J Photochem Photobiol B Biol.
2018;180:98–108.
38. Mori S, Chang JT, Andrechek ER, et al. Oncogene. 2009;28:2796–2805.
39. Khwaja S, Fatima K, Hassanain M, et al. Eur J Med Chem. 2018;151:51–61.
40. Zhao TQ, Zhao YD, Liu XY, et al. Eur J Med Chem. 2018;151:327–338.
41. Yi JJ, Wang ZY, Bai HN, et al. RSC Adv. 2016;6:5278–5287.
42. Zhou MY, Ji SB, Wu ZJ, et al. Eur J Med Chem. 2018;96:92–97.
43. Huang RZ, Liang GB, Huang XC, et al. Eur J Med Chem. 2017;138:979–992.
44. Dyshlovoy SA, Fedorov SN, Kalinovsky AI, et al. Marine Drugs. 2012;10:1212–1224.
45. Li ZC, Xu XG, Huang Y, et al. Int J Biol Sci. 2012;8:394–405.
46. Ma LY, Zheng YC, Wang SQ, et al. J Med Chem. 2015;58:1705–1716.
47. Wang B, Zhao B, Pang LP, et al. Pharm Res. 2017;122:66–77.
48. Trott O, Olson AJ. J Comput Chem. 2010;31:445–461.
27. Barakat A, Islam MS, Ghawas HM, et al. RSC Adv. 2018;8:14335.
28. Islam MS, Ghawas HM, El-Senduny FF, et al. Bioorg Chem. 2019;82:423–430.
29. Barakat, A.; Islam, M. S.; Al Majid, A. M.; Ghawas, H. M.; Badria, F. F.; Elshaier, Y. A.
16