Design, synthesis, and in vitro/in vivo anti-cancer activities of novel (20s)-10,11-methylenedioxy-camptothecin heterocyclic derivatives

Article abstract of DOI:10.3390/ijms21228495

A novel camptothecin analogue, (20S)-10,11-methylenedioxy-camptothecin (FL118), has been proven to show significant antitumor efficacy for a wide variety of solid tumors. However, the further development of FL118 is severely hindered due to its extremely poor water solubility and adverse side effects. Here, two series of novel 20-substituted (20S)-10,11-methylenedioxy-camptothecin coupled with 5-substituted uracils and other heterocyclic rings through glycine were synthesized. All the derivatives showed superior cytotoxic activities in vitro with IC50 values in the nanomolar range. Among them, 12e displayed higher cytotoxic activities in several cancer cell lines with better water solubility than FL118. Our results further showed that, like FL118, 12e inhibited cell proliferation resulting from cell cycle arrest and apoptosis by blocking the anti-apoptotic gene transcription of survivin, Mcl-1, Bcl-2, and XIAP in both A549 cells and NCI-H446 cells. Furthermore, 12e did not show any inhibitory activity on Topo I, which is involved in hematopoietic toxicity. In vivo, 12e showed similar antitumor efficacy to FL118 but lower toxicity. Our findings indicate that 12e is a promising therapeutic agent for cancer treatment, and the core structure of FL118 represents a promising platform to generate novel FL118-based antitumor drugs.

Full text of DOI:10.3390/ijms21228495

International Journal of
Molecular Sciences
Article
Design, Synthesis, and In Vitro/In Vivo Anti-Cancer
Activities of Novel (20S)-10,11-Methylenedioxy-
Camptothecin Heterocyclic Derivatives
Xiufen Dai 1,†, Guanzhao Wu 1,†, Yixuan Zhang 1, Xiaomin Zhang 1,2, Ruijuan Yin 1, Xin Qi 1,*,
Jing Li 1,2,* and Tao Jiang 1,2
1
Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy,
Ocean University of China, Qingdao 266003, China; fantasydxf@163.com (X.D.);
wuguanzhao09@hotmail.com (G.W.); zyxlljn@163.com (Y.Z.); xiaominzhang91@163.com (X.Z.);
yinruijuan@ouc.edu.cn (R.Y.); jiangtao@ouc.edu.cn (T.J.)
2
Open Studio for Druggability Research of Marine Natural Products, Laboratory for Marine Drugs and
Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China
*
†
Correspondence: qixin_ouc@ouc.edu.cn (X.Q.); lijing_ouc@ouc.edu.cn (J.L.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 24 September 2020; Accepted: 9 November 2020; Published: 11 November 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A novel camptothecin analogue, (20S)-10,11-methylenedioxy-camptothecin (FL118),
has been proven to show significant antitumor efficacy for a wide variety of solid tumors. However,
the further development of FL118 is severely hindered due to its extremely poor water solubility
and adverse side effects. Here, two series of novel 20-substituted (20S)-10,11-methylenedioxy-
camptothecin coupled with 5-substituted uracils and other heterocyclic rings through glycine were
synthesized. All the derivatives showed superior cytotoxic activities in vitro with IC50 values in the
nanomolar range. Among them, 12e displayed higher cytotoxic activities in several cancer cell lines
with better water solubility than FL118. Our results further showed that, like FL118, 12e inhibited
cell proliferation resulting from cell cycle arrest and apoptosis by blocking the anti-apoptotic gene
transcription of survivin, Mcl-1, Bcl-2, and XIAP in both A549 cells and NCI-H446 cells. Furthermore,
12e did not show any inhibitory activity on Topo I, which is involved in hematopoietic toxicity.
In vivo, 12e showed similar antitumor efficacy to FL118 but lower toxicity. Our findings indicate that
12e is a promising therapeutic agent for cancer treatment, and the core structure of FL118 represents a
promising platform to generate novel FL118-based antitumor drugs.
Keywords: Camptothecin derivatives; FL118; 12e; anticancer agent; molecular design
1. Introduction
In recent years, the incidence of malignant tumors has become a widespread public health concern.
Every year, more than 10 million new cancer cases and nearly seven million deaths are reported
worldwide [
over one hundred clinically approved drugs available [
(1, Figure 1), a quinoline alkaloid as an inhibitor of topoisomerase I, is a well-known drug isolated
from the Chinese tree Camptotheca by Wall et al. in 1966 [ ]. Over the past several decades, many CPT
1]. Chemotherapy is one of the most widely used modalities for cancer treatment, with
2
]. Among them, camptothecin (CPT) [
1]
3
derivatives were synthesized and tested, and its semisynthetic analogues such as topotecan (4, Figure 1)
and irinotecan (6, Figure 1) are approved by the USA Food and Drug Administration (FDA) for the
treatment of various cancers, such as lung cancer [4–6].
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www.mdpi.com/journal/ijms

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Figure 1. Structures of camptothecin derivatives.
In 2012, (20S)-10,11-methylenedioxy-camptothecin (FL118) (3, Figure 1), with similar structure to
SN-38 (active metabolite of irinotecan) (5, Figure 1) and topotecan, was identified by using genetically
modified cancer cell models via high throughput screening of chemical compound libraries [
Compared to other camptothecin derivatives, FL118 has been proven to possess much higher anticancer
activities in many different cancer types both in vitro and in vivo ]. Studies have demonstrated that
the presence of targeted Topo I is not critical for the antitumor activity of FL118, although it has the
same core structure with camptothecin [ ]. In contrast, the inhibition of Topo I by FL118 may mainly be
7].
[7
7
involved in the side effects of hematopoietic toxicity and possibly diarrhea as well. In fact, FL118 can
function as a single anticancer drug that targets multiple different drug-resistant mechanisms. It has
been reported that FL118 selectively inhibits gene promoter activities and endogenous expressions of
anti-apoptotic proteins, such as survivin, XIAP, cIAP2 and Mcl-1 [8,9], contributing to cell apoptosis
induction and antitumor activity [10]. Furthermore, FL118 performs those functions independent of p53
status (wild type, mutant, or null) in cells, and thus it can effectively control cancer with p53 function
incapacity, in which most DNA damage drugs (if not all) show a marked lack of efficiency [7,11].
Nevertheless, further relevant research of FL118 is severely hindered due to its extremely poor water
solubility and adverse side effects [10,12].
It has been revealed that anticancer activity of FL118 is highly dependent on its primary structure
and steric configuration [10], suggesting that FL118 may be a promising drug platform for novel
derivatives based on its core structure. In early research, it was once believed that the intact
six-membered α-hydroxy lactone E-ring of CPT was indispensable for antitumor activity, because
a free 20-hydroxy group favors lactone ring opening by forming intra-molecular hydrogen bonds
(H-bonds), which was the main cause of failure and side effects of camptothecin [13]. However,
further investigations demonstrated that modification of the free hydroxyl group at 20-position via
an ester bond could be a promising way to improve antitumor efficacy in vitro and in vivo, and to
reduce gastrointestinal toxicity [14–19], since esterification of the 20-hydroxy group should disfavor
ring opening [20]. In this regard, esterification of 20-hydroxyl group of FL118 could eliminate the
intramolecular hydrogen bonding and enhance the steric hindrance of carbonyl group of E-ring.
A series of results, such as 20(S)-O-acyl esters [21], 20(S)-O-carbonate linked tripeptide conjugates [22],
20(S)-O-linked glycoconjugates [23], and 20(S)-sulfonylamidine derivatives [18,19], have proved the
importance of esterified 1-derivatives as potential anticancer agents. Esterification of the 20-hydroxyl
group of 1 can also function as prodrugs to release the CPTs in vivo, and enhance solubility, plasma
stability, and pharmacokinetic character of CPTs [20]. Given these considerations, we designed a series
of novel 5-substituted uracil and heterocycles esters of FL118 and evaluated their in vitro and in vivo
antitumor activity. Results showed that these FL118 derivatives displayed impressive antitumor effects
in vitro. More importantly, compound 12e showed high antitumor efficacy in both NCI-H446 and
A549 xenograft mouse model without apparent toxicity, which is in line with our research expectation.

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Mechanically, 12e was revealed to have similar molecular mechanism to FL118 via inhibiting the
transcription and expression of multiple anti-apoptotic proteins rather than Topo I activity.
2. Results
2.1. Chemistry
The chemical reactivity of 20-hydroxy group of FL118 is much lower, because this 20-hydroxy
group is a tertiary alcohol with strong steric hindrance. Esterification of this hydroxy group under
standard conditions is the easiest method for masking it. Therefore, we prepared prodrugs of FL118
as ester derivatives. The synthesis of FL118 was accomplished using a Friedlander condensation
according to published procedures [10]. The synthetic routes to target compounds (11a–11c and
12a–12f) were outlined in Scheme 1. Briefly, the 20-hydroxy group of FL118 was converted to N-Boc
protected amino esters (7) using a combination of DIC (N, N0-diisopropyl carbodiimide) and DMAP
(4-dimethylaminopyridine) in a 95% yield. Subsequently, compound 7 was treated with trifluoroacetic
acid in dichloromethane to afford the desired amine intermediates 8 in 2 h. Compound 9 was reacted
with bromoacetic acid for 5 h to generate a carboxylic group. Condensation of the intermediates 8
with the uracil fragments by DMAP and DIC, afforded the hybrids compounds 11a–11c in 74% to
79% yields, respectively. In addition, a wide range of heterocyclic components containing pyrrole,
piperidine, and substituent piperazine were all efficiently coupled with intermediates 8, which was
first reacted with CDI (N, N0-Carbonyldiimidazole) as the linker, to provide compounds 12a–12f in
36% to 78% yields. All newly synthesized compounds were purified by column chromatography and
their structures were confirmed by 1H NMR, 13C NMR, and HRMS.
Scheme 1. Synthesis of FL118 derivatives. Reagents and conditions: (
EDCI, DCM, 4 h, 95%; ( ) CF3COOH, CH2Cl2, 2 h, 95%; ( ) BrCH2COOH, KOH, H2O, 60 C, 7 h,
) CDI, appropriate
a) Boc-Gly-OH, DMAP, DIC,
◦
b
c
45–56%; (d e
) 50-substituted uracil 10(N)-acetic acid, DMAP, DIC, DCM, 8 h, 74–79%; (
2◦ amine, Et3N, DCM, 0 ◦C, 8 h, 36–78%.
2.2. 12e Inhibits the Proliferation of Various Tumor Cells
The proliferation inhibitory activities of these synthesized compounds cells were assessed by SRB
assay. All the compounds displayed strong cytotoxicity on human non-small cell lung cancer cells
A549 with wild type p53, the IC50 values were between 1.37–38.71 nM after 72 h treatment (Figure 2A).
Noteworthily, 12e exhibited strongest cytotoxic activity with 10.28-fold potential than FL118. Hence,

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12e was selected for further studies. Furthermore, 12e was found to have better water solubility
than FL118.
Figure 2. 12e inhibits lung cancer cell growth. (
of synthesized compounds for 72 h. Cell proliferation was determined using SRB assay. (
cancer cells were treated with indicated concentrations of 12e for 72 h. Cell proliferation was determined
using SRB assay. The data are presented as means SD from three independent experiments. ( ) 12e
inhibits the colony formation. Cells were treated with 12e (0.01–100 nM) for 14 days, and stained
with Giemsa. Then colonies were photographed and counted. ( ) Quantification of the number of
colonies. Colonies consisting of 50 cells were scored. Data are presented as means SD from three
independent experiments. * p < 0.05, ** p < 0.01 vs. control. ( ) 12e affected cell cycle distribution.
Cells were treated with 12e (0.01-100 nM) for 24 h, then were collected, and stained by PI. The DNA
A) A549 cells were treated with various concentrations
B) Various
±
C
D
≥
±
E
content of cells was determined with the MoFlo XDP flow cytometry system. (F) Histograms show the
percentage of cells in G0/G1, G2/M, and S phase after treatment with 12e.

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The cytotoxicity of 12e on various human cancer cells is summarized in Figure 2B. It was
demonstrated that 12e displayed strong inhibitory activity on these selected cancer cells with IC50
values in the range of 1.37–122.48 nM after 72 h of treatment. Among these cancer cells, A549 cells
were most sensitive to 12e with IC50 value at 1.37 nM. Thus, A549 cells were used to further explore
the anticancer effect and mechanism of 12e in the following research. Given that there is no new drug
for small cell lung cancer nearly 30 years [24], the NCI-H446, a human small cell lung cancer line
with mutant p53, was also chosen in our research. The IC50 value of 12e on NCI-H446 was 9.48 nM,
which was very potent compared to most compounds reported.
Plate clone formation assay is the gold standard for measuring the cytotoxicity of compounds on
cancer cells in vitro [25]. Accordingly, 12e showed significant inhibition on the colony-forming abilities
of A549 and NCI-H446 cells in a dose-dependent manner (Figure 2C,D). Compared to the control group,
the inhibition rates of 12e (0.01–1 nM) after 14 days of treatment were 22.02%, 28.81%, and 45.20% in
NCI-H446 cells, 27.80%, 34.88%, and 53.41% in A549 cells, respectively. When the concentration of 12e
was more than 10 nM, the colony-forming was inhibited thoroughly both in A549 and NCI-H446 cells.
In order to explore whether the cell cycle arrest contributes to 12e-induced proliferation inhibition,
we further analyzed the cell cycle distribution and found that 12e induced S phase arrest in A549
cells and NCI-H446 cells (Figure 2E). In the control group, the cells in S phase represented 33.86%
(NCI-H446) and 39.82% (A549), and it increased to 34.95% and 47.56%.in the presence of 1 nM 12e.
While in the presence of 10 and 100 nM 12e, the cells in S phase rose to 100% (Figure 2F). These results
indicate that 12e suppresses the proliferation of lung cancer cells by arresting the cell cycle in the S
phase, which is similar to effect of FL118 on the cell cycle [26,27].
2.3. 12e Induces Cell Apoptosis of A549 Cells and NCI-H446 Cells
Next, we determine whether S phase arrest induced by 12e resulted in cell apoptosis. In apoptosis,
the nucleus undergoes a series of changes including segregation of nucleoli and condensation of
chromatin [28]. The nuclear morphology was evaluated after Hoechst 33342 staining. As shown in
Figure 3A, the number of cell nuclei that exhibited brighter blue fluorescence, shrinkage, or DNA
fragmentation (shown by arrows) was increased significantly after exposure to 12e in A549 cells and
NCI-H446 cells dose-dependently. Furthermore, the levels of γ-H2AX, an essential marker of DNA
double-strand breaks, was increased significantly in a dose-dependent manner (Figure 3B), suggesting
that 12e induced DNA double-strand breaks, which was consistent with the influence of FL118 on
DNA strand [29].
Cysteinyl aspartate specific proteinase-9 (caspase-9) is the apical caspases in the intrinsic apoptosis
pathways, and cysteinyl aspartate specific proteinase-3 (caspase-3) is considered to be the most
important component to the effectors caspases. The cleaving and activation of the caspase-9/caspase-3
is a hallmark of the intrinsic apoptosis [30]. In addition, the poly-adenosine diphosphate-ribose
polymerase (PARP) protein is a substrate of caspase-3. Cleaved PARP seems to be an early marker of
apoptosis in cells. Bax, a member of the Bcl-2 family, can promote apoptosis mediated by mitochondria.
As shown in Figure 3B, we observed obvious increases of cleaved caspase-9, cleaved caspase-3, cleaved
PARP, and pro-apoptotic protein bax in the presence of 12e. Meanwhile, the levels of caspase-8,
an important component of the death signal receptor pathway of apoptosis [31], had no change after
exposure to 12e treatment. Taken together, 12e induces A549 and NCI-H446 cell apoptosis involved in
mitochondrial pathway rather than death receptor pathway.
In order to quantify the extent of apoptosis, flow cytometry analysis was performed to detect
12e-induced apoptosis rates by Annexin V-FITC and PI staining. Annexin V-FITC and PI double
staining can distinguish apoptotic cells, necrotic cells as well as normal cells. The specific binding
of phosphatidylserine exposed on the cell surfaces undergoing apoptosis with Annexin V can be
used to detect apoptotic cells. The late apoptotic cells and necrotic cells were assessed by propidium
iodide (PI) staining [32]. As shown in Figure 3C, after treatment by 12e (0, 1, 10, and 100 nM) for 24 h,
the rates of early and late apoptosis (AV+/PI-) were 6.76%, 12.57%, 18.52%, and 20.32% in NCI-H446

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cells, and 6.84%, 12.55%, 15.61%, and 24.17% in A549 cells, respectively. These results support that 12e
induces cell apoptosis.
Figure 3. 12e promotes apoptosis of lung cancer cell lines. (
treated with 12e (0–100 nM) for 24 h, then stained with Hoechst 33342, the photos were taken using
microscopy (magnification, 200 ). The brighter blue fluorescence (indicted arrows), show apoptotic
cells. ( ) Western blotting analysis of the expression level of apoptosis-related proteins in indicated
cells after treated with 12e. -tubulin was chosen as the internal control. ( ) Apoptosis rate of cancer
cells induced by 12e. Cells were treated with indicated concentrations of 12e for 24 h, then stained with
A) NCI-H446 and A549 cells were
×
B
β
C
Annexin V-FITC/PI, and determined by flow cytometry. (
cells, * p < 0.05, ** p < 0.01 vs. control.
D) Quantification of the Annexin-V positive

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2.4. 12e does not Impair Cell Proliferation Signaling in A549 Cells and NCI-H446 Cells
Cell proliferations, apoptosis and differentiations are regulated by cell signal transduction pathway.
In various cancer cells, stat3, AKT and erk1/2 were abnormally activated, which are molecules in three
main signaling pathway, relating to the cell proliferation and survive [33–35]. To investigate which
cell signaling transduction pathway was involved in 12e-induced cell apoptosis, we investigated the
expressions and activations of stat3, AKT and erk1/2 by western blot assay. As shown in Figure 4,
after treatment with 1, 10, and 100 nM 12e for 24 h, the levels of stat3, AKT, and erk1/2 had no significant
change, and the phosphorylation levels of stat3, AKT, and erk1/2 were not appreciably decreased on
NCI-H446 and A549 cells. These results indicate that 12e-induced apoptosis is probably not associated
with the inactivation of cell proliferation upstream signaling.
Figure 4. 12e does not impair cell proliferation upstream signaling in lung cancer cell lines. NCI-H446
and A549 cells were treated with 12e (0–100 nM) for 24 h, the levels of proliferation-related proteins
were detected by western blotting. β-tubulin served as the loading control.
2.5. 12e Reduces the Transcription and Expression of Multiple Anti-Apoptotic Proteins
The IAP family and Bcl-2 family proteins play important roles in suppressing apoptosis of
cells [36]. It was reported that FL118 inhibited the transcriptions and expressions of survivin, XIAP and
Mcl-1 [8,9]. We then tested whether 12e affected the expression levels of these anti-apoptotic proteins.
After treatment with 1 to 100 nM of 12e for 24 h, the protein levels of survivin, Bcl-2, Mcl-1, and XIAP
were down-regulated both in A549 and NCI-H446 cells in a dose-dependent manner (Figure 5A).
Accordingly, the RT-PCR assay showed that the mRNA levels of survivin, Bcl-2, Mcl-1 and XIAP were
decreased significantly (Figure 5B). All these data suggest that 12e inhibits the genetic transcription of
multiple anti-apoptotic proteins and their subsequent expression. Early studies have demonstrated that
camptothecin (CPT) inhibits the synthesis of DNA and protein, due to inhibition of DNA topoisomerase
I (Topo I) activity [37]. Previous studies showed that the inhibition of Topo I activity by FL118 did
not play a major role in its antitumor activity [7,10]. To determine whether 12e inhibited DNA Topo
I activity, a Topo I -Mediated Relaxation of pBR322 DNA Assay was performed. The pBR322 DNA
was presented as supercoiled state (SC) without treatment of Topo I, and relaxed state (RLX) with
the treatment of Topo I. After co-treated with CPT, the relaxation activity of Topo I was inhibited and
SC DNA was reversely increased distinctly, while the pBR322 DNA was still presented as RLX after
co-treated with FL118, or 12e (Figure 5C). Intriguingly, FL118 exhibited a bit weak inhibitory activity
on Topo I than 12e, as 12e did not show any inhibitory activity on it. Meanwhile, we observed that
compounds 12a and 12c showed no inhibitory activity on Topo I. These results suggest that 12e inhibits

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transcriptions and expressions of multiple anti-apoptotic proteins, thereby inducing cell apoptosis of
A549 and NCI-H446 cells independent of Topo I activity.
Figure 5. 12e treatment reduces the transcription and expression of multiple anti-apoptotic proteins
in lung cancer cell lines. (
the expression levels of survivin, Bcl-2, Mcl-1 and XIAP were detected by western blotting.
was chosen as the internal control. ( ) Upon exposure to 12e (0–100 nM) for 24 h, the relative mRNA
level of survivin, Bcl-2, Mcl-1 and XIAP in NCI-H446 and A549 cells were determined using RT-PCR
technique. * p < 0.05, ** p < 0.01 vs. control. ( ) Topo I- Mediated relaxation of pBR322 DNA Assay
A
) NCI-H446 and A549 cells were treated with 12e (0–100 nM) for 24 h,
β-tubulin
B
C
with or without indicated compounds. The pBR322 DNA was presented as supercoiled (SC) without
treatment of Topo I, and relaxed (RLX) with the treatment of Topo I. CPT was used as a positive control.
2.6. 12e Suppresses the Growth of Lung Cancer Cells Xenograft In Vivo
Next, we studied the in vivo antitumor activity of 12e using animal xenograft models of human
A549 and NCI-H446 cancer cells. We compared the antitumor activities of 12e and FL118 at a dose
of 10 mg/kg by intragastric administration once per week. As shown in Figure 6, 12e exhibited a
distinct reduction in tumor volume compared to vehicle in A549 and NCI-H446 xenografts with similar
antitumor activity to FL118 (Figure 6A,B). The inhibitory rates of 12e and FL118 were 48.8% and 59.8%
(A549 xenografts), and 70.3% and 75.4% (NCI-H446 xenografts) respectively. During the period of

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drug administration, the mice body weights of FL118 group lost obviously both in A549 and NCI-H446
xenograft models compared with control group, while 12e did not induce an obvious loss of mice body
weight (Figure 6C,D). The results indicated that 12e has weaker toxicity than FL118. Furthermore,
just like FL118 12e down-regulated the expression of survivin, Mcl-1, Bcl-2 and XIAP in A549 and
NCI-H446 xenograft tumors (Figure 6E). These findings indicate that 12e suppresses lung cancer
growth in vivo through same mechanism as FL118, but with weak toxicity.
Figure 6. 12e suppresses tumor growth in vivo. A549 or NCI-H446 bearing mice were intragastrically
administrated with FL118, 12e or vehicle, and the tumor volumes and body weights of mice were
determined. (
xenografts. ( ) Body weights of A549 bearing mice. (
) Western blotting analyzes the expression levels of survivin, Bcl-2, Mcl-1and XIAP in the excised
tumor tissues. β-tubulin was chosen as the internal control. * p < 0.05, ** p < 0.01 vs. control.
A
) The tumor volumes of A549 xenografts. (
B) The tumor volumes of NCI-H446
C
D
) Body weights of NCI-H446 bearing mice.
(
E
3. Discussion
In this study, novel 20-substituted FL118 derivatives coupled with 5-substituted uracils and other
heterocyclic rings through glycine were synthesized. All the derivatives showed superior cytotoxic
activities in vitro, with IC50 values between 1.37–38.71 nM after 72 h treatment in A549 cell lines.

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Among them, 12e displayed higher cytotoxic activities on various human cancer cells including A549
and NCI-H446 cells. In addition, 12e was found to have better water solubility and more potential
cytotoxic effect than FL118. Cell cycle arrest and apoptosis were the two main causes of growth
inhibition [38]. Moreover, 12e suppressed the proliferation of NCI-H446 and A549 cells by arresting the
cell cycle at the S phase, which resulted in cell apoptosis. This result is consistent with previous studies
that FL118 induced S phase arrest in several types of human cancer cell line [19
33342 staining, western blot and flow cytometry analysis, our data showed that 12e induced apoptosis
in NCI-H446 and A549 lung cells via up-regulation of H2AX and bax, down-regulation of survivin,
,20]. With Hoechst
γ
Bcl-2, Mcl-1 and XIAP with cleavage and activation of caspase-3, caspase-9, and PARP. PI3K/AKT,
JAK/STAT3, and RAS/ERK1/2 signaling pathways contribute to a variety of processes that are critical in
mediating many aspects of cellular function including cell growth and survival, abnormalities of these
pathways have been reported in many human tumors, and inhibitors against these pathways promote
cell apoptosis [39]. However, there is no change in the activations and expressions of AKT, stat3 and
erk1/2 after 12e treatment, indicating that 12e plays a role in the downstream of these signaling pathway
related to cell growth and survival. As expected, 12e was verified to inhibit the genetic transcription of
multiple anti-apoptotic proteins, which is in line with the report that FL118 binds to similar promoter
region of the survivin, Mcl-1, Bcl-2 and XIAP genes, and thereby inhibiting these genes transcription.
Moreover, we confirmed that FL118 exhibited weaker inhibitory activity on Topo I than 12e, as 12e did
not show any inhibitory activity on Topo I. Inhibition of Topo I by FL118 is predominantly involved in
hematopoietic toxicity [25], but not in FL118 antitumor activity, which may be an explanation for the
lower toxicity of 12e. Previous studies indicated that FL118 treatment in its oral formulation has a
maximum tolerance dose of 10 mg/kg in human tumor-bearing SCID with the weekly
Similarly, we found that 12e showed high antitumor efficacy in both NCI-H446 and A549 mouse
models at a dose of 10 mg/kg with the weekly 4 schedule. More importantly, 12e did not cause
×
4 schedule [19].
×
apparent toxicity in xenograft mice compared to FL118, which is in line with our original intention of
molecular design.
In summary, through esterification of the 20-hydroxy group of FL118 with 5-substituted uracils
and N-containing heterocycles via a glycine linker, we successfully obtained a new compound 12e
with similar antitumor activity to FL118 while lower toxicity. In addition, mechanism research reveals
that 12e inhibits transcriptions and expressions of anti-apoptotic protein survivin, Bcl-2, Mcl-1 and
XIAP in line with molecular mechanism of FL118. Our findings indicate that the core structure of
FL118 represents a promising platform for the generation of novel FL118 derivatives, which lays the
foundation for further antitumor drug development in the future.
4. Materials and Methods
4.1. Chemistry
4.1.1. Materials and Methods
All reagents and solvents were reagent grade or were purified by standard methods before
use. Melting points were determined in open capillaries and are uncorrected. 1H and 13C NMR
spectra were recorded at 400 MHz and 100 MHz on a Bruker AM-400 spectrometer using TMS as
reference ( Bruker Daltonics Inc., Billerica, MA, USA). Chemical shifts (δ values) and coupling constants
(J values) are given in ppm and Hz, respectively. Anhydrous tetrahydrofuran (THF) was obtained by
distillation from sodium-benzophenone ketyl; dry methylene chloride was obtained by distillation from
phosphorus pentoxide. All reactions requiring anhydrous conditions were performed under a positive
nitrogen flow, and all glassware were oven dried and/or flame dried. Isolation and purification of the
compounds were performed by flash column chromatography on silica gel (200–300 mesh) produced
by Qingdao Marine Chemical Factory, Qingdao (China). Analytical thin-layer chromatography (TLC)
was conducted on Fluka TLC plates (silica gel 60 F254, aluminum foil).

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4.1.2. Synthesis of FL118
FL118 was synthesized according to our previous procedures [10], namely in three steps from the
commercially available piperonal.
4.1.3. Synthesis of (S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]
indolizino[1,2-b]quinolin-7-yl (tert-butoxycarbonyl)glycinate (7)
FL118 (0.50 mmol), Boc-Gl◦y-OH (0.60 mmol), and DMAP (1.02 mmol) were dissolved in anhydrous
DCM (50 mL) under N2 at 0 C. DIC (1.20 mmol) was added dropwise into the reaction mixture.
The mixture was stirred for 1 h at 0 ◦C and then stirred overnight at room temperature. The mixture
was washed with 100 mM HCl aqueous solution (3
×
100 mL), water (3
×
100 mL), and saturated brine
(
3 × 100 mL). The organic layer was dried over anhydrous Na2SO4 and purified by silica gel column
chromatography (Acetone/DCM = 1:20 to 10:1, v/v) (95% yield). mp 241–242 ◦C; HRMS (ESI): calcd for
1
C28H27N3O9 550.5360, found 550.1806. H NMR (500 MHz, DMSO)
δ
8.34 (s, 1H), 7.36 (s, 2H), 7.02
(
d, J = 70.1 Hz, 1H), 6.24 (s, 2H), 5.45 (s, 2H), 5.08 (d, J = 25.9 Hz, 2H), 3.87 (dd, J = 54.0, 17.3 Hz, 2H),
2.10 (s, 2H), 1.32 (d, 6H), 0.94 (d, 6H). 13C NMR (125 MHz, DMSO)
δ 169.98, 167.64, 157.03, 156.29,
151.82, 149.97, 149.09, 146.81, 146.63, 145.88, 130.60, 128.58, 125.99, 118.23, 104.79, 103.46, 103.07, 95.30,
79.08, 76.70, 66.61, 50.46, 42.34, 41.06, 30.69, 28.20, 23.63, 7.93.
4.1.4. Synthesis of (S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]
indolizino[1,2-b]quinolin-7-yl glycinate (8)
Compound 7 (0.36 mmol) was dissolved in TFA (20 mL), and the solution was stirred for 1 h at
room temperature. The solution was evaporated and purified by silica gel column chromatography
(Acetone/DCM = 1:10 to 1:1, v/v) (95% yield). mp > 250 ◦C; HRMS (ESI): calcd for C23H19N3O7 450.4190,
1
found 450.1288. H NMR (500 MHz, DMSO)
δ
8.44 (s, 1H), 7.45 (s, 1H), 7.40 (s, 1H), 7.16 (s, 1H), 6.25
(s, 2H), 5.48 (d, J = 16.5 Hz, 2H), 5.16 (d, J = 20.9 Hz, 2H), 4.26 (d, J = 17.9 Hz, 1H), 4.06 (d, J = 17.9 Hz
,
1H), 2.21 – 2.09 (m, 2H), 0.98 167.34, 167.15, 156.99,
−
0.89 (m, 3H). 13C NMR (125 MHz, DMSO)
δ
151.97, 149.97, 149.22, 146.89, 146.86, 145.20, 130.85, 128.80, 126.18, 118.33, 104.77, 103.62, 103.12, 95.17,
78.01, 66.75, 50.61, 30.57, 23.63, 7.93.
4.1.5. General Procedure for the Synthesis of Compounds 11a–11c
Compound
8 (0.13 mmol), 5-substituted uracil acid 10a–10c (1.1 mmol), and DMAP (0.20 mmol)
were dissolved in anhydrous DCM (20 mL) at 0 ◦C. DIC◦(0.20 mmol) was added dropwise into the
reaction mixture. The mixture was stirred for 1 h at 0 C and then stirred for overnight at room
temperature. The mixture was washed with 100 mM HCl aqueous solution (3
×
100 mL), water
(3 × 100 mL), and saturated brine (3 × 100 mL). The organic layer was dried over anhydrous Na2SO4
and purified by silica gel column chromatography (Acetone/DCM = 1:20 to 10:1, v/v) to yield the
desired compound, 11a–11c, respectively.
(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]
indolizino[1,2-b]quinolin-7-yl (2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycinate (11a)
The general synthetic method described above afforded compound 11a as a yellow solid (77%).
◦
mp > 250 C; HRMS (ESI): calcd for C29H23N5O10 602.5280, found 602.1506. 1H NMR (500 MHz,
DMSO)
(s, 3H), 5.11 (s, 2H), 4.35 (s, 2H), 4.22 (s, 1H), 4.09 (s, 1H), 2.15 (s, 2H), 0.89 (s, 3H). 13C NMR (125 MHz,
DMSO) 169.16, 167.91, 167.52, 164.22, 156.91, 151.84, 151.37, 150.10, 149.12, 146.86, 145.38, 130.58,
δ 11.21 (s, 1H), 8.71 (s, 1H), 8.38 (s, 1H), 7.46 (d, J = 31.5 Hz, 3H), 7.01 (s, 1H), 6.26 (s, 2H), 5.46
δ
128.75, 127.84, 126.08, 118.48, 114.29, 105.07, 103.50, 103.05, 101.07, 94.70, 76.84, 66.79, 50.51, 49.67,
30.86, 7.97.

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(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (2-(5-chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycinate (11b)
The general synthetic method described above afforded compound 11b as a yellow solid (79%),
1
mp 242-243 ◦C; HRMS (ESI): calcd for C29H22ClN5O10 636.9700, found 636.1151. H NMR (500 MHz,
DMSO)
(s, 2H), 5.11 (s, 2H), 4.36 (s, 2H), 4.24 (dd, J = 18.3, 5.5 Hz, 1H), 4.09 (dd, J = 17.9, 5.3 Hz, 1H), 2.19
(m, 2H), 0.90 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, DMSO)
169.11, 167.61, 167.51, 159.95, 156.91,
δ
11.79 (s, 1H), 8.74 (s, 1H), 8.38 (s, 1H), 8.03 (s, 1H), 7.42 (s, 2H), 7.01 (s, 1H), 6.26 (s, 2H), 5.46
−
2.09
δ
151.84, 150.51, 150.09, 149.12, 146.90, 146.86, 145.39, 144.01, 130.55, 128.74, 126.07, 118.45, 106.38, 105.08,
103.48, 103.04, 94.70, 76.86, 66.79, 50.50, 49.99, 40.72, 30.85, 7.97.
(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl)glycinate (11c)
The general synthetic method described above afforded compound 11c as a yellow solid (74%),
1
mp 242-243 ◦C; HRMS (ESI): calcd for C29H22FN5O10 620.5184, found 620.1409. H NMR (500 MHz,
DMSO)
(s, 2H), 5.09 (s, 2H), 4.29 (d, J = 18.1 Hz, 2H), 4.26
0.90 (t, J = 6.8 Hz, 3H). 13C NMR (125 MHz, DMSO)
δ
8.73 (s, 1H), 8.37 (s, 1H), 7.97 (d, J = 6.4 Hz, 1H), 7.41 (s, 2H), 7.01 (s, 1H), 6.26 (s, 2H), 5.46
4.17 (m, 1H), 4.11 4.02 (m, 1H), 2.19 2.07 (m, 2H),
169.12, 167.68, 167.51, 156.91, 151.84, 150.07,
−
−
−
δ
150.04, 149.11, 146.90, 146.84, 145.40, 140.65, 131.35, 131.07, 130.54, 128.70, 126.05, 118.46, 105.04, 103.47,
103.05, 94.71, 76.85, 66.79, 50.48, 49.86, 40.72, 30.86, 7.97.
4.1.6. General Procedure for the Synthesis of Compounds 12a–12f
Compound
8 (0.11 mmol) and Triethylamine (0.02 mmol) was dissolved in anhydrous DCM
(50 mL) under N2 at 0 ◦C. Then CDI (0.14 mmol) in anhydrous DCM (10 mL) was then added. After
stirring at room temperature for 1 h, various heterocycles (0.154 mmol) were added into the solution,
and the reaction mixture was stirred overnight at room temperature. The mixture was washed with
water (3
×
100 mL), and saturated brine (3
×
100 mL). The organic layer was dried over anhydrous
Na2SO4 and purified by silica gel column chromatography (Acetone/DCM = 1:8 to 5:1, v/v) to yield the
desired compound, 12a–12f, respectively.
(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (morpholine-4-carbonyl)glycinate (12a)
The general synthetic method described above afforded compound 12a as a yellow solid (78%).
1
mp > 250 ◦C; HRMS (ESI): calcd for C28H26N4O9 563.5350, found 563.1758. H NMR (500 MHz, DMSO)
δ
8.42 (s, 1H), 7.46 (s, 1H), 7.42 (s, 1H), 7.14 (t, J = 5.7 Hz, 1H), 7.05 (s, 1H), 6.27 (d, J = 4.8 Hz, 2H), 5.45
(s, 2H), 5.15 (s, 2H), 4.02 (dd, J = 17.7, 5.7 Hz, 1H), 3.90 (dd, J = 17.8, 5.8 Hz, 1H), 3.48 (t, J = 4.7 Hz, 4H),
3.25 (dt, J = 13.5, 6.6 Hz, 4H), 2.12 (td, J = 13.8, 7.0 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz,
DMSO)
δ 170.50, 167.64, 157.76, 156.94, 151.85, 150.20, 149.12, 146.92, 146.68, 145.54, 130.58, 128.76,
126.05, 118.48, 105.01, 103.53, 103.06, 95.08, 76.45, 66.76, 66.35, 50.49, 44.21, 42.44, 30.93, 7.93.
(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (piperidine-1-carbonyl)glycinate (12b)
The general synthetic method described above afforded compound 12b as a yellow solid (76%),
1
mp >250 ◦C; HRMS (ESI): calcd for C29H28N4O8 560.5634, found 560.1875. H NMR (500 MHz, DMSO)
δ
8.42 (s, 1H), 7.46 (s, 1H), 7.42 (s, 1H), 7.14 (t, J = 5.7 Hz, 1H), 7.05 (s, 1H), 6.27 (d, J = 4.8 Hz, 2H), 5.45
(s, 2H), 5.15 (s, 2H), 4.02 (dd, J = 17.7, 5.7 Hz, 1H), 3.90 (dd, J = 17.8, 5.8 Hz, 1H), 3.48 (t, J = 4.7 Hz
4H), 3.25 (dt, J = 13.5, 6.6 Hz, 4H), 2.12 (td, J = 13.8, 7.0 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR
(125 MHz, DMSO) 172.96, 170.72, 167.68, 157.47, 156.95, 151.81, 150.21, 149.06, 146.63, 145.65, 130.58,
,
δ
128.71, 126.01, 118.43, 105.23, 103.52, 96.32, 95.26, 76.38, 72.85, 66.73, 65.67, 50.58, 44.69, 42.68, 30.66,
25.80, 24.51, 8.24.

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(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (pyrrolidine-1-carbonyl)glycinate (12c)
The general synthetic method described above afforded compound 12c as a yellow solid (76%),
1
mp > 250 ◦C; HRMS (ESI): calcd for C28H26N4O8 547.5360, found 547.1809. H NMR (500 MHz, DMSO)
δ
8.34 (d, J = 3.2 Hz, 1H), 7.36 (s, 1H), 7.17 (t, J = 5.8 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.21 (d, J = 2.3 Hz,
2H), 5.41 (dd, J = 30.3, 16.6 Hz, 2H), 4.94 (dd, J = 22.9, 12.8 Hz, 2H), 4.02 (dd, J = 17.7, 5.7 Hz, 1H), 3.96
−
−
3.83 (m, 1H), 3.71 (d, J = 14.6 Hz, 1H), 3.25 (dt, J = 13.5, 6.6 Hz, 4H), 2.12 (td, J = 13.8, 7.0 Hz, 2H), 1.87
1.79 (m, 1H), 1.53 170.76,
1.30 (m, 5H), 0.98 (dd, J = 21.3, 6.8 Hz, 3H). 13C NMR (125 MHz, DMSO)
167.64, 156.97, 156.57, 151.86, 150.24, 149.16, 146.97, 146.75, 145.45, 135.04, 130.65, 128.86, 126.13, 120.43,
118.63, 105.09, 103.61, 103.06, 95.00, 76.34, 66.82, 50.56, 45.72, 42.16, 31.04, 25.45, 7.89.
−
δ
(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (4-methylpiperazine-1-carbonyl)glycinate (12d)
The general synthetic method described above afforded compound 12d as a yellow solid (36%),
1
mp 243-244 ◦C; HRMS (ESI): calcd for C29H29N5O8 576.5780, found 576.2073. H NMR (500 MHz,
DMSO)
2H), 5.05 (s, 2H), 4.03 (d, J = 17.9 Hz, 1H), 3.92 (d, J = 17.9 Hz, 1H), 3.36 (s, 4H), 2.59 (s, 4H), 2.36 (s, 3H),
2.08 (dt, J = 24.0, 7.0 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO)
170.45, 167.84,
δ 8.36 (s, 1H), 7.37 (s, 1H), 7.34 (s, 1H), 7.10 (s, 1H), 6.21 (d, J = 2.3 Hz, 2H), 5.42 (q, J = 16.8 Hz,
δ
157.51, 157.15, 151.99, 149.78, 149.17, 146.67, 146.48, 146.10, 130.94, 128.51, 126.09, 118.22, 104.60, 103.48,
103.11, 66.58, 53.86, 53.63, 45.17, 44.38, 42.33, 42.24, 30.70, 7.86.
(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (2-methylpiperidine-1-carbonyl)glycinate (12e)
The general synthetic method described above afforded compound 12e as a yellow solid (78%),
1
mp 240–242 ◦C; HRMS (ESI): calcd for C30H30N4O8 575.5900, found 575.2121. H NMR (500 MHz,
DMSO)
6.20 (d, J = 5.0 Hz, 2H), 5.41 (dd, J = 30.3, 16.6 Hz, 2H), 4.94 (dd, J = 22.9, 12.8 Hz, 2H), 4.07
(m, 1H), 3.97 3.85 (m, 1H), 3.71 (d, J = 14.6 Hz, 1H), 2.69 (dt, J = 26.3, 12.7 Hz, 1H), 2.14 2.01 (m, 2H),
1.87 1.79 (m, 1H), 1.53 1.30 (m, 5H), 0.98 (dd, J = 21.3, 6.8 Hz, 3H), 0.93
0.83 (m, 3H). 13C NMR
(125 MHz, DMSO) 172.97, 170.74, 157.28, 156.98, 151.79, 150.51, 150.40, 149.09, 147.02, 146.36, 130.61,
δ
8.28 (d, J = 3.2 Hz, 1H), 7.32 – 7.28 (m, 1H), 7.25 (d, J = 10.6 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H),
−
3.97
−
−
−
−
−
δ
128.86, 126.05, 118.53, 105.27, 103.56, 103.03, 96.34, 76.40, 72.86, 65.69, 55.37, 50.62, 45.60, 38.35, 30.68,
18.75, 15.74, 8.24.
(S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[30,40:6,7]indolizino[1,2-b]
quinolin-7-yl (thiomorpholine-4-carbonyl)glycinate (12f)
The general synthetic method described above afforded compound 12f as a yellow solid (70%),
mp > 250 ◦C; HRMS (ESI): calcd for C28H26N4O8S 579.5860, found 579.1544. 1H NMR (500 MHz,
DMSO)
d, J = 4.9 Hz, 2H), 5.46 (s, 2H), 5.17 (d, J = 9.6 Hz, 2H), 4.01 (dd, J = 17.7, 5.7 Hz, 1H), 3.89 (dd, J = 17.7
5.7 Hz, 1H), 3.59 (ddd, J = 12.9, 9.1, 4.3 Hz, 3H), 3.29 3.25 (m, 1H), 2.84 2.80 (m, 1H), 2.46 (t, J = 4.9 Hz
3H), 2.13 (tt, J = 14.2, 7.2 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, DMSO)
170.56, 167.66,
δ 8.43 (s, 1H), 7.47 (s, 1H), 7.42 (s, 1H), 7.17 (t, J = 5.8 Hz, 1H), 7.09 (d, J = 5.9 Hz, 1H), 6.27
(
,
−
−
,
δ
157.18, 156.97, 151.85, 150.23, 149.13, 146.94, 146.69, 145.65, 130.58, 128.78, 126.05, 118.42, 105.03, 103.55,
103.06, 95.20, 76.49, 66.74, 50.52, 46.54, 45.21, 42.70, 30.91, 26.55, 24.08, 7.97.
4.2. Biological Evalutation
4.2.1. Cell Culture
Human colorectal cancer cell lines HCT-8, gastric cancer cell line MGC80-3, leukemia cell
lines HL-60 and K562, pancreatic cancer cell line ASPC-1 and PNAC-1, hepatoma cell line HepG2,
neuroglioma cell line U87, neuroblastoma cell line SH-SY5Y, NSCLC cell line A549, and SCLC cell

Int. J. Mol. Sci. 2020, 21, 8495
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line NCI-H446 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences (Shanghai, China). NCI-H446, HCT-8, MGC80-3, and ASPC-1 were cultured in 1640 medium
(Gibco, Grand Island, NY, USA), which were supplemented with 10% FBS (Gibco, Grand Island, NY,
USA), additional 25 mg/L glucose and 1 mM sodium pyruvate (Solarbio, Beijing, China). HL-60
and HepG2 were maintained in 1640 supplemented with 10% FBS. A549 cells were maintained in
F12K medium (Gino, Hangzhou, China), which were supplemented with 10% FBS. K562 cells were
maintained in IMDM medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS. PNAC-1
cells were maintained in DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 10%
FBS. All cells were maintained in 5% CO2 at 37 ◦C and passaged every 2–4 days.
4.2.2. Cell Proliferation Assay
Cell proliferation was measured by the SRB (Sigma, St. Louis, MO, USA) or 3-(4,5-Dimethylthiazol-
2-yl)-2,5-Diphenyltetrazolium Bromide reagent (MTT, Solarbio, Beijing, China) assay. Cells were
seeded into 96-well plates, and treated with various concentrations of the indicated samples for 72 h.
MTT (10
dissolved and quantitated by spectrophotometry at a wavelength of 570 nm. Inhibition rate of each
sample was calculated from the A570 nm values as follows: Inhibition rate (%) = (A570 nm control
A570 nm sample)/A570 nm control 100%. For SRB assay, the cells are immobilized with TCA and
µ
L, 5 mg/mL) was added and incubated at 37 ◦C for another 4 h. The formazan product was
−
×
tinted with SRB stain. The SRB stain that bound to cells was dissolved and quantitatively analyzed at a
wavelength of 515 nm. The cytotoxicity of compounds was expressed as IC50. All experiments were
repeated at least three times.
4.2.3. Hoechst 33342 Staining
NCI-H446 cells and A549 cells were treated with 12e (0–100 Nm) for 24 h, then were stained with
Hoechst 33342 (5 µg/mL, Sigma, St. Louis, MO, USA). The nuclear morphology was taken photos
under fluorescence microscope.
4.2.4. Clonogenic Assay
NCI-H446 or A549 cells were seeded at 500 cells/well in 6-well plates, treated with various
concentrations of 12e for 14 days. Fixed by methanol at room temperature and stained with Giemsa
(Sigma, St. Louis, MO, USA) solution, colonies were defined as
counted and photographed.
≥
50 cells/colony. Finally, colonies were
4.2.5. Annexin V-FITC/PI Double-Staining Assay
The apoptosis was performed using the Annexin V-FITC/PI apoptosis detection kit
(Absin Bioscience Inc., Shanghai, China). Briefly, cells (3
105) were incubated with 12e (0–100 nM)
for 24 h, and then harvested by centrifugation, washed with ice-cold PBS twice, and resuspended
in binding buffer. Staining was started by adding Annexin V-FITC (5 L) and PI (5 L) followed by
×
µ
µ
incubation for 10 min at room temperature in the dark. Then, samples were immediately analyzed by
NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA).
4.2.6. Cell Cycle Analysis
NCI-H446 and A549 cells were incubated with 12e (0–100 nM) for 24h. After that, the cells were
collected and washed in PBS and fixed in ice-cold 70% (v/v) ethanol overnight at
was resuspended in PBS and stained with a mixture of RNase (10 g/mL, Solarbio, Beijing, China) and
PI (50 g/mL, Sigma, St. Louis, MO, USA) for 20 min in the dark. Cell cycle distribution analysis was
performed using a MoFlo XDP flow cytometry system (Beckman Coulter, Boulevard Brea, CA, USA).
−
20 ◦C. The cell pellet
µ
µ

Int. J. Mol. Sci. 2020, 21, 8495
15 of 18
4.2.7. Western Blotting
Cells were incubated with various concentrations of 12e for 24 h and then washed twice in cold
phosphate buffered saline (PBS). Cells were lysed with lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl,
1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.5% NP-40, 1 mM propidium iodide
(PI), 1mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)) and placed on ice for 1 h
with occasional vortex. Centrifugation followed at 10,000 rpm for 10 min and each cell lysate (50 µg) was
subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred
to nitrocellulose membranes (Pall, New York, NY, USA). Blots were blocked with 5% skim milk in TBST
for 1 h at room temperature, then incubated with indicated primary antibodies (Cleaved Caspase-3,
Cleaved Caspase-9, Cleaved PARP, Caspase-8, bax, Bcl-2, Mcl-1, survivin, γH2AX, stat3, p-stat3,
erk1/2, p-erk1/2, AKT, and p-AKT, 1:2000, Cell Signaling Technology, Danvers, MA, USA) overnight
at 4 ◦C, followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase-conjugated
immunoglobulin G (IgG) and visualized with enhanced chemiluminescence.
4.2.8. RT-PCR
Total RNA was extracted from cells treated with indicated concentrations of 12e for 24 h
using RNAiso Plus (TaKaRa, Tokyo, Japan). Total RNA (2
using 5 all in one RT mastermix (Abmgood, Zhenjiang, China) following the manufacturer’s
instructions. Individual reverse transcription reactions of 20 L were then diluted to 200
with ddH2O. 20 L of diluted RT reaction was used for real-time qPCR using the iTaq SYBR
µg per sample) was converted to cDNA
×
µ
µL
µ
Green Supermix with ROX (Bio-Rad, Hercules, CA, USA). The sequences of primers used in
real time qPCR reactions were as follows: 50-TGGCGTAAGATGATGGA-30 (survivin forward) and
50-TAGGGACGACGATGAAA-30 (survivin reverse), 50-AACACGTACTTGTGCG-30 (XIAP forward)
and 50-ACTTTGATCTGGCTCA-30 (XIAP reverse), 50-AAAGCCTGTCTGCCAAAT-30 (Mcl-1 forward)
and 50-TATAAACCCACCACTCCC-30 (Mcl-1 reverse), 50-GCCTTCTTTGAGTTCG-30 (Bcl-2 forward)
and 50-CAGCCTCCGTTATCC-30 (Bcl-2 reverse).
β-actin was used as an internal control. Triplicate
qPCR reactions were performed for each of the samples. The real-time qPCR condition is 95 ◦C for
2 min as a pre-denature step, followed by 40 PCR cycles at 95 ◦C for 10 s, 60 ◦C for 45 s and 72 ◦C for 30 s.
The data were analyzed using the Applied Biosystems 7300 Real Time PCR System (Thermo Scientific,
Waltham, MA, USA) and normalized to β-actin.
4.2.9. DNA Relaxation Assay
DNA relaxation assay was performed as described [40] with slight modification. In brief, a total
of 20 µL reaction containing pBR322 DNA (0.5 µg), Topo I (1 U), 10 × DNA Topo I Buffer (2 µL), 0.1%
◦
BSA (2
µ
L) with or without the compound was incubated at 37 C for 30 min. The reaction was
terminated by adding 2
Then gels were stained with 3
(Protein Simple, San Jose, CA, USA).
µ
L of DNA 10
×
loading buffer and subjected to electrophoresis in 1% agarose.
×
GelRedTM Nucleic Acid Gel Stain and visualized by FluorChem E
4.2.10. In Vivo Studies
Male BALB/c-nu mice (4 to 6 weeks of age) were purchased from Hunan SJA Laboratory Animal
Co., Ltd. (Changsha, China). All animal experiments were approved by the Institutional Animal
Care and Use Committee of Ocean University of China and according to standard institutional
guidelines. 2 × 106 NCI-H446 or A549 cells were injected subcutaneously into the right flank of nude
mice. The tumor volumes of these mice were checked to assess tumor growth. When the tumors
reached about 100 mm3, the tumor-bearing mice were randomly allocated to various experimental
group (n = 5, per group), and treated by intragastric administration with FL118, 12e, or vehicle.
The formulation for FL118 and 12e in this study used the formulation recipe which contains FL118 or

Int. J. Mol. Sci. 2020, 21, 8495
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12e (1 mg/mL), DMSO (5%), Tween-80 (4%), HS-15 (4%), and saline (87%). The corresponding vehicle
solution in the basic formulation recipe contains DMSO (5%), Tween-80 (4%), and HS-15 (4%) in saline.
The mice body weights were weighed three times a week. The lengths (L) and widths (W) of
tumors were measured three times a week using electronic calipers and the volumes (V) was calculated
as following formula: V = (L
×
W2)/2. When the tumor volume reached about 1000 mm3, tumors
were excised, weighed, ground, and then disrupted on ice for 30 min in loading buffer, and boiled for
10 min. Protein levels were analyzed by western blotting. The antitumor effect of the compounds was
determined by inhibition rate (IR), which calculated according to the following equation: IR (%) =
(tumor volume of control
d0) × 100%.
−
tumor volume of compound)/(tumor volume of control-tumor volume of
4.2.11. Data Analysis
One-way ANOVA with Tukey’s post hoc test was used for statistical analysis of the data, and
values were expressed as mean ± SD. Differences of p < 0.05 were considered statistically significant.
Author Contributions: Conceptualization, J.L.; methodology, X.Q.; software, X.Q.; validation, X.D., G.W., Y.Z.,
X.Z. and R.Y.; formal analysis, X.D. and G.W.; investigation, X.D., G.W., Y.Z., X.Z. and R.Y.; resources, J.L.; data
curation, X.Q.; writing—original draft preparation, X.D. and G.W.; writing—review and editing, J.L. and X.Q.;
visualization, X.D. and G.W.; supervision, J.L.; project administration, X.Q. and T.J.; funding acquisition, J.L.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China [No. 81673450],
National Natural Science Foundation of China-Shandong Joint Fund (U1906212, U1706213), the National Key R&D
Program of China (2019YFC0312504), National Science and Technology Major Project of China (2018ZX09735-004),
the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology
(Qingdao) (2018SDKJ0403). Shandong Provincial Natural Science Foundation (major basic research projects)
[ZR2019ZD18], Shandong Provincial Natural Science Foundation (PhD Fund Project) [ZR2018BH039].
Acknowledgments: The authors are grateful for financial support granted by the National Natural Science
Foundation of China [No. 81673450], National Natural Science Foundation of China-Shandong Joint Fund
(U1906212, U1706213), the National Key R&D Program of China (2019YFC0312504), National Science and
Technology Major Project of China (2018ZX09735-004), the Marine S&T Fund of Shandong Province for Pilot
National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0403). Shandong Provincial Natural
Science Foundation (major basic research projects) [ZR2019ZD18].
Conflicts of Interest: The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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