Design, synthesis and biological evaluation of novel 7H-benzo [c] [1, 3] dioxolo [4, 5-f] chromen-7-one derivatives with potential anti-tumor activity

Article abstract of DOI:10.1016/j.bioorg.2020.104381

In this study, a series of novel 7H-benzo [c] [1,3] dioxolo [4, 5-f] chromen-7-one derivatives were obtained by structural modification of the lead compounds with Fissitungfine B. A total 15 compounds were designed, synthesized and evaluated as inhibitors of tumor. These target compounds have the novel chemical structures that named three six-membered rings including one lactone six-membered ring. In vitro assay, the results showed that the target compounds have a broad spectrum and strong of anti-tumor activity. Such as the target compound 4n to MCF-7 was IC₅₀ = 0.35 ± 0.01 μM, to A-549 was IC₅₀ = 0.37 ± 0.01 μM, to Hela was IC₅₀ = 0.56 ± 0.02 μM, to MDC-803 was IC₅₀ = 0.53 ± 0.02 μM and COLO-205 was IC₅₀ = 0.50 ± 0.02 μM in vitro. At the same time, in vivo anti-tumor activity assay results showed that the target compounds had a good inhibitory effect on tumor growth. Among them, the target compound 4n had the best anti-tumor activity, it could inhibit tumor growth well at a low dose. The target compound 4n could be used as a candidate drug for further research and development, in order to be used as early as application in the clinical treatment of tumors.

Full text of DOI:10.1016/j.bioorg.2020.104381

Journal Pre-proofs
Design, synthesis and biological evaluation of novel 7H-benzo [c] [1, 3] diox-
olo [4, 5-f] chromen-7-one derivatives with potential anti-tumor activity
Shiyang Zhou, Gangliang Huang
PII:
S0045-2068(20)31679-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104381
YBIOO 104381
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
21 July 2020
11 September 2020
13 October 2020
Please cite this article as: S. Zhou, G. Huang, Design, synthesis and biological evaluation of novel 7H-benzo [c]
[1, 3] dioxolo [4, 5-f] chromen-7-one derivatives with potential anti-tumor activity, Bioorganic Chemistry (2020),
doi: https://doi.org/10.1016/j.bioorg.2020.104381
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Design, synthesis and biological evaluation of novel 7H-benzo [c] [1, 3] dioxolo [4,
5-f] chromen-7-one derivatives with potential anti-tumor activity
Shiyang Zhoua,b, Gangliang Huanga,*
aChongqing Key Laboratory of Green Synthesis and Application, Active Carbohydrate Research
Institute, College of Chemistry, Chongqing Normal University, Chongqing 401331, China
bKey Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, College of
Chemistry and Chemical Engineering, Hainan Normal University, Haikou, Hainan 571158, China
E-mail: huangdoctor226@163.com
Abstract: In this study, a series of novel 7H-benzo [c] [1, 3] dioxolo [4, 5-f] chromen-7-one
derivatives were obtained by structural modification of the lead compounds with Fissitungfine B.
A total 15 compounds were designed, synthesized and evaluated as inhibitors of tumor. These target
compounds have the novel chemical structures that named three six-membered rings including one
lactone six-membered ring. In vitro assay, the results showed that the target compounds have a
broad spectrum and strong of anti-tumor activity. Such as the target compound 4n to MCF-7 was
IC50=0.35 ± 0.01 μM, to A-549 was IC50=0.37 ± 0.01 μM, to Hela was IC50=0.56 ± 0.02 μM, to
MDC-803 was IC50=0.53 ± 0.02 μM and COLO-205 was IC50=0.50 ± 0.02 μM in vitro. At the same
time, in vivo anti-tumor activity assay results showed that the target compounds had a good
inhibitory effect on tumor growth. Among them, the target compound 4n had the best anti-tumor
activity, it could inhibit tumor growth well at a low dose. The target compound 4n could be used as
a candidate drug for further research and development, in order to be used as early as application in
the clinical treatment of tumors.
Keywords: Fissitungfine B; design; synthesis; anti-tumor activity
1. Introduction
Malignant tumor was a disease caused by abnormal mechanism of controlling cell growth and
proliferation (Birbrair, et al., 2014). It was a common disease that can seriously threaten human
health. According to the world health organization (WHO), three out of five people in the world die
of cancer, diabetes, cardiovascular disease and chronic respiratory diseases (Cunningham, et al.,
1

2011). In 2008, the cancer killed 7.6 million people, accounting for 13% of global deaths (Handa,
et al., 2011). More than 70% of cancer deaths occurred in low and middle income countries, more
than 13.2 million people were predicted to die of cancer by 2030. Anti-tumor drugs were a class of
drugs for the treatment of tumor diseases. Simply speaking, there were chemotherapy drugs and
biological agents (Katsurano, et al., 2012). In recent years, the development of molecular oncology
and molecular pharmacology has gradually clarified the nature of tumor. The invention and
application of large-scale rapid screening, combinatorial chemistry, genetic engineering and other
advanced technologies have accelerated the process of drug development (Vogelstein, et al., 2013).
The research and development of anti-tumor drugs has entered a new era. At present, drug therapy
has become one of the important means for the clinical treatment of cancer (Murphy, 2014). Due to
the high incidence and mortality of cancer, the sales of anti-tumor drugs were also increasing year
by year. After years of development, many important advances have been made in the research and
development of anti-tumor drugs (Eyers & Murphy, 2016). However, in the face of the most serious
threat to human life and health, solid tumors, which account for more than 90% of malignant tumors.
The still lack effective and specific drugs, which on the one hand reflects the difficulty in the
development of anti-tumor drugs (Linden, et al., 2013). On the other hand, it also means that the
development of anti-tumor drugs still needs the application of new ideas, new technologies and new
methods.
With the development of modern medicine, natural drugs have been gradually recognized and
developed as anti-tumor drugs. In recent years, there have been more and more researches on the
anti-tumor effect of calm drugs. The researches on the anti-tumor active ingredients and anti-tumor
mechanism of natural drugs have begun to take shape in different parts of the world (Cho, et al.,
2010). The discovery of many potent anticancer compounds in plants could be attributed directly or
indirectly to the history of plant anticancer in traditional medicine. The first plant-derived drugs to
enter the clinic were the alkaloids of vinblastine and vincristine. Catharanthus roseus has been used
to treat type 2 diabetes in many countries (Gangjee, et al., 2013). At first, the oral hypoglycemic
effect of Catharanthus roseus was mainly studied. Later, it was discovered by chance that
Catharanthus roseus could reduced white blood cell count and inhibit bone marrow regeneration in
mice. However, there have been adverse events related to the drug's ability to reduce white blood
cell count and prevent bone marrow regeneration, which may result in limited clinical use of the
2

drug. Another example of a more direct anticancer ingredient derived from traditional medicine was
the acetabulum toxin isolated from the root of the malus (Shi & Guang, 2018). Taxol was a natural
secondary metabolite isolated from the bark of gymnosperms of taxus cuspidate. In the biological
activity screening, it was found that the extract containing paclitaxel had high biological activity on
the inhibition of mouse tumor cells in vitro, further studies were conducted to isolate the anti-tumor
active ingredients.
As a Chinese endemic medicinal plant, only distributed in Hainan Island (Hu, et al., 2007), up
to now, there is only a preliminary investigation on the chemical composition of Fissistigma
tungfangense (Zhang, et al., 2007). Previous chemical studies of Fissistigma tungfangense have led
to the separation of a number of compounds (Hwangs et al., 2009), including oxymorphines,
aporphines, phenanthrenes, morphinandienones, flavonoids, protoberberines, and chalcones. Early
pharmacological studies have shown that these compounds have anti-tumor, anti-platelet
aggregation anti-inflammatory and immunosuppressive activities (Ge, et al., 2013). Fissitungfine A
and Fissitungfine B (Figure 1) were extracted from the tropical medicinal plant Fissistigma
tungfangense (Qi, et al., 2016). It was a natural product with strong anti-tumor activity, but its
content in plants was very low. On the basis of the structure of the lead compound Fissitungfine B,
a series of 7H-benzo [c] [1, 3] dioxolo [4, 5-f] chromen-7-one derivatives were designed and
synthesized by using the bioisostere and modification with alkyl groups (Figure 2 (a)). In terms of
spatial structure, the designed compounds has a similar spatial structure to Fissitungfine B. In terms
of drug structure-activity relationship (SAR), the anti-tumor activity (Hwangs et al., 2009; Hong C,
et al. 2019 ; Hong C, Y, et al. 2020) of the designed compounds may be better than that of the lead
compound Fissitungfine B. Because the substituents designed on the benzene ring could better bind
to the receptor, thus achieving a better effect of inhibiting the growth of tumor cells. In the process
of synthesis, target compounds were synthesized by acyl chloride reaction, esterification reaction
and heck reaction (Scheme 1). The synthetic route was characterized by simple operation and high
total yield. In the process of screening the anti-tumor activity in vitro and in vivo, the experimental
results showed that these compounds had a good anti-tumor activity. These compounds could be
used as the potential anti-tumor drug candidates and their value remains to be further studied.
3

NH
NH
H3CO
H3CO
O
O
O
O
O
O
O
O
Fissitungfine A
Fissitungfine B
IC50 (μM)
A-549
3.19
Compounds
Hela
MCF-7
2.92
MGC-803
3.01
COLO-205
3.21
Fissitungfine A
Fissitungfine B
2.30
4.01
5.21
6.31
4.98
6.70
Figure 1 The chemical structure of Fissitungfine A and B.
R2
NH
O
O
O
O
O
Design
O
O
Synthesis
O
O
R1
R1: -H, -NH2, -NO2
R2: -H, -CH2CH3, -CH(CH3)2, -COCH3
Fissitungfine B
Target compounds 4a-4o
(a)
The benzene ring 2
R2
O
Replace the site 2
R2:-H, -CH3, -CH2CH3,
O
-CH(CH3)2 and -COCH3
O
The mother
nucleus
O
structure
R1
The benzene ring 1
Replace the site 1
R1:-H, -NH2 and -NO2
(b)
Figure 2 The design and structure-activity relationship (SAR) of target compounds 4a-4o.
2. Results and discussion
2.1. Design and synthesis of the target compounds 4a-4o
Fissitungfine A and Fissitungfine B were extracted from the tropical medicinal plant
Fissistigma tungfangense. They were natural product that showed a broad spectrum and strong of
anti-tumor activity (MCF-7, A-549, Hela, MDC-803 and COLO-205), and IC50 were at the μM level
in vitro (Figure 1). In this study, Fissitungfine B was used as the lead compound to replace the
substituents on the benzene ring 1 and benzene ring 2 on the basis of retaining the basic skeleton
4

(Figure 2 and Figure 4). During the structural design of the target compounds, a total of 15 target
compounds 4a-4o were designed. In the design of the target compounds, we chose -H, -NH2 and -
NO2 as substituents for R1 on the benzene ring 1, the -H, -CH3, -CH2CH3, -CH(CH3)2 and -COCH3
as substituents for R2 on the benzene ring 2. This kind of drug design, which retains the parent
nucleus, provides certain guarantee for the success of new drug research and development, and also
shortens the cycle and reduces the cost of research and development. The structure was designed to
alter the physical and chemical properties of the target compounds, including logP and pKa and the
toxicity of the target compounds. The anti-tumor activity in vitro and in vivo were screened to obtain
the target compounds with a good anti-tumor activity and low toxicity. On this basis, the further
pharmacokinetic, clinical and other related studies were conducted. In the synthetic route design
process, we chose an efficient and simple synthetic route (Scheme 1). Using substituted benzoic
acid (compounds 1a-1c) as starting material, the target compounds 4a-4o were synthesized by acyl
chloride reaction, esterification reaction and heck reaction in three steps. The acyl chloride reaction
was conducted with substituted benzoic acid (compounds 1a-1c) as starting material. In this step,
the methylene chloride (CH2Cl2) as solvent, N, N’-dimethyl formamide (DMF) used as catalyst and
thionyl chloride (SOCl2) as chloride reagent. In the first step of reaction, it only takes 2 hours for
refluxing reaction to complete the acyl chloride reaction, and the acyl chloride reaction yield was
relatively high, up to more than 98%. The esterification reaction, the substitutedchlorobenzoyl
chloride (compounds 2a-2c) and substituted sesamol as reactants, methylene chloride (CH2Cl2) as
solvent. During the esterification reaction process, there was no need to add any catalyst and heat
the reaction. The esterification reaction could be completed at room temperature and stirring 2 hours.
In the process of esterification reaction, a good yield could also be obtained, and the yield of
intermediate 3a-3o were more than 95%. The heck reaction, tetrahydrofuran (THF) as solvent,
anhydrous potassium carbonate as acid applying agent and palladium acetate (Pd(OAc)2) used as
catalyst for the reaction. The heck reaction could be completed after 5 hours of reflux reaction of
intermediate 3a-3o. In the heck reaction, the yield of the synthesized target compounds 4a-4o
reached 74% to 88%. According to the design of synthesis route, we successfully synthesized the
target compounds 4a-4o. In the process of synthesis, the conditions of each steps of the reaction
were optimized, and the yield of the target compounds were a good under the optimal reaction
conditions. The synthesis route has few reaction steps and only three steps to complete the synthesis
5

of the target compounds. Each steps of the synthesis operation was relatively simple, the reaction
conditions was mild and the total yield was high.
R2
O
Cl
Cl
a
b
O
COCl
COOH
O
O
R1
R1
Cl
R1
1a-1c
2a-2c
3a-3o
R2
O
O
c
R1: -H, -NH2, -NO2
R2: -H, -CH2CH3, -CH(CH3)2, -COCH3
O
O
R1
4a-4o
Scheme 1 The synthetic route of target compounds 4a-4o. Reagents and conditions: (a) SOCl2,
CH2Cl2, DMF, reflux 2 hours; (b) substituted sesamol, CH2Cl2, stirring 2 hours; (c) THF, K2CO3,
Pd(OAc)2, reflux 5 hours.
2.2. Biological activities of the target compounds 4a-4o
2.2.1. Biological activity screening in vitro
Anti-tumor drugs was a class of drugs that could directly kill or inhibit the growth or
proliferation of tumor cells. The mechanisms include inhibition of nucleic acid or protein synthesis,
interference with macromolecular metabolism, interference with microtubule system and inhibition
of topoisomerase. In vitro tests were mainly used to screen candidate compounds, to preliminarily
understand the mechanism of action, the type and intensity of sensitive tumor, and to provide
references for subsequent in vivo tests. In vitro anti-tumor activity screening of the target compounds
4a-4o were performed by MTT method to determine the proliferation inhibitory activity on a variety
of human tumor cell lines. Determine the anti-tumor activity selectivity of the target compounds
among different tumor lines, thus providing a basis for in vivo nude mouse model experiment. In
the anti-tumor tests in vitro, human breast cancer cells (MCF-7), lung adenocarcinoma cells (A-
549), cervical cancer cells (Hela), gastric adenocarcinoma cells (MDC-803) and colon cancer cancer
6

cells (COLO-205) were used as the inhibitory targets. The human fibroblast (Hum. Fib.) cell line
was used as control for toxicity. The inhibitory activity was measured by half maximal inhibitory
concentration (IC50), it's commonly used as a measure of drug effectiveness. The adriamycin was
used as positive drug and dimethylsulfoxide (DMSO) was used as the blank control. The anti-tumor
in vitro results of biological activity tests were shown in Table 1. The data of this study were the
statistical software was versions SPSS13.0, the analyzed by linear regression and the results of data
analysis show a linear fit. In Table 1, the test results showed that target compounds 4d, 4i, 4m and
4n had better inhibitory effect on breast cancer cells (MCF-7), lung adenocarcinoma cells (A-549),
cervical cancer cells (Hela), gastric adenocarcinoma cells (MDC-803) and colon cancer cancer cells
(COLO-205). In addition to the target compounds 4d, 4i, 4m and 4n, other target compounds also
showed good anti-tumor activity in vitro, with IC50 at the level of one to tens of micromoles. Among
them, the inhibitory activity of the target compound 4n to breast cancer cells (MCF-7) was
IC50=0.35 ± 0.01 μM, to lung adenocarcinoma cells (A-549) was IC50=0.37 ± 0.01 μM, to cervical
cancer cells (Hela) was IC50=0.56 ± 0.02 μM, to gastric adenocarcinoma cells (MDC-803) was
IC50=0.53 ± 0.02 μM and to colon cancer cancer cells (COLO-205) was IC50=0.50 ± 0.02 μM. It
could be seen from the data in Table 1 that these target compounds had a broad spectrum and strong
of anti-tumor activity in vitro. At the same time, most of these compounds showed no obvious
cytotoxicity effects on human fibroblast, except compounds 4f and 4j (Table 1). By analyzing the
preliminary anti-tumor activity data in vitro, the target compounds 4d, 4i, 4m and 4n could be used
to evaluate the anti-tumor activity in vivo. According to the results of in vitro activity and
substituents (R1 and R2), the structure-activity relationship (SAR) of drugs could be established. At
the same time, the physical chemical constants (logp and pKa) of the target compounds were
determined (Table 1). From the data of physical and chemical constants, it could be seen that these
target compounds were fat-soluble compounds, which were conducive to the absorption of
compounds. Meanwhile, the pKa of the target compounds were 7.0-9.4, and its absorption site was
concentrated in the intestinal tract. These physical and chemical constants were helpful for further
study of pharmacokinetics.
7

Table 1 The target compounds 4a-4o of inhibitory activity in vitro.
Compounds
R1
R2
logp
pKa
IC50 (μM)a ± SD
Hum. Fib.b
MCF-7
A-549
Hela
MDC-803
COLO-205
4a
H
H
2.91
3.41
3.80
4.13
2.24
2.15
2.60
3.03
3.36
1.46
1.63
2.05
2.50
2.71
1.14
8.3
8.2
8.2
8.1
7.8
9.4
9.3
9.3
9.0
8.5
7.6
7.5
7.5
7.3
7.0
5.34 ± 0.28
2.57 ± 0.08
1.03 ± 0.04
0.61 ± 0.02
7.24 ± 0.36
8.13 ± 0.35
2.65 ± 0.09
0.89 ± 0.03
0.42 ± 0.02
12.36 ± 0.34 11.23 ± 0.31 10.23 ± 0.30 ＞100c
4b
H
CH3
4.31 ± 0.12
2.12 ± 0.06
0.74 ± 0.03
4.11 ± 0.12
2.01 ± 0.06
0.78 ± 0.03
3.59 ± 0.10
1.56 ± 0.05
0.60 ± 0.03
＞100
＞100
＞100
4c
H
CH2CH3
CH(CH3)2
COCH3
H
4d
H
4e
H
10.66 ± 0.36 14.33 ± 0.42 13.26 ± 0.40 12.04 ± 0.36 ＞100
4f
NH2
NH2
NH2
NH2
NH2
NO2
NO2
NO2
NO2
NO2
11.24 ± 0.62 17.63 ± 0.81 23.67 ± 0.92 21.09 ± 0.88 19.34 ± 0.86 76.32 ± 4.02
4g
CH3
5.67 ± 0.30
2.10 ± 0.08
0.81 ± 0.02
5.88 ± 0.34
1.69 ± 0.06
0.79 ± 0.02
8.56 ± 0.37
4.33 ± 0.11
1.10 ± 0.04
8.11 ± 0.31
4.13 ± 0.11
0.81 ± 0.04
7.55 ± 0.32
3.24 ± 0.19
0.90 ± 0.04
＞100
＞100
＞100
4h
CH2CH3
CH(CH3)2
COCH3
H
4i
4j
23.13 ± 0.92 35.27 ± 1.33 42.03 ± 1.89 40.37 ± 1.78 38.96 ± 1.76 94.26 ± 4.51
4k
3.06 ± 0.13
1.22 ± 0.05
0.62 ± 0.03
0.35 ± 0.01
4.78 ± 0.02
0.81 ± 0.03
Noned
3.84 ± 0.16
1.06 ± 0.04
0.44 ± 0.02
0.37 ± 0.01
5.00 ± 0.02
0.86 ± 0.03
None
5.34 ± 0.30
2.10 ± 0.08
0.79 ± 0.03
0.56 ± 0.02
7.24 ± 0.42
1.45 ± 0.08
None
5.04 ± 0.28
2.03 ± 0.08
0.88 ± 0.03
0.53 ± 0.02
7.01 ± 0.40
1.38 ± 0.08
None
4.80 ± 0.25
1.24 ± 0.05
0.74 ± 0.04
＞100
＞100
＞100
4l
CH3
4m
CH2CH3
CH(CH3)2
COCH3
4n
0.450 ± 0.02 ＞100
4o
6.57 ± 0.38
1.09 ± 0.07
None
＞100
＞100
None
Adriamycin
DMSO
——
——
——
——
——
——
——
——
a Data represent the mean values from at least eight independent experiments each in triplicate (n≥8).
b Human fibroblast (Hum. Fib.) cell line was used as control for toxicity.
c Without any noticeable toxicity against human fibroblast.
d None=No biological activity.
8

2.3. Anti-tumor in vivo
In vivo anti-tumor activity test was used to confirm the target compounds killing or inhibiting
effect on specific types of tumor cells, and to explore the dosage, route, frequency and cycle of the
target compounds pharmacological effect. The Kunming mice were subcutaneously inoculated with
sarcoma S180 and lung cancer H22 tumor strains according to the standard methods of anti-tumor
activity test in vivo. In vivo anti-tumor activity tested results were important indicators to evaluate
the efficacy of target compounds. The relative tumor proliferation rate T/C (%) was used as the
evaluation index, and the evaluation criteria were: T/C (%) >40 % was invalid, T/C (%) ≤40% and
P < 0.05 was considered effective by statistical analysis. In the process of screening for anti-tumor
activity in vitro, the target compounds 4d, 4i, 4m and 4n were screened for breast cancer cells
(MCF-7), lung adenocarcinoma cells (A-549), cervical cancer cells (Hela), gastric adenocarcinoma
cells (MDC-803) and colon cancer cancer cells (COLO-205) showed good inhibitory effect and a
broad spectrum anti-tumor activity. On the basis of anti-tumor activity in vitro, the anti-tumor
activity of target compounds 4d, 4i, 4m and 4n in vivo was further evaluated. In the process of in
vivo anti-tumor activity testing, the low, medium and high doses (10 mg. kg-1. d-1, 50 mg. kg-1. d-1
and 100 mg. kg-1. d-1) of the target compounds 4d, 4i, 4m and 4n were respectively used for relevant
tests (Figure 3). In vivo anti-tumor evaluation method, we chose to measure the change of tumor
volume after direct administration. The data of this study were the statistical software was versions
SPSS13.0, the analyzed by linear regression and the results of data analysis show a linear fit. In
Figure 3, the in vivo anti-tumor activity test data showed that the target compounds 4d, 4i, 4m and
4n had a good inhibitory effect on tumor growth in vivo. The low, medium and high doses (10 mg.
kg-1. d-1, 50 mg. kg-1. d-1 and 100 mg. kg-1. d-1) , the relative tumor proliferation rates T/C (%) of
these target compounds on sarcoma S180 and lung cancer H22 were both lower than 40%, which
was considered as effective tests. In Figure 3, it could be found that the compounds 4d, 4i, 4m and
4n showed a good inhibitory effect on tumor growth at low dose (Figure 3a, 10 mg .kg-1. d-1),
medium dose (Figure 3b, 50 mg. kg-1. d-1) and high dose (Figure 3c, 100 mg. kg-1. d-1). At the same
dose, the anti-tumor activity of the target compounds were comparable to or better than that of the
positive control (adriamycin). For example, the anti-tumor activity of the target compound 4n was
significantly higher than that of the positive control at the same dose. The target compounds 4d, 4i,
4m and 4n showed good activity in vivo anti-tumor of evaluation, which may be related to
9

itsphysical chemical constants (logp and pKa). The good physical and chemical constants were
conducive to the digestion and absorption of drugs, and improve the efficacy of drugs. In addition,
these target compounds may have better spatial structure and substituents, which was beneficial for
the target compounds to bind to receptor proteins better, thus showing good anti-tumor activity in
vivo. At the same time, we also investigated the weight changes of the target compound 4n in nude
mice (Figure 4). The results showed that there was little change in body weight between the two
models, suggesting that compound 4n was active and had relatively few side effects. Could be seen
from the test data in vivo, the significance of these target compounds should be further, particularly
as target compound 4n can be used as an anti-tumor drug candidate for mechanism and
pharmacokinetic study. It is also the job next to further more research direction, for an early into
clinical trials make necessary experimental basis and theoretical basis.
25
20
15
10
5
1.6
1.4
1.2
1
(b)
(a)
S180
H22
S180
H22
0.8
0.6
0.4
0.2
0
0
i
n
i
n
i
n
i
c
d
n
d
4
m
4
m
4
c
4
4
4
4
4
y
y
m
m
a
a
i
i
r
r
d
d
A
A
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
(c)
S180
H22
i
n
i
n
d
4
m
4
c
4
4
y
m
a
i
r
d
A
Figure 3 In vivo anti-tumor activity test. The data show the mean±SEM (n=8). (a): low dose (10
mg. kg-1. d-1); (b) medium dose (50 mg .kg-1. d-1); (c) high dose (100 mg. kg-1. d-1).
23
22
4n-S180
4n-H22
21
20
1 2 3 4 5 6 7 8 9 10
d
10

Figure 4 The body weight change of nude mice after compound 4n treatment.
2.4. Structure-activity relationship (SAR) of study
By combining the results of in vitro and in vivo anti-tumor activity tests and analyzing the
relationship between the structures of the new compounds. We could obtain some useful key
structural information characteristics that affect the anti-tumor activity of the target compounds. The
parent structure of the synthesized new compound remained unchanged and the sites of the
difference were different substituents on benzene ring 1 and benzene ring 2 (Figure 2 (b)). The
structure-activity relationship (SAR) of study were summarized as follows: (1) The anti-tumor
activity of the target compounds whose substituent group on benzene ring 1 was the electron-
absorbing group was better than that of the electron-donating group (-NO2 > -H > -NH2). (2) The
anti-tumor activity of the target compounds with the substituent of benzene ring 2 as the electron-
donor group was better than that of the electron-sucking group. (3) With the increase of carbon
chain the anti-tumor activity of the electron-donor group in benzene ring 2 increases continuously
(-H < -CH3 < -CH2CH3 < -CH(CH3)2). The study of SAR provides necessary theoretical guidance
for further structural optimization in the future. To provides guarantee for the rapid and convenient
acquisition of target compounds with better anti-tumor activity, lower toxic effects and better
pharmacokinetic properties.
2.5. Lethal dose 50% (LD50) of study
Acute toxicity refers to the toxic effect caused by a single (or multiple within 24 hours)
exposure of the organism (human or laboratory animals) to foreign compounds, or even death. In
toxicology, the lethal dose (LD50) was the dose of hazardous substances, toxic substances, or
ionizing radiation that can kill half of the test population. The lower the LD50 value was the stronger
the toxicity of the exogenous chemical. Conversely, the higher the LD50 value, the lower the toxicity.
Determination of LD50 to understand the toxicity intensity, nature and possible target organs of the
tested substance, to provide a basis for further toxicity test dose and toxicity observation index
selection, and according to LD50 toxicity grading. As could be seen from the LD50 test results in
Table 2, the acute toxicity of the target compounds were tested by intragastric administration and
the toxicity of the target compounds 4d, 4i, 4m and 4n were lower than that of the positive control
group (adriamycin). According to the classification of acute toxicity, this kind of target compounds
11

belongs to low toxicity compound, which meets the standard of new drug development. The results
of in vitro anti-tumor activity data and acute toxicity data showed that there was a certain linear
relationship between them. The better the anti-tumor activity of the target compound, the higher the
toxicity. This linear relationship was consistent with the basic characteristics of general anti-tumor
drugs, which was necessary to further study the mechanism of action of such drugs.
Table 2 The LD50 test result.
Compounds
Method of administration
LD50 ± SD (mg. kg-1)a
821.4 ± 4.2
4d
i.g.
i.g.
i.g.
i.g.
i.g.
4i
846.7 ± 4.6
4m
786.5 ± 3.7
4n
763.6 ± 3.3
Adriamycin
568.4 ± 2.3
a LD50 values are displayed as means±standard deviations, n≥8.
3. Conclusion
We reported that a novel series of 7H-benzo [c] [1, 3] dioxolo [4, 5-f] chromen-7-one
derivatives were designed, synthesized and evaluated as inhibitors of tumor. The Fissitungfine B as
the lead compound, using the principle of modifying by bioisostere formation and modification with
alkyl groups. In the process of synthesis, target compounds were synthesized by acyl chloride
reaction, esterification reaction and heck reaction. In vitro assay, the results showed that the target
compounds have a broad spectrum and strong of anti-tumor activity, such as the target compounds
4d, 4i, 4m and 4n, especially of target compound 4n to human breast cancer cells (MCF-7) was
IC50=0.35 ± 0.01 μM, to lung adenocarcinoma cells (A-549) was IC50=0.37 ± 0.01 μM, to cervical
cancer cells (Hela) was IC50=0.56 ± 0.02 μM, to gastric adenocarcinoma cells (MDC-803) was
IC50=0.53 ± 0.02 μM and to colon cancer cancer cells (COLO-205) was IC50=0.50 ± 0.02 μM. In
vivo anti-tumor activity test results showed that the target compounds 4d, 4i, 4m and 4n had a good
inhibitory effect on tumor growth. At the same time, the results of acute toxicity tests showed that
the target compounds 4d, 4i, 4m and 4n were of low toxicity. In general, the target compound 4n
could inhibit tumor growth well at a low dose and could be used as a candidate drug for further
research and development. At the same time, the results of SAR research and analysis indicated that
12

these target compounds have the potential of further structural optimization to find more effective
anti-tumor activity target compounds.
4. Experimental section
4.1. Chemistry section
Chemistry materials and general methods. The reagents (chemicals) were purchased and used
without further purification. The chemical reagent used was purchased from Aladdin (Shanghai),
with a purity ≥ 97%. Nuclear magnetic resonance (NMR) spectroscopy was performed using a
Bruker AMX-400 spectrometer (IS as TMS). The melting point (m.p.) was determined by SGW X-
4B micro melting point instrument. Mass spectroscopy was performed with an Agilent 6460 system.
HPLC analysis of all final biologically test compounds was carried out using an Agilent 1260 series
HPLC system. The purity was determined by reversed-phase HPLC and is ≥95% for all biologically
tested compounds.
4.1.1. A general method for synthesis of compounds 2a to 2c
The 2-chlorobenzoic acid (compound 1a, 15.66 g, 0.10 mol) was putted into 250 mL round
bottom flask, then 50 mL methylene chloride (CH2Cl2) as solvent and 5 drops of N, N’-dimethyl
formamide (DMF) as the catalyst for the reaction were added. The flask was placed in the magnetic
mixer, stirring until the reaction liquid became clarified. The thionyl chloride (18.75 mL, 0.20 mol)
was constantly dropped into the flask under stirring, at the same time the drop speed rate and reaction
temperature were controlled (≤25 ℃ and ≥ 20 min). While the reactants were added, the reaction
lasted 2 hours under refluxing. The methylene chloride and excessive thionyl chloride were removed
under vacuum. The mixture was dried ,which can get the crude product of 2-chlorobenzoyl chloride
(compound 2a). The crude product was atmospheric distillation, collect the product of 238 ℃, and
give pure product of 2-chlorobenzoyl chloride (compound 2a) as a colorless transparent liquid. The
general method was used to synthesis of compounds 2b to 2c and all were of colorless transparent
liquid.
The 2-chlorobenzoyl chloride (compound 2a, 12.66 mL, 0.10mol) was putted into 250 mL
round bottom flask, then 50 mL methylene chloride (CH2Cl2) as solvent for the reaction were added.
The flask was placed in the magnetic mixer (≤25 ℃), and a magnetic mixer was used to stir until
the reaction liquid became balanced. Dissolve the sesamol (13.81 g, 0.10 mol) in 20 mL of
13

methylene chloride (CH2Cl2) and constantly dropped into the flask under stirring, and the drop speed
rate and reaction temperature were controlled (≤25 ℃ and ≥20min). After the reactants were added,
the reaction needs stir for 2 hours. After the stirring reaction was completed, the solvent methylene
chloride (CH2Cl2) was removed by vacuum distillation. The mixture was dried, which could get the
crude product of benzo [d] [1, 3] dioxol-5-yl-2-chlorobenzoate (compound 3a). The crude product
was recrystallised with toluene, filtered, and dried in vacuum to give pure product of compound 3a
as a white powder. The general method was used to synthesis of compounds 3b to 3o and all were
of white powder.
4.1.3. A general method for synthesis of the target compounds 4a to 4o
The benzo [d] [1, 3] dioxol-5-yl-2-chlorobenzoate (compound 3a, 27.67 g, 0.10 mol) was
putted into 250 mL round bottom flask. Then 50 mL tetrahydrofuran (THF) as solvent, anhydrous
potassium carbonate (K2CO3, 6.89 g, 0.05 mol) as acid applying agent and 0.03% palladium acetate
(Pd(AcO)2) used as catalyst for the reaction were added. After the reactants were added, the reaction
lasted for 5 hours under refluxing. When the reflux reaction was completed, the mixture was filtered
at a high temperature, removing unreacted anhydrous potassium carbonate (K2CO3) and catalyst
palladium acetate (Pd(AcO)2), the filtrate was collected, cooled and white powder were precipitated.
The white powder was filtered and vacuum dried to obtain the crude product of 7H-benzo [c] [1, 3]
dioxolo [4, 5-f] chromen-7-one (compound 4a). The crude product compound 4a was recrystallised
from petroleum ether (boiling range 60-90℃), filtered, and vacuum dried to give the pure product
of compound 4a as white powder. The general method was used to synthesis of compounds 4b and
4o as all were of white powder.
1
compound 4a white powder, yield 84%, m.p. 168-170℃; H NMR (400MHz, DMSO-d6) δ: 6.26
(2H, d, J = 15.5 Hz, -CH2-), 6.98 (1H, d, J = 8.5 Hz, Ph-H), 7.13 (1H, d, J = 8.5 Hz, Ph-H), 7.40-
7.60 (2H, 7.45 (ddd, J = 8.1, 7.4, 1.4 Hz, Ph-H), 7.55 (ddd, J = 8.0, 7.4, 1.4 Hz, Ph-H)), 7.76 (1H,
ddd, J = 8.1, 1.4, 0.5 Hz, Ph-H), 8.30 (1H, ddd, J = 8.0, 1.4, 0.5 Hz, Ph-H); 13C NMR (100MHz,
DMSO-d6) δ: 107.8, 113.6, 117.3, 118.0, 127.0, 129.7, 132.7, 132.9, 139.0, 139.4, 149.4, 149.9,
155.2, 167.8; HR-ESI-MS m/z: calcd for C14H8O4｛［M+H］+｝240.0424, found 240.2145; Anal.
calcd for C14H8O4: C, 70.00; H, 3.36; O, 26.64; found: C, 70.02; H, 3.35; O, 26.63%.
1
compound 4b white powder, yield 83%, m.p. 165-167℃; H NMR (400MHz, DMSO-d6) δ: 2.35
(3H, s, -CH3), 6.26 (2H, d, J = 15.5 Hz, -CH2-), 6.72 (1H, s, Ph-H), 7.41 (1H, ddd, J = 8.1, 7.6, 1.3
14

Hz, Ph-H), 7.50-7.66 (2H, 7.53 (ddd, J = 8.2, 7.6, 1.4 Hz, Ph-H), 7.62 (ddd, J = 8.1, 1.4, 0.5 Hz, Ph-
H)), 7.69 (1H, ddd, J = 8.2, 1.3, 0.5 Hz, Ph-H); 13C NMR (100MHz, DMSO-d6) δ: 15.9, 101.6,
108.1, 111.8, 121.5, 122.8, 124.5, 126.8, 127.0, 132.4, 133.1, 144.6, 145.7, 162.1; HR-ESI-MS m/z:
+
calcd for C15H10O4｛［M+H］｝254.0573, found 254.2416; Anal. calcd for C15H10O4: C, 70.86; H,
3.96; O, 25.17; found: C, 70.85; H, 3.96; O, 25.18%.
1
compound 4c white powder, yield 81%, m.p. 163-165℃; H NMR (400MHz, DMSO-d6) δ: 1.16
(3H, s, -CH3), 2.83 (2H, q, J = 7.2 Hz, CH2-), 6.24 (2H, d, J = 15.5 Hz, CH2-), 6.43 (1H, s, Ph-H),
7.44 (1H, ddd, J = 8.1, 7.6, 1.3 Hz, Ph-H), 7.50-7.62 (2H, 7.53 (ddd, J = 8.2, 7.6, 1.4 Hz, Ph-H),
7.60 (ddd, J = 8.1, 1.4, 0.5 Hz, Ph-H)), 7.69 (1H, ddd, J = 8.2, 1.3, 0.5 Hz, Ph-H); 13C NMR
(100MHz, DMSO-d6) δ: 16.2, 21.8, 101.6, 109.8, 110.6, 121.4, 124.0, 125.4, 126.8, 126.9, 131.8,
133.1, 143.6, 144.2, 146.0, 161.5; HR-ESI-MS m/z: calcd for C16H12O4｛［M+H］+｝268.0732,
found 268.2689; Anal. calcd for C16H12O4: C, 71.64; H, 4.51; O, 23.86; found: C, 71.65; H, 4.51;
O, 23.84%.
1
compound 4d white powder, yield 80%, m.p. 160-162℃; H NMR (400MHz, DMSO-d6) δ: 1.16
(6H, s, -CH3), 3.31 (1H, d, J = 6.9 Hz, -CH-), 6.22 (2H, d, J = 15.5 Hz, -CH2-), 6.29 (1H, s, Ph-H),
7.44 (1H, ddd, J = 8.1, 7.6, 1.3 Hz, Ph-H), 7.50-7.63 (2H, 7.53 (ddd, J = 8.2, 7.6, 1.4 Hz,Ph-H),
7.61 (ddd, J = 8.1, 1.4, 0.5 Hz, Ph-H)), 7.67 (1H, ddd, J = 8.2, 1.3, 0.5 Hz, Ph-H); 13C NMR
(100MHz, DMSO-d6) δ: 22.9, 27.4, 101.6, 109.3, 110.4, 121.6, 125.4, 128.7, 128.8, 132.5, 133.1,
143.8, 145.0, 147.1, 161.6; HR-ESI-MS m/z: calcd for C17H14O4｛［M+H］+｝282.0898, found
282.2954; Anal. calcd for C17H14O4: C, 72.33; H, 5.00; O, 22.67; found: C, 72.35; H, 5.00; O,
22.65%.
1
compound 4e white powder, yield 78%, m.p. 166-168℃; H NMR (400MHz, DMSO-d6) δ: 2.32
(3H, s, -CH3), 6.43 (2H, d, J = 12.5 Hz, -CH2-), 7.49-7.60 (2H, 7.51 (ddd, J = 7.9, 7.4, 1.3 Hz, Ph-
H), 7.58 (ddd, J = 7.7, 7.4, 1.4 Hz, Ph-H)), 7.65 (1H, ddd, J = 7.9, 1.4, 0.5 Hz, Ph-H), 7.74 (1H, s,
Ph-H), 7.98 (1H, ddd, J = 7.7, 1.3, 0.5 Hz, Ph-H); 13C NMR (100MHz, DMSO-d6) δ: 30.9, 101.6,
110.2, 110.4, 120.7, 122.1, 125.5, 126.7, 126.8, 132.7, 133.1, 144.0, 146.2, 150.6, 162.0, 201.3;
HR-ESI-MS m/z: calcd for C16H10O5｛［M+H］+｝282.0525, found 282.2513; Anal. calcd for
C16H10O5: C, 68.09; H, 3.57; O, 28.34; found: C, 68.07; H, 3.58; O, 28.35%.
1
compound 4f white powder, yield 88%, m.p. 156-158℃; H NMR (400MHz, DMSO-d6) δ: 6.12
(2H, d, J = 15.5 Hz, -CH2-), 6.64 (1H, dd, J = 8.5, 1.2 Hz, Ph-H), 6.81 (1H, d, J = 8.5 Hz, Ph-H),
15

7.12 (1H, d, J = 8.5 Hz, Ph-H), 7.56-7.61 (2H, 7.58 (dd, J = 1.2, 0.4 Hz, Ph-H), 7.60 (dd, J = 8.5,
0.4 Hz, Ph-H)); 13C NMR (100MHz, DMSO-d6) δ: 101.9, 104.8, 106.6, 112.5, 114.9, 118.3, 130.3,
136.3, 143.9, 144.0, 148.4, 150.4, 162.0; HR-ESI-MS m/z: calcd for C14H9NO4 ｛［M+H ］+ ｝
255.0536, found 255.2296; Anal. calcd for C14H9NO4: C, 65.88; H, 3.55; N, 5.49; O, 25.07; found:
C, 65.86; H, 3.57; N, 5.47; O, 25.09%.
1
compound 4g white powder, yield 85%, m.p. 154-156℃; H NMR (400MHz, DMSO-d6) δ: 2.31
(3H, s, -CH3), 6.13 (2H, d, J = 15.5 Hz, -CH2-), 6.54 (1H, dd, J = 8.5, 1.2 Hz, Ph-H), 6.64 (1H, s,
Ph-H), 7.53 (1H, dd, J = 1.2, 0.4 Hz, Ph-H), 7.60 (1H, dd, J = 8.5, 0.4 Hz, Ph-H); 13C NMR
(100MHz, DMSO-d6) δ: 15.9, 101.6, 104.8, 107.3, 111.8, 115.0, 119.1, 123.0, 130.3, 135.2, 144.2,
144.8, 145.2, 150.4, 161.4; HR-ESI-MS m/z: calcd for C15H11NO4｛［M+H］+｝269.0683, found
269.2568; Anal. calcd for C15H11NO4: C, 66.91; H, 4.12; N, 5.20; O, 23.77; found: C, 66.90; H,
4.13; N, 5.21; O, 23.76%.
compound 4h white powder, yield 83%, m.p. 150-152℃; 1H NMR (400MHz, DMSO) δ: 1.15 (3H,
s, -CH3), 2.87 (2H, q, J = 7.2 Hz, -CH2-), 6.15 (2H, d, J = 15.5 Hz, -CH2-), 6.42 (1H, s, Ph-H), 6.54
(1H, dd, J = 8.5, 1.2 Hz, Ph-H), 7.56 (1H, dd, J = 1.2, 0.4 Hz, Ph-H), 7.63 (1H, dd, J = 8.5, 0.4 Hz,
Ph-H); 13C NMR (100MHz, DMSO) δ: 16.2, 21.8, 101.6, 104.7, 108.8, 110.6, 115.0, 118.7, 124.5,
130.3, 134.7, 140.5, 143.7, 145.3, 150.5, 161.4; HR-ESI-MS m/z: calcd for C16H13NO4｛［M+H］
+ ｝283.0841, found 283.2836; Anal. calcd for C16H13NO4: C, 67.84; H, 4.63; N, 4.94; O, 22.59;
found: C, 67.83; H, 4.64; N, 4.95; O, 22.58%.
1
compound 4i white powder, yield 80%, m.p. 148-150℃; H NMR (400MHz, DMSO-d6) δ: 1.17
(6H, s, -CH3), 3.16 (1H, d, J = 6.9 Hz, -CH-), 6.13 (2H, d, J = 15.5 Hz, -CH2-), 6.25 (1H, s, Ph-H),
6.54 (1H, dd, J = 8.5, 1.2 Hz, Ph-H), 7.52-7.61 (2H, 7.53 (dd, J = 1.2, 0.4 Hz, Ph-H), 7.60 (dd, J =
8.5, 0.4 Hz, Ph-H)); 13C NMR (100MHz, DMSO-d6) δ: 22.9, 27.4, 101.6, 104.9, 108.6, 109.4, 115.0,
119.1, 129.2, 130.3, 135.3, 143.2, 143.5, 146.7, 150.4, 161.5; HR-ESI-MS m/z: calcd for C17H15NO4
｛［M+H］+｝297.1007, found 299.1065; Anal. calcd for C17H15NO4: C, 68.68; H, 5.09; N, 4.71;
O, 21.53; found: C, 68.67; H, 5.09; N, 4.73; O, 21.52%.
1
compound 4j white powder, yield 77%, m.p. 162-164℃; H NMR (400MHz, DMSO-d6) δ: 2.32
(3H, s, -CH3), 6.34 (2H, d, J = 12.5 Hz, -CH2-), 6.60 (1H, dd, J = 8.5, 1.2 Hz, Ph-H), 7.55 (1H,
dd, J = 8.5, 0.4 Hz, Ph-H), 7.65 (1H, s, Ph-H), 7.71 (1H, dd, J = 1.2, 0.4 Hz, Ph-H); 13C NMR
(100MHz, DMSO-d6) δ: 30.9, 101.6, 105.7, 110.5, 115.0, 118.9, 120.6, 130.3, 135.5, 143.9, 146.1,
16

150.0, 150.4, 162.0, 201.3; HR-ESI-MS m/z: calcd for C16H11NO5｛［M+H］+｝297.0633, found
297.2661; Anal. calcd for C16H11NO5: C, 64.65; H, 3.73; N, 4.71; O, 26.91; found: C, 64.64; H,
3.76; N, 4.70; O, 26.90%.
1
compound 4k white powder, yield 80%, m.p. 182-184℃; H NMR (400MHz, DMSO-d6) δ: 6.31
(2H, d, J = 15.5 Hz, -CH2-), 7.02 (1H, d, J = 8.6 Hz, Ph-H), 7.12 (1H, d, J = 8.6 Hz, Ph-H), 7.99
(1H, dd, J = 8.5, 0.5 Hz, Ph-H), 8.06 (1H, dd, J = 8.5, 1.8 Hz, Ph-H), 8.36 (1H, dd, J = 1.8, 0.5 Hz,
Ph-H); 13C NMR (100MHz, DMSO-d6) δ: 101.9, 107.7, 111.6, 112.2, 119.4, 124.7, 127.5, 129.3,
135.9, 143.5, 144.5, 149.3, 151.0 161.1; HR-ESI-MS m/z: calcd for C14H7NO6 ｛［M+H］+ ｝
285.0271, found 285.2113; Anal. calcd for C14H7NO6: C, 58.96; H, 2.47; N, 4.91; O, 33.66; found:
C, 58.97; H, 2.46; N, 4.90; O, 33.67%.
1
compound 4l white powder, yield 79%, m.p. 180-182℃; H NMR (400MHz, DMSO-d6) δ: 2.35
(3H, s, -CH3), 6.31 (2H, d, J = 15.5 Hz, -CH2-), 6.75 (1H, s, Ph-H), 7.99-8.07 (2H, 8.00 (dd, J = 8.5,
0.5 Hz, Ph-H), 8.06 (dd, J = 8.5, 1.8 Hz, Ph-H)), 8.32 (1H, dd, J = 1.8, 0.5 Hz, Ph-H); 13C NMR
(100MHz, DMSO-d6) δ: 15.9, 101.6, 107.7, 111.8, 119.3, 123.0, 124.7, 128.2, 129.3, 134.9, 143.7,
144.7, 146.0, 151.2, 159.9; HR-ESI-MS m/z: calcd for C15H9NO6｛［M+H］+｝299.0434, found
299.2382; Anal. calcd for C15H9NO6: C, 60.21; H, 3.03; N, 4.68; O, 32.08; found: C, 60.20; H, 3.04;
N, 4.67; O, 32.09%.
compound 4m white powder, yield 77%, m.p. 179-181℃; 1H NMR (400MHz, DMSO-d6) δ: 1.19
(3H, t, J = 7.2 Hz, -CH3), 2.82 (2H, q, J = 7.2 Hz, -CH2-), 6.33 (2H, d, J = 15.5 Hz, -CH2-), 6.49
(1H, s, Ph-H), 7.99-8.07 (2H, 8.00 (dd, J = 8.5, 0.5 Hz, Ph-H), 8.06 (dd, J = 8.5, 1.8 Hz, Ph-H)),
8.32 (1H, dd, J = 1.8, 0.5 Hz, Ph-H); 13C NMR (100MHz, DMSO-d6) δ: 16.3, 21.8, 101.6, 109.3,
110.6, 119.4, 123.7, 124.7, 127.8, 129.3, 134.4, 143.7, 144.2, 147.2, 151.2, 159.9; HR-ESI-MS m/z:
calcd for C16H11NO6｛［M+H］+｝313.0587, found 313.2653; Anal. calcd for C16H11NO6: C,
61.35; H, 3.54; N, 4.47; O, 30.64; found: C, 61.34; H, 3.53; N, 4.48; O, 30.63%.
1
compound 4n white powder, yield 75%, m.p. 177-179℃; H NMR (400MHz, DMSO-d6) δ: 1.21
(6H, s, -CH3), 3.27 (1H, d, J = 7.0 Hz, -CH-), 6.30 (2H, d, J = 15.5 Hz, -CH2-), 6.46 (1H, s, Ph-H),
7.99-8.07 (2H, 8.00 (dd, J = 8.5, 0.5 Hz, Ph-H), 8.06 (dd, J = 8.5, 1.8 Hz, Ph-H)), 8.32 (1H, dd, J =
1.8, 0.5 Hz, Ph-H); 13C NMR (100MHz, DMSO-d6) δ: 22.9, 27.3, 101.6, 109.4, 110.2, 119.5, 124.7,
126.2, 129.2, 123.0, 135.0, 143.5, 144.5, 147.4, 151.2, 160.1; HR-ESI-MS m/z: calcd for C17H13NO6
｛［M+H］+｝327.0746, found 327.2921; Anal. calcd for C17H13NO6: C, 62.39; H, 4.00; N, 4.28;
17

O, 29.33; found: C, 62.38; H, 4.01; N, 4.27; O, 29.34%.
1
compound 4o white powder, yield 74%, m.p. 191-193℃; H NMR (400MHz, DMSO-d6) δ: 1.21
(6H, s, -CH3), 3.27 (1H, d, J = 7.0 Hz, -CH-), 6.30 (2H, d, J = 15.5 Hz, -CH2-), 6.46 (1H, s, Ph-H),
7.99-8.07 (2H, 8.00 (dd, J = 8.5, 0.5 Hz, Ph-H), 8.06 (dd, J = 8.5, 1.8 Hz, Ph-H)), 8.32 (1H, dd, J =
1.8, 0.5 Hz, Ph-H); 13C NMR (100MHz, DMSO-d6) δ: 30.9, 101.6, 109.9, 110.5, 119.3, 120.6, 124.7,
126.3, 129.3, 135.2, 144.0, 146.3, 150.2, 151.2, 161.1, 200.9; HR-ESI-MS m/z: calcd for C16H9NO7
｛［M+H］+｝327.0375, found 327.2482; Anal. calcd for C16H9NO7: C, 58.72; H, 2.77; N, 4.28;
O, 34.22; found: C, 58.71; H, 2.78; N, 4.29; O, 34.21%.
4.2. Biological activities section
4.2.1. Biological activity screening in vitro
Cells in logarithmic phase were collected (human breast cancer cells (MCF-7), lung
adenocarcinoma cells (A-549), cervical cancer cells (Hela), gastric adenocarcinoma cells (MDC-
803) and colon cancer cancer cells (COLO-205) and cell suspension concentration was adjusted.
Then inoculated into 96-well plates, 200 µL for each hole and cells density was adjusted to 104 holes
by paving plates. The culture plates were placed at 37 ℃ in 5% CO2 incubator to make the cells
stick to the wall for 24 hours. The prepared concentration gradients of the samples to be tested were
repeated 6 times for each gradient. The 20 µL was taken to each hole of the experimental group,
and a blank group and a control group were set for further cultivation for 48 hours. When the culture
was completed, the supernatant was carefully aspirated, the 80 µL fresh RPMI 1640 was added,
then the 20 µL MTT solution (5 mg.ml-1, 0.5% MTT) was added and the incubation for 4 hours.
The culture solution was centrifuged and then the supernatant was sucked off 150 µL of dimethyl
sulfoxide (DMSO) was added into each hole, and low-speed oscillations were set on the shaker for
10 min to fully dissolve the crystals. The optical density (OD) of each hole were measured by
enzyme linked immunosorbent assay (ELISA) at 490 nm. At the same time, the zeroing holes
(medium, MTT, DMSO) were set and the control holes (cells, the same concentration of drug
solvent, culture medium, MTT, DMSO) were set, and each group was set to have 3 multiple holes.
The data processing: The cell inhibition rate was calculated by formula 1-1. The data of this study
were analyzed by linear regression, and the statistical software was versions SPSS13.0.
18

ODcontrol group -ODspecimen
ODcontrol group
………………………………1-1
Cellinhibtion rate＝
100%
4.2.2. Animals
The Kunming type nude mice weighing approximately 200-250 g at the beginning of the
experimental procedure were used. Four rats were housed per cage (size 26×41 cm2); animals were
fed with standard laboratory diet and tap water ad libitum, kept at 23±1 °C with a 12 h light/dark
cycle, light at 7 a.m. All animal procedures were performed in accordance with the Guidelines for
Care and Use of Laboratory Animals of Chongqing Normal University and approved by the Animal
Ethics Committee of Chongqing Normal University.
4.2.3. Biological activity evaluation in vivo
According to the standard method of anti-tumor activity test in vivo, the Kunming type nude mice
were selected. The logarithmic growth phase of tumor cells (sarcoma S180 and lung cancer H22
tumor strains) was taken and cell suspensions were prepared for subcutaneous inoculation. The
animals were randomly assigned to the drug after the tumor grew to 100-300 mm3. There were three
dose treatment groups of 10 mg. kg-1. d-1, 50 mg. kg-1. d-1 and 100 mg. kg-1. d-1, a positive control
group (50 mg. kg-1. d-1) and a blank control group, and take it orally once a day for 10 days. The
anti-tumor effect of the sample was observed dynamically by measuring tumor diameter. The
measurement frequency of tumor diameter depends on the growth of transplanted tumor, usually
2-3 times per week. The trial will also look at indicators related to drug safety, such as animal weight
gain and mortality, and comparing these data between the treatment group and the standard
treatment control group is important for determining drug safety and development prospects.The
relative tumor proliferation rate (T/C, %) was used as the evaluation index of anti-tumor activity in
vivo. The relevant formulas were shown in equations 1-2, 1-3 and 1-4. The data of this study were
analyzed by linear regression, and the statistical software was versions SPSS13.0.
TRTV
………………………………………………………………1-2
T/C（%）
100%
CRTV
TRTV: The treatment group RTV.
CRTV: The blank control group RTV.
19

1

6
Tumor volume，V  ab2or abc
……………………………………1-3
2
a: Length.
b: Width.
c: Height.
V
t
………………………………………………1-4
Relative tumor volume，RTV 
V0
V0：Tumor volume was measured during general administration.
Vt：Tumor volume at each measurement.
4.2.4. Acute toxicity test
The Kunming mice were kept in the laboratory animal room for observation for 3-5 days, so
that they could adapt to the environment. After it was proved that they were healthy animals, they
were randomly divided into half males and half females, and the weight of the mice was 18-22 g.
Mice were fasted on an empty stomach for 16 hours, during the time, drinking water could be
properly fed. Intragastric administration was used, and the gavage volume of each dose group and
positive control group were the same, with a volume of 0.40 mL (20 mL. kg-1). The tested
compounds was prepared into different dose groups with dimethylsulfoxide (DMSO) as solvent,
adriamycin as positive control group. After oral administration of the subject, the subject shall be
observed for 7 or 14 days. If death continues on the 4th day after administration, the patient shall be
observed for 14 days, or extended to 28 days if necessary. Record the number of deaths, refer to the
table to obtain lethal dose 50% (LD50), and record the time of death and poisoning manifest.
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The Project was Sponsored by Postgraduate Research and Innovation Project of Hainan
Normal University (Hsyx2018-8)，and Open Foundation Project of Key Laboratory of Tropical
Medicinal Resource Chemistry of Ministry of Education (RDZH2019002). The work was also
supported by Scientific Research Foundation for the Returned Overseas Chinese Scholars, State
20

Education Ministry (No. 2015-1098), Chongqing Key Research Project of Basic Science & Frontier
Technology (No. cstc2017jcyjBX0012), Foundation Project of Chongqing Normal University (No.
14XYY020), Chongqing General Research Program of Basic Research and Frontier Technology
(No. cstc2015jcyjA10054), and Chongqing Normal University Postgraduate's Research and
Innovation Project (No. YKC17004), China.
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Highlights
1. A series of derivatives were obtained by structural modification of Fissitungfine B.
2. The target compounds had a good inhibitory effect on tumor growth.
3. The target compound 4n could be used as a candidate drug.
Graphical abstract
22

R2
O
NH
O
O
O
O
O
Design
O
Synthesis
O
O
R1
R1: -H, -NH2, -NO2
R2: -H, -CH2CH3, -CH(CH3)2, -COCH3
Fissitungfine B
Target compounds 4a-4o
Declaration of interests
☑ The authors declare that they have no known competing financialinterestsor personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
23