Design, synthesis, and cytotoxic evaluation of novel scopoletin derivatives

Article abstract of DOI:10.1111/cbdd.13120

A series of scopoletin derivatives were designed and synthesized by introducing α-aminoacetamide, acrylamide and β-aminopropamide, respectively, to 3-position of scopoletin, and their chemical structures were confirmed by ESI-MS, IR, ¹H NMR, an

Full text of DOI:10.1111/cbdd.13120

PROFESSOR LI CHEN (Orcid ID : 0000-0002-6211-7672)
Article type
: Research Letter
Design, synthesis and cytotoxic evaluation of novel scopoletin
derivatives
1
,†
1,†
1
2
1 *
1**
Wei Shi , Jinlu Zhang , Na Bao , Fuqin Guan , Li Chen , Jianbo Sun
1
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China.
Institute of Botany, Jiangsu Province and Chinese Academy of Science, Nanjing, 210014, China.
2
*Corresponding authors: Li Chen, chenliduo@sohu.com; Jianbo Sun, sjbcpu@gmail.com
†
Contributed to this work equally
Abstract: A series of scopoletin derivatives were designed and synthesized by introducing α-aminoacetamide,
acrylamide and β-aminopropamide respectively to 3-position of scopoletin and their chemical structures were
1
13
confirmed by ESI-MS, IR, H NMR and C NMR spectra. All target compounds were evaluated in vitro against four
human cancer cell lines (MDA-MB-231, MCF-7, HepG2 and A549) by MTT method. Cytotoxic assay showed that
compounds 7a, 7b, 7e, 7f, 8a and 8e exhibited more potent cytotoxicities compared to scopoletin. Besides, we
have further evaluated the growth inhibitory activities of these selected compounds against normal tissue cell
lines HFL-1. Although compound 8a showed the strongest anti-proliferative activity in vitro, it exhibited strong
cytotoxicity on normal cells HFL-1, which limited its further study. Compound 7a and 7b exhibited higher
anti-proliferative activity against MDA-MB-231 and HepG2 cells and weak cytotoxicity on HFL-1, which suggested
that 7a and 7b might be ideal anticancer candidates. The SARs showed that the introduction of the acrylamide
and its analogues β-aminopropamide could significantly improve activity, while the α-aminoacetamide failed to
enhance potency obviously. Therefore, the mechanism of compound 7a and 7b is worthy of further research and
the structure of compound 8a should be further optimized.
Key words: scopoletin, α-aminoacetamide, acrylamide, β-aminopropamide, cytotoxic activity
Coumarins, widely present in higher plants, animals and microbial metabolites, play a major role in agricultural
and pharmaceutical industries (1). Most of them and their derivatives possess a variety of biological activities
including anticancer, antioxidant, antidepressant and anti-inflammatory (2, 3).
Scopoletin (6-methoxy-7-hydroxycoumarin), a phenolic coumarin present in many medicinal plants, has been
proven to possess versatile pharmacological properties, such as anti-tumor, anti-inflammatory and hypouricemic
activities (4, 10, 11). In recent years, the mechanism of antitumor effects of scopoletin has been reported. It was
able to induce cell cycle arrest and trigger apoptosis via activation of caspase-3 and inhibition of the endothelial
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cell growth in human prostate tumor cells and leukemia cells (5-7). More important, scopoletin also has no
cytotoxicity against normal cells. However, its low antitumor activity in vitro and in vivo and a high elimination
rate in vivo limited its clinical application. Therefore, scopoletin is regarded as an ideal lead compound and our
group have designed and synthesized a great deal of derivatives (8, 9). Some scopoletin derivatives exhibited
higher antitumor activities in vitro and in vivo than scopoletin (Fig. 1). For example, those scopoletin derivatives
which cinnamic acid was introduced into 7-position of scopoletin or 3-position of 3-amino scopoletin were
screened for in vitro cytotoxic activity against five human tumor cell lines (MCF-7, MDA-MB-231, A549, HCT-116
and Hela) with doxorubicin as the positive control (8). Among them, compound SC-III3 (Fig. 1),
(
E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)acrylamide,
showed
high
potent
anticancer activity at low concentrations and had no cytotoxicity to normal liver cells in vitro and significantly
dose-independently suppressed tumor growth without causing the decreasing of the mean body weight of mice
in vivo (10).
Fig. 1
The mechanism of SC-III was that it could induce cytotoxicity in hepatocellular carcinoma through ROS mediated
3
DNA damage and ataxiatelangiectasia-mutated nuclear protein kinase activation (10). In addition, SC-III also led
3
to the autophagy of human hepatoma HepG2 cells through inducing mitochondrial dysfunction, depleting ATP
and activating AMPK-mTOR pathway (11). Further study of the structure-activity relationships (SARs) of these
active scopoletin derivatives revealed that almost all the compounds have α, β-unsaturated carbonyl group (the
Michael-acceptor), and as we mentioned above, a great number of studies reported that this group could interact
with nucleophilic residues in the body which suggested it might be a pharmacophore (12-19). For example,
irreversible acrylamide-based inhibitors, the Michael-acceptor compounds, could form covalent bonds with
cysteine or other nucleophilic residues in the protein to get the conjugate-addition products. The irreversible
acrylamide inhibitors have a lot of potential advantages including enhanced pharmacological potency and
selectivity and prolonged pharmacodynamics (17-19). However, the high reactivity of the acrylamide group may
cause rapid metabolism or nonspecific reaction with off targets (19). In order to solve this problem, a protection
strategy for the acrylamide would be beneficial. It is reported that the β-aminoethyl ketones could be
bioconverted through a β-elimination reaction to the α,β-unsaturated ketones, and then reacted with
nucleophiles, such as thiols of cysteines (20-22). According to this strategy, Carml et al (20) converted the
acrylamide to the β-aminopropamide which could undergo a β-elimination reaction to release the acrylamide,
and then reacted with thiols of cysteine residues in the intracellular environment (Fig. 2).
Fig. 2
Based on these findings and our previous researches, for one thing, in this paper we hypothesized that
introduction of acrylamide and β-aminopropamide to 3-position of scopoletin could obtain novel anticancer
reagents. For another, in order to compare the effects of different linker on the activity of scopoletin, the
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α-aminoacetamide was also introduced to 3-position of scopoletin. Therefore, a series of scopoletin derivatives
by introducing α-aminoacetamide, acrylamide and β-aminopropamide respectively to 3-position of scopoletin
were synthesized and screened for cytotoxicity four tumor cell line using scopoletin as a positive control (Fig. 3).
Then, the most potent cytotoxic compounds 7a, 7b, 7e, 7f, 8a and 8b were assayed for their anti-proliferative
activity indicated by IC50 values.
Fig. 3
Results and discussion
Chemistry
The synthetic pathway of target compounds 7a-7i was summarized in Scheme 1. According to the previous
reported method (23, 24), compound 2 was synthesized from 2,4,5-trimethoxybenzaldehyde 1 by treatment with
dry aluminum chloride in dry DCM and followed acid hydrolysis. Compound 2 was condensed with glycine in
acetic anhydride using sodium acetate as base. Burk and Allen’s one-pot protocol (25) was applied to convert
acetamide 3 to Boc-protected-3-amino-scopoletin 4. Compound 5 was synthesized from 4 and acetic anhydride
by treatment with DMAP and pyridine in dry DCM at room temperature. The intermediate 6 was afforded via
removing the Boc-group using 20% TFA/DCM. Compound 7a was synthesized in one-step reaction from 6 and
acryloyl chloride using trimethylamine as acid-binding agent, which was hydrolyzed by potassium carbonate in
methanol at reflux to get compound 7b. The next step was that 7b was reacted with various secondary amines in
CH CN/THF (v/v=3:1) at reflux to get the target compounds 7c-7i.
3
Scheme 1
The synthetic route of target compounds 8a-8i was outlined in Scheme 2. Compound 6 was treated with
chloroacetyl chloride in dry DCM at room temperature using trimethylamine as acid-binding agent to get
compound 8a, which was reacted with various secondary amines by treatment with anhydrous potassium
carbonate and potassium iodide in 2-butanone, and then hydrolyzed by potassium carbonate in methanol to give
the target compounds 8b-8i.
Scheme 2
Pharmacology
All target compounds 7a-7i and 8a-8i were preliminarily evaluated in vitro for their cytotoxicity to human breast
cancer cells MDA-MB-231 and MCF-7 and human liver cancer cells HepG2 respectively by using the standard MTT
method adopting scopoletin as a positive control. All data were given in Table 1. The results showed that several
compounds (the inhibition rate greater than 50%) exhibited strong growth inhibitory activities against the tested
cells, such as 7a, 7b, 7e, 7f, 8a and 8e.
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Table 1 Growth inhibition (%) obtained from the single dose (100 μM, 72h) test.
Growth inhibition (%)
Compd.
MDA-MB-231
93.55
94.64
2.77
HepG2
81.61
90.63
16.59
7.36
71.15
62.29
NE
MCF-7
83.75
89.25
23.05
19.49
89.91
91.36
7.35
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
14.16
46.03
54.91
NE
g
h
i
NE
NE
7.91
22.78
94.68
24.94
5.87
18.12
84.72
5.82
NE
12.85
87.52
19.26
12.92
8.61
a
b
c
d
e
f
25.58
57.21
11.96
8.72
NE
61.97
NE
78.84
2.67
g
h
i
19.33
23.21
34.81
3.67
12.28
18.21
74.31
2.63
28.26
NE
scopoletin
8.47
NE = no effect at 100 μM.
Compounds 7a, 7b, 7e, 7f, 8a and 8e were next assayed for their anti-proliferative activity against human breast
cancer cells MDA-MB-231, human liver cancer cells HepG2 and human lung cancer cells A549 indicated by IC50
values, which were summarized in Table 2. It was found that these compounds displayed significant
anti-proliferative activity compared to scopoletin. Compound 7a, 7b and 7f exhibited higher anti-proliferative
activity against MDA-MB-231 and HepG2 cells, while the compound 7e and 8e manifested lower activity against
three human tumor cells. Especially, compound 8a displayed the strongest anti-proliferative activity against most
human cancer lines with IC50 values ranging from 2.195 to 3.961 μM. Based on these results, we have further
assayed the growth inhibitory activities of six active compounds against the human normal tissue (human lung
fibroblasts) cell lines and the datum are summarized in Table 3. The results showed that 7a and 7b displayed
weak cytotoxicity on HFL-1, which suggested that 7a and 7b might be ideal anticancer candidates. Their
mechanism of anti-proliferative activity is worthy of further research. Compound 8a has a good activity in vitro
while it exhibits strong cytotoxicity on normal cells HFL-1, which has limited its further study. Considering this
reason, the structure of compound 8a should be further optimized in the future. From the SARs we may conclude
that the introduction of the acrylamide and its analogue β-aminopropamide can significantly improve cytotoxicity,
while the α-aminoacetamide fails to enhance potency obviously. This preliminary result indicates that
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β-aminopropamide may be converted into Michael receptor acrylamide to play biological activity. In addition, the
activity of chloroacetamide at 3-position of scopoletin was superior to the α-aminoacetamide at 3-position of
scopoletin. When the β-aminopropamide was introduced, the piperidine and 4-methylpiperidine analogues (7e
and 7f) exhibited good activity. At the same time, when the α-aminoacetamide was introduced, the piperazine
analogue (8e) showed good activity.
Table 2 IC50 values of compounds 7a, 7b, 7e, 7f, 8a and 8e as determind based on MTT assay
IC (μM)
50
Compd.
MDA-MB-231
HepG2
A549
>100
a
7
7
7
7
8
8
a
b
e
f
8.882±0.93
8.710±0.94
4.80±0.11
58.20±2.16
14.15±0.71
3.721±0.61
30.97±1.49
>100
21.96±2.29
25.46±1.42
8.579±0.48
2.195±0.21
71.86±2.45
>100
>100
>100
>100
a
e
3.961±0.89
41.16±1.73
>100
b
scopoletin
a
b
Values represent mean ± standard deviation for at least two separate experiments performed in triplicate.
Positive control drug.
Table 3 Cytotoxicities (IC , μM) of six active compounds on normal cell HFL-1
50
Compd.
HFL-1
Compd.
7f
HFL-1
a
7a
7b
7e
47.04±1.45
18.56±1.66
2.56±0.87
21.25±1.28
42.17±0.98
166.2±2.18
8a
8e
a
Values represent mean ± standard deviation for at least two separate experiments performed in triplicate.
Conclusion
In summary,
a series of scopoletin derivatives by introducing α-aminoacetamide, acrylamide and
β-aminopropamide respectively to 3-position of scopoletin were synthesized and tested their anti-proliferative
activity against four human cancer cell lines (MDA-MB-231, MCF-7, HepG2 and A549). Cytotoxic assay showed
that compounds 7a, 7b, 7e, 7f, 8a and 8e exhibited high potent cytotoxicities compared to scopoletin. Among
them, compound 7a and 7b exhibited higher anti-proliferative activity against MDA-MB-231 and HepG2 cells and
weak cytotoxicity on normal cells HFL-1, which suggested that 7a and 7b might be ideal anticancer candidates.
Compound 8a showed the strongest anti-proliferative activity in vitro while it exhibited strong cytotoxicity on
normal cells HFL-1, which had limited its further study. From the SARs we may conclude that the introduction of
the acrylamide and its analogue β-aminopropamide can significantly improve activity, while the
α-aminoacetamide fails to enhance potency obviously. Therefore, the mechanism of compound 7a and 7b is
worthy of further research and the structure of compound 8a should be further optimized.
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Acknowledgements
This research was financially supported by College Students Innovation Project for the R&D of Novel Drugs and
National Found for Fostering Talents of Basic Science (NO. J1310032) and Natural Science Foundation of Jiangsu
Province.
Conflict of Interest statement
The authors confirm that this article content has no conflict of interest.
References
1.
2.
3.
4.
5.
Emami S., Dadashpour S. (2015) Current developments of coumarin-based anti-cancer agents in medicinal
chemistry. Eur J Med Chem;102:611-630.
Sandhu S., Bansal Y., Silakari O., Bansal G. (2014) Coumarin hybrids as novel therapeutic agents. Bioorg Med
Chem;22:3806-3814.
Thakur A., Singla R., Jaitak V. (2015) Coumarins as anticancer agents: a review on synthetic strategies,
mechanism of action and SAR studies. Eur J Med Chem;101:476-495.
Gnonlonfin G.J.B., Sanni A., Brimer L. (2012) Review scopoletin - a coumarin phytoalexin with medicinal
properties. Crit Rev Plant Sci;31:47-56.
Kim E.K., Kwon K.B., Shin B.C., Seo E.A., Lee Y.R., Kim J.K., Park J.W., Park B.H., Ryu D.G. (2005) Scopoletin
induces apoptosis in human promyeloleukemic cells, accompanied by activations of nuclear factor κB and
caspase-3. Life Sci;77:824-836.
6
.
.
Li Y., Dai Y., Liu M., Pan R., Luo Y.B., Xia Y.F., Xia X.F. (2009) Scopoletin induces apoptosis of fibroblast-like
synoviocytes from adjuvant arthritis rats by a mitochondrial-dependent pathway. Drug Dev Res;70:378-385.
Pan R., Gao X.H., Li Y., Xia Y.F., Dai Y. (2009) Anti-arthritic effect of scopoletin, a coumarin compound
occurring in Erycibe obtusifolia Benth stems, is associated with decreased angiogenesis in synovium. Fundam
Clin Pharmacol;24:477-490.
7
8
9
1
.
Li L.H., Zhao P., Hu J.L., Liu J.H., Liu Y., Wang Z.Q., Xia Y.F., Dai Y., Chen L. (2015) Synthesis, in vitro and in vivo
antitumor activity of scopoletin-cinnamic acid hybrids. Eur J Med Chem;93:300-307.
Shi W., Hu J.L., Bao N., Li D.A., Chen L., Sun J.B. (2017) Design, synthesis and cytotoxic activities of
scopoletin-isoxazole and scopoletin-pyrazole hybrids. Bioorg Med Chem Lett;27:147-151.
.
0. Zhao P., Chen L., Li L.H., Wei Z.F., Tong B., Jia Y.G., Kong L.Y., Xia Y.F., Dai Y. (2014) SC-III3, a novel scopoletin
derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and
ataxiatelangiectasia-mutated nuclear protein kinase activation. BMC Cancer;14:987-999.
1. Zhao P., Dou Y.N., Chen L., Li L.H., Wei Z.F., Yu J.T., Wu X., Dai Y., Xia Y.F. (2015) SC-III3, a novel scopoletin
derivative, induces autophagy of human hepatoma HepG2 cells through AMPK/mTOR signaling pathway by
acting on mitochondria. Fitoterapia;104:31-40.
1
1
2. Jackson P.A., Widen J.C., Harki D.A., Brummond K.M. (2017) Covalent modifiers: a chemical perspective on
the reactivity of α,β-unsaturated carbonyls with thiols via Hetero-Michael addition reactions. J Med
Chem;60:839-885.
This article is protected by copyright. All rights reserved.

1
1
1
1
3. Engel J., Lategahn J., Rauh D. (2016) Hope and disappoint: covalent inhibitors to overcome drug resistance in
non-small cell lung cancer. Med Chem Lett;7:2-5.
4. Bauer R.A. (2015) Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and
designed therapies. Drug Discov Today;20:1061-1073.
5. Miller R.M., Paavilainen V.O., Krishnan S., Serafimova I.M., Taunton J. (2013) Electrophilic fragment-based
design of reversible covalent kinase inhibitors. J Am Chem Soc;135:5298-5301.
6. Krishnan S., Miller R.M., Tian B., Mullins R.D., Jacobson M.P., Taunton J. (2014) Design of reversible,
cysteine-targeted Michael acceptors guided by kinetic and computational analysis.
J Am Chem
Soc;136:12624-12630.
1
1
1
2
7. Liu Q.S., Sabnis Y., Zhao Z., Zhang T.H., Buhrlage S.J., Jones L.H., Gray N.S. (2013) Developing irreversible
inhibitors of the protein kinase cysteinome. Chem Biol;20:146-159.
8. Carmi C., Mor M., Petronini P.G., Alfieri R.R. (2012) Clinical perspectives for irreversible tyrosine kinase
inhibitors in cancer. Biochem Pharmacol;84:1388-1399.
9. Barf T., Kaptein A. (2012) Irreversible protein kinase inhibitors: balancing the benefits and risks. J Med
Chem;55:6243-6262.
0. Carmi C., Galvani E., Vacondio F., Rivara S., Lodola A., Russo S., Aiello S., Bordi F., Costantino G., Cavazzoni A.,
Alfieri R.R., Ardizzoni A., Petronini P.G., Mor M. (2012) Irreversible inhibition of epidermal growth factor
receptor activity by 3-aminopropanamides. J Med Chem;55:2251-2264.
2
1. Wenzel N.I., Chavain N., Wang Y.L., Friebolin W., Maes L., Pradines B., Lanzer M., Yardley V., Brun R., Mende
C.H., Biot C., Toth K., Charvet E.D. (2010) Antimalarial versus cytotoxic properties of dual drugs derived from
4
-aminoquinolines and Mannich bases: interaction with DNA. J Med Chem;53:3214-3226.
2. Han C., Wan L.D., Ji H.B., Ding K., Huang Z.J., Lai Y.S., Peng S.X., Zhang Y.H. (2014) Synthesis and evaluation of
-anilinopyrimidines bearing 3-aminopropamides as potential epidermal growth factor receptor inhibitors.
2
2
Eur J Med Chem;77:75-83.
2
2
2
3. Zhou J.P., Wang L., Wei L.J., Zheng Y., Zhang H.B., Wang Y.B., Cao P., Niu A., Wang J., Dai Y. (2012) Synthesis
and antitumor activity of acopoletin derivatives. Lett Drug Des Discov;9:397-401.
4. Liu W.K., Hua J., Zhou J.P., Zhang H.B., Zhu H.Y., Cheng Y.H., Gust R. (2012) Synthesis and in vitro antitumor
activity of novel scopoletin derivatives. Bioorg Med Chem Lett;22:5008-5012.
5. Burk M.J., Allen J.G. (1997) A mild amide to carbamate transformation. J Org Chem;62:7054-7057.
Supporting Information
1
Experimental section, ESI-MS and H NMR of synthetic compounds
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Figure Legends
Fig. 1. Structures of scopoletin and its derivatives
Fig. 2. The mechanism between acrylamide and β-aminopropamide
Fig. 3. Design of scopoletin-substituted analogues
Scheme 1. Synthesis of target compounds 7a-7i. Reagents and conditions: (a) AlCl3 (6eq), dry CH Cl , CTAB
2
2
(
0.002eq), reflux; (b) glycine (2eq), Ac O, AcONa (4eq), reflux, (c) Boc O (2eq), DMAP (0.2eq), THF, reflux; then
2 2
hydrazine hydrate (0.3eq), CH OH, rt; (d) Ac O (1.3eq), DMAP (0.2eq), pyridine (2.5eq), dry CH Cl , rt; (e)
3
2
2
2
trifluoroacetic acid, CH Cl , rt; (f) acryloyl chloride (1.5eq), Et N (2.5eq), dry CH Cl , rt; (g) K CO (1.1eq), CH OH,
2
2
3
2
2
2
3
3
reflux; (h) various secondary amines (2eq), CH CN/THF (v/v=3:1), reflux.
3
Scheme 2. Synthesis of target compounds 8a-8i. Reagents and conditions: (i) Et N (1.5eq), chloroacetyl chloride
3
(
1.3eq), dry CH Cl , rt; (b) various secondary amines (1.3eq), K CO (2.5eq), KI (0.1eq), 2-butanone, reflux; then
2 2 2 3
K CO (1.1eq), CH OH, reflux.
2
3
3
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