Design, synthesis and cytotoxic activities of scopoletin-isoxazole and scopoletin-pyrazole hybrids

Article abstract of DOI:10.1016/j.bmcl.2016.11.089

12 novel scopoletin-isoxazole and scopoletin-pyrazole hybrids were designed, synthesized and their chemical structures were confirmed by HR-MS, IR,¹H NMR and¹³C NMR spectra. The anticancer activities of the newly synthesized compounds were evaluated in vitro against three human cancer cell lines including HCT-116, Hun7 and SW620 by MTT assay. The screening results showed that six compounds (9a, 9c, 9d, 12a, 18b and 18d) exhibited potent cytotoxic activities with IC₅₀values below 20?μM. Besides, we have further evaluated the growth inhibitory activities of six compounds against the human normal tissue cell lines HFL-1. Especially, compound 9d displayed significant anti-proliferative activity with IC₅₀values ranging from 8.76?μM to 9.83?μM and weak cytotoxicity with IC₅₀value of 90.9?μM on normal cells HFL-1, which suggested that isoxazole-based hybrids of scopoletin were an effective chemical modification to improve the anticancer activity of scopoletin.

Full text of DOI:10.1016/j.bmcl.2016.11.089

Accepted Manuscript
Design, synthesis and cytotoxic activities of scopoletin-isoxazole and scopole-
tin-pyrazole hybrids
Wei Shi, Jinglin Hu, Na Bao, Dongang Li, Li Chen, Jianbo Sun
PII:
S0960-894X(16)31262-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.089
BMCL 24484
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
18 October 2016
28 November 2016
30 November 2016
Please cite this article as: Shi, W., Hu, J., Bao, N., Li, D., Chen, L., Sun, J., Design, synthesis and cytotoxic activities
of scopoletin-isoxazole and scopoletin-pyrazole hybrids, Bioorganic & Medicinal Chemistry Letters (2016), doi:
http://dx.doi.org/10.1016/j.bmcl.2016.11.089
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Design, synthesis and cytotoxic activities of scopoletin-isoxazole
and scopoletin-pyrazole hybrids
Wei Shi, Jinglin Hu, Na Bao, Dongang Li, Li Chen *, Jianbo Sun **
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009,
China.
*Corresponding author: Li Chen
Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang
Nanjing 210009, P. R. China
Tel: +86 25 83271447
E-mail address: chenli627@cpu.edu.cn
**Corresponding author: Jianbo Sun
Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang
Nanjing 210009, P. R. China
Tel: +86 25 83271415
E-mail address: sjbcpu@gmail.com

ABSTRACT
12 novel scopoletin-isoxazole and scopoletin-pyrazole hybrids were designed, synthesized and
1
their chemical structures were confirmed by HR-MS, IR, H NMR and 13C NMR spectra. The
anticancer activities of the newly synthesized compounds were evaluated in vitro against three
human cancer cell lines including HCT-116, Hun7 and SW620 by MTT assay. The screening
results showed that six compounds (9a, 9c, 9d, 12a, 18b and 18d) exhibited potent cytotoxic
activities with IC50 values below 20 µM. Besides, we have further evaluated the growth inhibitory
activities of six compounds against the human normal tissue cell lines HFL-1. Especially,
compound 9d displayed significant anti-proliferative activity with IC50 values ranging from 8.76
µM to 9.83 µM and weak cytotoxicity with IC50 value of 90.9 µM on normal cells HFL-1, which
suggested that isoxazole-based hybrids of scopoletin were an effective chemical modification to
improve the anticancer activity of scopoletin.
Key words: scopoletin, pyrazole, isoxazole, cytotoxic activity

Coumarins, including naturally occurring compounds and versatile synthetic scaffolds, play
an important role in pharmaceutical industries.1 Most of coumarins and their derivatives display a
broad range of biological and pharmacological activities, such as anticancer, anti-inflammatory,
antioxidant, antidepressant, anti-HIV, etc.2 Among these, their anticancer effect was extensively
examined, and a large number of studies showed that coumarins target a series of pathways in
cancer pathogenesis, such as kinase inhibition, cell cycle arrest, angiogenesis inhibition, Hsp90
inhibition, etc.2-10 For example, novobiocin (Fig. 1), a coumarin glycosides and the first Hsp90
C-terminal inhibitors, could induce degradation of Hsp90 client proteins. However, its anticancer
activity is relatively weak which requires further structural modification to provide more potent
compounds.4 Based on these findings, Blagg et al had designed and synthesized a series of
novobiocin derivatives which showed potent cytotoxic activities on several different cancer cells.
Based on the structure-activity relationships, the amide aromatic side chain was introduced to
3-position of novobiocin which should exhibit hydrophobic character. In addition, the noviose
sugar at 7-position could be replaced with simplified mimics such as alkyl amines which could
simultaneously maintain or increase solubility and activity.5-10
Encouraged by these successes, we recently have focused on the design and synthesis of
another coumarin (scopoletin) derivatives. Scopoletin (6-methoxy-7-hydroxycoumarin) (Fig. 1) is
a phenolic coumarin isolated from many plants.11 Previous studies indicated that scopoletin
possessed anti-tumor and anti-angiogenesis activities by inducing cell cycle arrest and promoting
apoptosis in human prostate tumor cells and leukemia cell line through activation of caspase-3 and
inhibition of the endothelial cell growth.12-15 Besides, scopoletin also has no cytotoxicity to normal
cells. However, its low activity, poor bioavailability and metabolically labile in vivo have limited

its clinical application. Therefore, scopoletin is being an ideal lead compound and a great number
of derivatives with diverse chemical structures have been designed and synthesized. Hitherto,
from the previous structure-activity relationships (SARs), we found that the structural
modification sites of scopoletin were mostly focused on 3-position and 7-position. When the
aromatic hydrophobic groups were introduced to 3-position or 7-position of scopoletin, these
scopoletin derivatives’ anticancer activities were improved than before. Besides, these groups
should have certain steric bulk because these positions could exist big hydrophobic pockets.16-18
Based on these successes, our group previously reported dozens of novel scopoletin derivatives
(Fig. 1) which cinnamic acid was introduced into 7-position of scopoletin or 3-position of 3-amino
scopoletin and some of them showed higher anticancer activities in vitro and in vivo than
scopoletin.19, 20
Fig. 1. Structures of novobiocin derivatives and scopoletin derivatives
Considering that heterocyclic structure, bearing atoms of at least two different elements as a
member of its ring, is the core skeleton of a lot of natural products and synthetic anticancer drugs.
Especially, pyrazole and isoxazole (Fig. 2), the class of five membered heterocycles, contain two

double bonds and two heteroatoms (N and O) at adjacent positions and have higher efficiency and
lower toxicity which have drawn considerable attention of the researchers in the past few
decades.21-24 Harish Kumar et al.25 and Kamalneet Kaur et al.26 provided comprehensive reviews
concerning these two important heterocycles and their derivatives in anticancer research. In these
reviews, we surprised to notice that many natural and synthetic compounds’ anticancer activities
could be improved when pyrazole and isoxazole were introduced, which suggested that the
pyrazole and isoxazole could be active pharmacophore. Based on these former findings and the
previous SARs, in this paper we hypothesized that introduction of pyrazole and isoxazole
respectively to scopoletin to form scopoletin-pyrazole and scopoletin-isoxazole hybrids may be
promising to obtain novel anticancer agents. According to our group previous research, the
pyrazole and isoxazole were introduced to 7-position of scopoletin by esters. In addition, to further
enhance the activity and solubility of scopoletin, two hydrophilic heterocycles morpholine and
isoxazole were introduced respectively to 7-position and 3-position of scopoletin (Fig. 2). All
target compounds were screened for their cytotoxicities against three human tumor cell lines using
sunitinib as a positive control and assayed for their anti-proliferative activity indicated by IC50
values.
Fig. 2. Design of scopoletin-substituted pyrazole or isoxazole hybrids

The synthetic route of target compounds 9a-9d is outlined in Scheme 1. According to the
previous synthetic method,16-17 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 treated with malonic acid in pyridine at room temperature using aniline as catalyst affording
compound 3, which was then decarboxylated by refluxing in pyridine to give scopoletin 4.
Compounds 6a-6d were synthesized in one-step reaction from ethyl acetoacetate and various
substituted benzoyl chloride by treatment with dry magnesium chloride and pyridine as catalyst in
dry DCM, which were cyclized with hydroxylamine hydrochloride in methanol and then followed
alkaline hydrolysis to get the intermediates 8a-8d. Finally, intermediates 8a-8d were treated with
scopoletin 4 in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) and
4-dimethyl-aminopyridine (DMAP) in dry DCM to obtain target hybrids 9a-9d.
Scheme 1. Synthesis of target compounds 9a-9d. Reagents and conditions: (a) AlCl3, CH2Cl2,
CTAB, reflux; (b) Malonic acid, Aniline, pyridine; (c) pyridine, reflux; (d) various substituted
benzoyl chloride, MgCl2, CH2Cl2, pyridine; (e) Hydroxylamine hydrochloride, ethanol, reflux; (f)
NaOH, MeOH/THF/H2O(1:1:3); (g) EDCI, DMAP, CH2Cl2.
The synthetic pathway of target compounds 12a-12d as shown in Scheme 2 was started from
6a-6d. Compounds 6a-6d were treated with hydrazine hydrate in ethanol at reflux affording

compounds 10a-10d, which were then hydrolyzed with borontribromide in dry DCM at -10℃ to
get the intermediates 11a-11d. Compounds 12a-12d were synthesized by the coupling reaction of
scopoletin 4 and intermediates 11a-11d in the presence of EDCI and DMAP in dry DCM at room
temperature.
Scheme 2. Synthesis of target compounds 12a-12d. Reagents and conditions: (h) Hydrazine
hydrate, aceticacid, reflux; (i) BBr3, CH2Cl2, -10℃; (j) EDCI, DMAP, CH2Cl2.
The synthesis of compounds 18a-18d is summarized in Scheme 3. Compound 2 was
condensed with glycine in acetic anhydride using sodium acetate as base. Burk and Allen’s
one-pot protocol27 was applied to convert the acetamide 13 to Boc-protected 3-amino-scopoletin
15. Compound 16 was synthesized from 15 and 1-(2-chloroethyl)morpholine by treatment with
anhydrous potassium carbonate and potassium iodide in 2-butanone at reflux. The key
intermediate 17 was afforded after removing the Boc-group using 20% TFA/DCM. The next step
was that 17 and 8a-8d were condensed using EDCI and DMAP in dry DCM at room temperature
to obtain target hybrids 18a-18d.

Scheme 3. Synthesis of target compounds 18a-18d. Reagents and conditions: (k) glycine, Ac2O,
AcONa, reflux; (l) Boc2O, THF, DMAP, reflux; (m) Hydrazine hydrate, MeOH; (n)
1-(2-chloroethyl)morpholine, K2CO3, KI, 2-butanone, reflux; (o) Trifluoroacetic acid, CH2Cl2; (p)
EDCI, DMAP, CH2Cl2.
All target compounds and scopoletin were evaluated for growth inhibitory activities against
human colorectal carcinoma cell lines HCT-116 and SW620, and human hepatoma cell line Huh7
respectively via using the standard MTT method adopting sunitinib as a positive control. The
concentrations of the scopoletin derivatives with 50% inhibition (IC50) on the cells were
determined, and the results are summarized in Table 1. The results showed that compounds 9a, 9c,
9d, 12a, 18b and 18d displayed significant enhanced anti-proliferative activity compared to
scopoletin and compounds 9d and 18d possess similar growth inhibition compared to sunitinib.
Especially, compound 9d displayed significant anti-proliferative activity against most human
cancer lines with IC50 values ranging from 8.76 to 9.83 µM. Based on these results, we have
further evaluated the growth inhibitory activities of six compounds (9a, 9c, 9d, 12a, 18b and 18d)
against the human normal tissue (human lung fibroblasts) cell lines HFL-1 and the datum are
summarized in Table 2. The result showed that compound 9d displayed weak cytotoxicity with
IC50 value of 90.9 µM on HFL-1, which suggested 9d may be an ideal anticancer candidate with
weak toxicity on normal cells or tissues. From the SARs we may conclude that the introduction of

the isoxazole can improve cytotoxic activity, while the pyrazole fail to enhance potency. In
addition, isoxazole at 7-position of scopoletin showed higher potency cytotoxicity than at
3-position of scopoletin. Electron withdrawing group (Cl and NO2) at 4-position of phenyl ring
will increase activity also. Thus, structure modification of scopoletin can greatly increase its
antitumor activity.
Table 1 Cytotoxicities (IC50, µM) of all target compounds.
Compd.
9a
HCT-116
10.9
>25
Huh7
12.4
>25
>25
8.76
16.2
>25
>25
>25
>25
14.7
>25
12.1
>25
3.03
SW620
12.7
>25
9b
9c
22.7
9.21
13.8
>25
23.4
9.83
17.1
>25
9d
12a
12b
12c
>25
>25
12d
>25
>25
18a
>25
>25
18b
15.6
20.9
10.3
>25
13.9
>25
18c
18d
10.5
>25
Scopoletin
Sunitinib
5.62
6.25
Table 2
9a
42.17
Cytotoxicities (IC50, µM) of six compounds on normal cell HFL-1
Compd.
9c
9d
12a
18b
18d
HFL-1
69.32
90.9
47.04
68.52
74.2
In summary, we synthesized a series of scopoletin-pyrazole and scopoletin-isoxazole hybrids
and tested their anti-proliferative activity against three cancer cell lines (HCT-116, Huh7 and

SW620). Cytotoxic assay indicated that compounds 9a, 9c, 9d, 12a, 12b and 12d showed potent
cytotoxicities compared to scopoletin. Especially, compound 9d displayed the similar
anti-proliferative activity with sunitinib in vitro with IC50 values of 9.21 µM on HCT-116 cells,
8.76 µM on Huh7 cells and 9.83µM on SW620 cells, respectively. Besides, compound 9d showed
weak cytotoxicity with IC50 value of 90.9 µM on normal cells HFL-1. From the SARs we may
conclude that the introduction of the isoxazole at 7-position or 3-position of the scopoletin can
enhance cytotoxicity activities. Furthermore, using election withdrawing group (Cl and NO2) at
4-position of phenyl ring will also increase activities. Based on these results, compound 9d can be
considered as a ideal candidate for further research and its mechanism of anti-proliferative activity
is in progress.
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 (NFFTBS).
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Graphical Abstract
12 novel scopoletin-pyrazole and scopoletin-isoxazole hybrids have been designed
and synthesized. The in vitro anti-tumor activity screening results showed that the
isoxazole-based hybrids of scopoletin are an effective chemical modification to
improve the anticancer activity of scopoletin.

Highlights
• 12 novel scopoletin-isoxazole and scopoletin-pyrazole hybrids have been designed
and synthesized.
• Their anti-tumor activities against HCT-116, Huh7 and SW 620 cells in vitro were
evaluated.
• Compound 9d displayed significant anti-proliferative activity on tumor cells and
weak cytotoxicity on normal cells.