Design, synthesis and biological evaluation of esculetin derivatives as anti-tumour agents

Article abstract of DOI:10.1039/c5ra06070b

Esculetin, a naturally catecholic coumarin, possess multiple pharmacological activities including anti-tumour, anti-inflammatory and anti-oxidant. However, the extensive phase II metabolism and rapid elimination from the human body significantly hinder esculetin and its derivatives as drug leads/candidates. To improve both the metabolic stability and the anti-tumour activity of esculetin via rational modification, a series of C-4 and C-8 substituted derivatives were designed and synthesized by perchloric acid catalysed von Pechmann reaction and Mannich reaction, respectively. The in vitro metabolic half-life in human liver S9 and anti-tumour activities in A549 and B16 cell lines of the newly synthesized compounds were assayed. Of these compounds, 8-(pyrrolidin-1-ylmethyl)-4-trifluoromethyl esculetin 15 was the most potent candidate compound, with almost a 20-fold increase in antiproliferative activity and a 3-fold prolonged half-life in human liver S9 compared with the parent compound 1. In addition, the potential structure-activity relationship and structure-metabolic stability relationship were discussed. Notably, the introduction of a nitrogen containing group as a hydrogen bond acceptor at the C-8 position of esculetin can improve both the metabolic stability and anti-tumour activity. All of these findings are very helpful for the structural modification of esculetin and other bioactive phenolic compounds to improve their phase II metabolic stability and bioactivity synchronously.

Full text of DOI:10.1039/c5ra06070b

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: P. Wang, Y. Xia, Y.
Yu, J. Lu, L. Zou, L. Feng, G. Ge and L. Yang, RSC Adv., 2015, DOI: 10.1039/C5RA06070B.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

View Article Online
DOI: 10.1039/C5RA06070B
Page 1 of 8
RSC Advances
RSC Advances
RSCPublishing
ARTICLE
Design, synthesis and biological evaluation of
esculetin derivatives as anti-tumour agents
Cite this: DOI: 10.1039/x0xx00000x
Ping Wang
Guang-Bo Ge
‡
, Yang-Liu Xia
‡, Yang Yu, Jun-Xia Lu, Li-Wei Zou, Lei Feng,
*
and Ling Yang
*
Received 00th January 2012,
Accepted 00th January 2012
Esculetin, a naturally catecholic coumarin, possess multiple pharmacological activities
including anti-tumour, anti-inflammatory and anti-oxidant. However, the extensive phase II
metabolism and rapid elimination from human body significantly hinder esculetin and its
derivatives as drug leads/candidates. To improve both the metabolic stability and the anti-
tumour activity of esculetin via rational modification, a series of C-4 and C-8 substituted
derivatives were designed and synthesized by perchloric acid catalysed von Pechmann reaction
and Mannich reaction, respectively. The in vitro metabolic half-life in human liver S9 and anti-
tumour activities in A549 and B16 cell lines of the newly synthesized compounds were
assayed. Of these compounds, 8-(pyrrolidin-1-ylmethyl)-4-trifluoromethyl esculetin 15 was the
most potent candidate compound, with almost a 20-fold increase in antiproliferative activity
DOI: 10.1039/x0xx00000x
www.rsc.org/
and a 3-fold prolonged half-life in human liver S9 compared with the parent compound 1. In
addition, the potential structure-activity relationship and structure-metabolic stability
relationship were discussed. Notably, the introduction of a nitrogen containing group as
hydrogen bond acceptor at the C-8 position of esculetin can improve both the metabolic
stability and anti-tumour activity. All of these findings are very helpful for the structural
modification of esculetin and other bioactive phenolic compounds to improve their phase II
metabolic stability and bioactivity synchronously.
and catecholic coumarins showed better anti-tumor activity than
Introduction
1
1,12
monohydroxy coumarins in several human tumour cell lines
.
Coumarins are a large class of natural and synthetic compounds that
have drawn much attention by their remarkable biological activities,
and several good druglikeness properties including excellent safety,
Esculetin (6,7-dihydroxycoumarin), naturally occurring
a
catecholic coumarin, widely distributed in many medicinal plants
such as Artemisia capillaries, Citrus limonia and Euphorbia
1
-5
high solubility and good permeability
.
Currently, several
1
3
lathyris . Esculetin and its derivatives have been reported to inhibit
the proliferation of a number of human malignant cell lines in vitro
and have demonstrated activity against several types of animal
6
7
8
coumarins including warfarin , daphnetin and armillarisin A are
clinically used drugs. Studies found that the existence of phenolic
groups and their position on the coumarin skeleton are very
1
4-17
tumours
. Moreover, it also enhances taxol-induced apoptosis in
9
,10
important for its anti-tumour activities . The structure-activity
relationships of various phenolic coumarins have revealed that the
catechol group is the key pharmacophore for anti-tumour activities,
1
8
human hepatocellular carcinoma cells (HepG2) . In addition,
esculetin has various bioactivities including lipoxygenase-
1
9
20
21
inhibition , free radical scavenging , DNA protection and cancer
2
2
chemoprevention . Hence, these cytotoxic coumarins represent an
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

View Article Online
DOI: 10.1039/C5RA06070B
RSC Advances
Page 2 of 8
ARTICLE
Journal Name
exploitable source of new anticancer agents, which might also help obtained by refluxing chloride 5 in the mixture of DMF and water
addressing side-toxicity and resistance phenomena. Although the with the yield of 83%.
pharmacological activities of esculetin and its analogs are recognized,
the extensive phase II metabolism and rapid elimination from human
body significantly hinder the development of these compounds as
2
3,24
drug leads/candidates
.
From the view of chemical structure, the catechol group is
not only the pharmacophore of coumarins but also the
susceptible group for the attack of phase II metabolic enzymes.
According to our previous study, the 7-OH of esculetin was the
major site for UGT metabolism, whereas 6-OH could hardly be
Scheme 1 Synthesis of compounds 2 ~ 6. Reagents and conditions:
a) HClO , rt, 6 ~ 8 h; (b) DMF/H O (1:3), reflux, 20 h.
(
4
2
2
5
glucuronidated . In addition, it has been presented earlier that
coumarin Mannich bases play a significant role as biologically
The structure of another series of derivatives combines the
biological active group of 6,7-dihydroxycoumarin and the amine
moiety as a new template. The synthesis of the coumarin derivatives
2
6,27
active compounds in various diseases
. Therefore, taking
into account of previous data, to improve both the metabolic
stability and the anti-tumour activities, in the present paper we
devoted our efforts to design and synthesize some 4-substituted
esculetins and 8-substituted esculetin Mannich bases as
possible anti-tumour agents examining their ability to inhibit
proliferation and their metabolic half-life in vitro. Furthermore,
the preliminary conclusions regarding the structure-activity
relationship of the metabolic stability and the anti-tumour
properties of esculetin derivatives were discussed, which can
provide helpful guidance for the structural modification of 6,7-
dihydroxycoumarins and other bioactive phenolic compounds.
8
~ 15 was accomplished according to the Mannich reaction as
depicted in Scheme 2. Examples of hydroxycoumarin condensed
with
formaldehyde
and amines
have
been
reported
26,27,33,34
previously
. The synthesis of compound 13 was given in a
35
previous publication . We have included them in our study in order
to explore the potential structure-activity relationships, within these
Mannich bases. In our case, esculetin/4-substituted esculetin,
formaldehyde and the corresponding secondary amine in 1:1.5:1.3
molar ratio were stirred in absolute ethanol or methanol at 25 ~ 60
o
C for 8 ~ 12 h to yield the desired Mannich bases derivatives. The
reactions were monitored by thin-layer chromatography. Esculetin
condensed with formaldehyde and amines to compound 8 ~ 11 with
the low yield and the poor regioselectivity. For example, esculetin
reacted with formaldehyde and 4-hydroxy piperidine to yield
compound 8 and compound 9 with the yield of 12% and 3%,
respectively. The structure of compound 8 and 9 (Fig. 1) was
Results and discussion
Chemistry
A sulphuric acid or perchloric acid catalysed von Pechmann reaction
is a common method to prepare 4-substituted coumarins in a
1
13
2
8,29
confirmed by H NMR, C NMR, HSQC, HMBC and mass spectral
analysis. Especially, the position of the aminomethyl group at
coumarin was unambiguously corroborated by HMBC experiment.
The HMBC spectrum of compound 8 showed two protons of C-11 (δ
laboratory scale
. Knoevenagel, Wittig and Perkin reactions have
3
0-32
also been used according to the different starting materials
.
However, we used a perchloric acid catalysed von Pechmann
reaction at room temperature, this was expected to diminish side-
reactions but may also have an effect on reaction time. Moreover,
the perchloric acid is practical to use because it does not give rise to
warming in contrast to sulphuric acid, which heats up in pouring the
reaction mixture into the water. The desired 4-substituted esculetin
derivatives were prepared as depicted in Scheme 1. The compound 2
4
.00 ppm) correlated with C-7 (δ 146.8 ppm), C-8 (δ 106.8 ppm) and
C-9 (δ 153.6 ppm), respectively. Meanwhile, the HMBC spectrum of
compound 9 showed two protons of C-11 (δ 4.44 ppm) correlated
with C-5 (δ 110.7 ppm), C-6 (δ 143.5 ppm) and C-10 (δ 112.9 ppm),
respectively. Moreover, structural modifications at the C-4 position
strongly affect the bioactivities of esculetins, so 4-substituted-
esculetins were used as raw material to prepare a series of 8-
substituted esculetin Mannich derivatives. Notably, 4-substituted
~
5 were prepared via the condensation reaction of 1,2,4-phenenyl
triacetate with properly substituted β-ketoesters in the presence of
perchloric acid with the good yield. Subsequently, compound 6 was
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5RA06070B
Page 3 of 8
Journal Name
RSC Advances
ARTICLE
esculetin condensed with formaldehyde and amines to compound 12 position was rapidly eliminated, with an elimination t1/2 value of
15 with the good yield and the excellent regioselectivity.
~
20.8 min. A simple structure–metabolic stability relationship
analysis showed that hydrophilic substitution in the C-4 position of
esculetin is more favorable to the improvement of metabolic stability.
All of these 8-substituted esculetin Mannich derivatives
demonstrated much higher metabolic stability than the parent
compound 1, with elimination t1/2 values from 102 to 176 min.
Among these analogs, compound 14 with a dimethylamino group
demonstrated the most metabolically stable with a 3-fold longer t1/2
compared with the parent compound 1. From the view of structural
features of these esculetin Mannich bases, the introduction of
dimethylamino group to the C-8 site of esculetin demonstrated to be
more effective strategy to enhance metabolic stability than the
introduction of pyrrolidinyl group and piperidinol group to this
position. It’s possible due to that the intramolecular hydrogen bond
energy of dimethylamino derivative is stronger than pyrrolidinyl
derivative and piperidinol derivative. Moreover, it's worth
mentioning that compound 9 exhibited a relatively short t1/2
compared with the other aminomethyl substituted esculetin
derivatives, although there may be a intramolecular hydrogen bond
formed between the aminomethyl group and 6-OH of esculetin. This
confirmed our previous observation that the 7-OH of esculetin was
the major site for UGT metabolism. On the other hand, the amine
containing substitutes at C-8 position partially increased the
solubility of these esculetin Mannich derivatives, which may
contribute to the reduction of the substrate affinity to metabolic
enzyme. Also the increased molecular volume could cause certain
steric hindrance for substrate to enter the active site of enzyme.
Taken together, all of these findings revealed that aminomethyl
Scheme 2 Synthesis of Compound 8 ~ 15
Fig. 1 The structure of compound 8 and compound 9
Metabolic stability assay in vitro
Metabolic stability refers to the susceptibility of compounds to group introduced to the C-8 position, of which the amine group
biotransformation in the context of selecting and/or designing drugs could interact with 7-OH of esculetin and form an intramolecular
with favourable pharmacokinetic properties. To identify the most hydrogen bond, might play a significant role in improving metabolic
promising drug candidates, thus reducing attrition at later stages and stability. This strategy could be utilized to guide the structure
increasing success rates, the in vitro half-life (t1/2) of all the modification towards improving phase II metabolic stability of 6,7-
synthesized esculetin derivatives were evaluated in pooled human dihydroxycoumarins.
liver S9 fraction in the presence of the cofactors for glucuronidation,
sulfation, and methylation, i.e., UDPGA, PAPS, and SAM. The
results were shown in Table 1. Among seven 4-substituted esculetin
derivatives, most of compounds demonstrated higher metabolic
stability than the parent compound 1 with an exception of compound
Table 1 In vitro anti-proliferative activity and t1/2 of esculetin
derivatives
B16
A549
t1/2 in HS9
(min)
Compd
IC50 ( μM)
IC ( μM)
50
3
. Compound 7 was the most metabolically stable with elimination
1
2
71.4 ± 8.3
29.2 ± 3.6
48.9 ± 10.3
17.2 ± 3.1
44.7
57.8
t1/2 value of 91.4 min, double longer compared with the parent
compound 1. In contrast, compound 3 with phenyl group at this
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

View Article Online
DOI: 10.1039/C5RA06070B
RSC Advances
Page 4 of 8
ARTICLE
Journal Name
aminomethyl esculetin derivatives, the anti-proliferative activity of
most of the synthesized compounds (with 28.7 μM to 3.8 μM of IC50
values for B16, and 14.1 μM to 1.9 μM for A549) significantly
increased compared with the parent compound 1 (IC50 71.4 μM and
3
4
5
6
7
8
9
16.8 ± 0.1
13.2 ± 0.2
18.4 ± 1.3
52.4 ± 1.4
26.7 ± 0.1
15.3 ± 1.3
16.5 ± 0.1
9.3 ± 2.5
9.6 ± 2.1
9.2 ± 1.8
24.4 ± 2.2
> 100
20.8
49.1
47.4
4
8.9 μM for B16 and A549, respectively). Concerning the structural
67.4
features of the esculetin Mannich bases, esculetin derivatives with a
pyrrolidinyl group in C-8 position of esculetin exhibited higher anti-
proliferative activity than the derivatives with a dimethylamino or
piperidinol group. Subsequently, it's worth mentioning that
compound 15 containing both trifluoromethyl and pyrrolidinyl group
was designed, and an almost 20-fold increase in anti-proliferative
activity was observed. Taken together, it seems that the aminomethyl
moiety of these compounds might play a significant role, meanwhile
the lipophilic substituents at C-4 position slightly enhanced the anti-
proliferative activity.
> 100
91.4
9.1 ± 0.1
6.5 ± 0.3
6.3 ± 0.4
14.1 ± 0.8
10.9 ± 2.3
5.9 ± 0.4
11.0 ± 3.4
1.9 ± 0.5
102.4
96.9
1
0
1
2
3
4
5
124.9
140.9
118.1
165.3
176.4
115.3
1
1
1
1
1
28.7 ± 1.3
12.1 ± 1.6
5.5 ± 2.1
Conclusion
16.6 ± 12.5
3.8 ± 1.2
In summary, to improve both the metabolic stability and the anti-
tumour activity of esculetins, a series of 4-substituted esculetins and
8
-substituted esculetin Mannich bases were designed and
Antiproliferative activity assays in vitro
synthesized by perchloric acid catalysed von Pechmann reaction and
Mannich reaction. All the newly synthesized compounds were
evaluated for their metabolic half-life in pooled human liver S9
fraction and in vivo anti-tumour activity in B16 and A549 cell lines.
The assay results showed that most of the synthesized compounds
displayed better anti-proliferative activity than the parent compound
Actually, in vitro cytotoxicity assays with cultured cells are widely
used to chemicals including cancer chemotherapeutics,
pharmaceuticals, biomaterials, natural toxins, antimicrobial agents
and industrial chemicals because they are rapid and economical. To
evaluate the anti-proliferative activity of all the synthesized
compounds, the IC50 values for the inhibition on human cancer cell
lines A549 (non-small cell lung carcinoma) and B16 (mouse
melanoma cell) were tested using SRB (Sulforhodamine B) assay
with esculetin 1 as the reference compound. The results shown in
Table 1 demonstrated that most of the synthesized compounds
displayed better anti-proliferative activity than the parent compound
1
with IC50 values in micromole range. Concerning the structural
features, aminomethyl group introduced to C-8 position, of which
the amine group could interact with 7-OH of esculetin and form an
intramolecular hydrogen bond, might play a significant role for both
the metabolic stability and the anti-tumour activity, while the
lipophilic substitution in C-4 position led to a slightly enhanced anti-
proliferative activity and hydrophilic substitution in C-4 position
was more favourable to the improvement of metabolic stability.
These findings could provide useful guidance for the structure
modification towards improving phase II metabolic stability.
1
with IC50 values in micromole range. Among seven 4-substituted
esculetin derivatives, compound 4 gave the best results with 13.2 μM
of IC50 value for B16 cell line and 9.6 μM for A549. Compound 2, 3
and 5, which possess CH , Ph and CH Cl in this position showed
3
2
moderate activities. Whereas compounds 6 and 7 with CH OH and
2
Experimental
CH COOH in this position exhibited relatively poor activities,
2
especially for A549 cell lines, and both of them with IC50 values
over 100 μM. A simple structure–activity relationship analysis
showed that the anti-proliferative activity closely related to the
lipophilic substituted groups at C-4 position, which showed a
contrasting effect on the metabolic stability. Among 8-substituted
Materials and methods
Compound 1 (Esculetin) and compound 7 (4-acetic acid esculetin)
was purchased from the Boyle company (shanghai, China).
Alamethicin, Brij 58, magnesium chloride, D-saccharic acid, 1,4-
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5RA06070B
Page 5 of 8
Journal Name
RSC Advances
ARTICLE
lactone, β-glucuronidase (EC No. 3.2.1.31), uridine 5’-diphospho- The resultant suspension was filtered and the collected solid was
glucuronic acid trisodium salt (UDPGA), 3′-phosphoade-nosin-5′- washed with water and dried, then crude compound was
phosphosulfate (PAPS), S-adenosyl-L-methionine (SAM) and recrystallized from methanol to afford product.
niflumic acid were purchased from Sigma-Aldrich (St. Louis, MO, 6,7-dihydroxy-4-methyl-2H-chromen-2-one (2). According to the
USA). Pooled human liver S9 fraction (HS9) was purchased from general procedure, 1,2,4-phenenyl triacetate was reacted with ethyl
Research Institute for Liver Diseases (Shanghai, China). acetoacetate in the presence of perchloric acid, and crude compound
sulforhodamine B (SRB) were purchased from Sigma-Aldrich (St. was recrystallized from methanol to afford compound 2 as light
1
Louis, MO, USA), A549 and B16 were purchased from the white solid, 87% yield. H NMR (400 MHz, DMSO-d ) δ: 2.31 (s,
6
Committee on Type Culture Collection of Chinese Academy of 3H, CH ), 6.09 (s, 1H, COCH=C), 6.73 (s, 1H, ArH), 7.00 (s, 1H,
3
1
3
Sciences (Shanghai, China).
ArH), 9.34 (s, br, 1H, ArOH), 10.19 (s, br, 1H, ArOH); C NMR
All reagents used in the synthesis were obtained commercially (100 MHz, DMSO-d ) δ: 18.68, 103.13, 109.89, 110.86, 111.96,
6
1
13
and used without further purification. The H NMR and C NMR 143.24, 148.17, 150.58, 153.68, 161.08; ESI-MS: M = 192, found
-
spectra were recorded in DMSO-d6 using a Bruker ARX 400 191.0 [M-H] .
1
13
spectrometer (400 MHz for H NMR and 100 MHz for C NMR), 6,7-dihydroxy-4-phenyl-2H-chromen-2-one (3). According to the
and chemical shifts were expressed as ppm against TMS as an general procedure, 1,2,4-phenenyl triacetate was reacted with ethyl
internal reference. The reactions were monitored by thin layer benzoylacetate in the presence of perchloric acid, and crude
chromatography (TLC) on glass-packed precoated silica gel GF254 compound was recrystallized from methanol to afford compound 3
1
plates and visualized in an iodine chamber or with a UV lamp. Flash as white solid, 91% yield. H NMR (400 MHz, DMSO-d ) δ: 6.12 (s,
6
column chromatography was performed using silica gel (200~300 1H, COCH=C), 6.80 (s, 1H, ArH), 6.84 (s, 1H, ArH), 7.50-7.58 (m,
mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. High- 5H, ArH) 9.43 (s, br, 1H, ArOH), 10.26 (s, br, 1H, ArOH); ESI-MS:
-
resolution mass spectral (HRMS) analyses were measured with M = 254, found 253.0 [M-H] .
Hybrid Ion Trap-Orbitrap Mass Spectrometer (LTQ Orbitrap XL, 6,7-dihydroxy-4-trifluoromethyl-2H-chromen-2-one
(4).
Thermo). According to the general procedure, 1,2,4-phenenyl triacetate was
Esculetin and its derivatives were analyzed by a ultra-fast liquid reacted with ethyl 4,4,4-trifluoroacetoacetate in the presence of
chromatography spectrometry system (Shimadzu, Kyoto, Japan), perchloric acid, and crude compound was recrystallized from
1
equipped with two LC-20AD pumps, a DGU-20A3 vacuum degasser, methanol to afford compound 4 as light yellow solid, 63% yield. H
a SIL-20ACHT auto-sampler, a CTO-20AC column oven, an SPD- NMR (400 MHz, DMSO-d ) δ: 6.71 (s, 1H, COCH=C), 6.86 (s, 1H,
6
M 20A diode-array detector (DAD), a CBM-20A communications ArH), 7.03 (s, 1H, ArH), 9.78 (s, br, 1H, ArOH), 10.61 (s, br, 1H,
1
3
bus module, a mass detector (2010EV) with an electrospray ArOH); C NMR (100 MHz, DMSO-d ) δ: 104.01, 105.05, 108.89,
6
ionization (ESI) interface, and a computer equipped with UFLC-MS 112.12, 120.94, 123.68, 144.03, 149.59, 152.08, 159.62; ESI-MS: M
-
Solution version 3.41 software. A Hedera C18 (150.0 mm×2.1 mm, = 246, found 245.1 [M-H] .
3
μm) analytical column was used and column temperature was kept 6,7-dihydroxy-4-chloromethyl-2H-chromen-2-one (5). According
at 40 °C. The mobile phase was acetonitrile (A) and 0.2% formic to the general procedure, 1,2,4-phenenyl triacetate was reacted with
acid water (B) at a flow rate of 0.4 ml/min, with a gradient: 0-6.0 ethyl 4-chloroacetoacetate in the presence of perchloric acid, and
min, 95% B-65% B; 6.0-9.0 min, 5% B; 9.0-15min, balance to 95% crude compound was recrystallized from methanol to afford
1
B.
compound 5 as white solid, 78% yield. H NMR (400 MHz, DMSO-
General procedure for the synthesis of 4-substituted esculetins (2
d
6
) δ: 4.90 (s, 2H, CH
Cl), 6.40 (s, 1H, COCH=C), 6.78 (s, 1H, ArH),
2
~
5)
7.12 (s, 1H, ArH), 9.48 (s, br, 1H, ArOH), 10.39 (s, br, 1H, ArOH).
-
-
To a mixture of 1,2,4-phenenyl triacetate (5.0 mmol) and properly ESI-MS: M = 225.5, found 224.9 [M-H] and 226.9 [M-H] .
substituted β-ketoesters (10.0 mmol) was added drop-wise perchloric 6,7-dihydroxy-4-hydroxymethyl-2H-chromen-2-one (6). Chloride
acid (5.0 mL) at room temperature and stirred for 6 ~ 8 h. After 5 (0.5 g, 2.2 mmol) was dissolved in a mixture of DMF (3 ml) and
completion of the reaction as indicated by TLC, the reaction mixture H O (10 ml) with stirring and then refluxed for 20 h. After
2
was poured slowly into a mixture of ice-water (100 mL) with stirring. completion of the reaction as indicated by TLC, the mixture was
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

View Article Online
DOI: 10.1039/C5RA06070B
RSC Advances
Page 6 of 8
ARTICLE
Journal Name
diluted with water. The resultant suspension was filtered and the reaction as indicated by TLC, the solvent was removed under
collected solid was washed with water and dried, then crude reduced pressure. The residue was applied to flash column
compound was recrystallized from methanol to afford chromatography (silicagel) to afford compound 10 with the yield of
1
1
hydroxymethyl 6 (380 mg, 83%) as light brown solid. H NMR (400 18 % as light brown solid. H NMR (400 MHz, DMSO-d ) δ: 1.85
6
MHz, DMSO-d ) δ: 4.63 (s, 2H, CH OH), 6.21 (s, 1H, COCH=C), (m, 4H, CH CH ), 2.88 (t, J = 4.8 Hz 4H, NCH CH ), 4.17 (s, 2H,
6
2
2
2
2
2
6
.75 (s, 1H, ArH), 6.94 (s, 1H, ArH), 9.31 (s, br, 1H, ArOH), 10.19 CH ), 5.97 (d, J=8.0 Hz, 1H, COCH=C), 6.82 (s, 1H, ArH), 7.76 (d,
2
+
13
(
s, br, 1H, ArOH); ESI-MS: M = 208, found 208.9 [M+H] .
General procedure for the synthesis of 8-substituted esculetin (2C), 49.88, 52.84(2C), 105.98, 107.16, 107.75, 109.05, 143.49,
Mannich bases (8 ~ 15)
144.90, 147.58, 156.57, 160.86; ESI-MS: M = 261, found 262.1
To a solution of the appropriate amines (0.013 mol) in 30 mL of [M+H] ; HRMS (ESI): C H NO calcd for [M + H] : 262.1079,
J=8.0 Hz, 1H, PhCH=C); C NMR (100 MHz, DMSO-d ) δ: 23.05
6
+
+
1
4
15
4
absolute methanol was added aqueous formaldehyde (37%) (0.015 found: 262.1072.
mol). After 0.5 h of gentle refluxing at 50 °C, esculetin/4-substituted 6,7-dihydroxy-8-((dimethylamino)methyl)-2H-chromen-2-one
esculetin was added (0.01 mol), dissolved in 50 mL of absolute (11) According to the general procedure, esculetin was reacted with
methanol. The duration of the reaction was 8 ~ 12 h and the reaction aqueous formaldehyde and aqueous dimethylamine. After
was monitored by TLC.
,7-dihydroxy-8-((4-hydroxypiperidin-1-yl)methyl)-2H-
completion of the reaction as indicated by TLC, the solvent was
removed under reduced pressure. The residue was applied to flash
6
chromen-2-one (8) and 5-((4-hydroxypiperidin-1-yl)methyl)-6,7- column chromatography (silicagel) to afford compound 11 with the
1
dihydroxy-2H-chromen-2-one (9). According to the general yield of 15 % as light brown solid. H NMR (400 MHz, DMSO-d ) δ:
6
procedure, esculetin was reacted with aqueous formaldehyde and 4- 2.49 (s, 6H, 2CH ), 4.03 (s, 2H, CH ), 6.03 (d, J=8.0 Hz, 1H,
3
2
hydroxy piperidine. After completion of the reaction as indicated by COCH=C), 6.86 (s, 1H, ArH), 7.79 (d, J=12.0 Hz, 1H, PhCH=C),
+
TLC, the solvent was removed under reduced pressure. The residue 8.16 (s, 1H, ArOH); ESI-MS: M = 235, found 236.1 [M+H] .
was applied to flash column chromatography (silicagel) to afford 6,7-dihydroxy-8-((4-hydroxypiperidin-1-yl)methyl)-4-methyl-
compound 8 and compound 9 with the yield of 12% and 3%, 2H-chromen-2-one (12). According to the general procedure, 4-
respectively.
methyl esculetin was reacted with aqueous formaldehyde and 4-
1
Compound 8: H NMR (400 MHz, DMSO-d ) δ: 1.48 (m, 2H, CH ), hydroxy piperidine. After completion of the reaction as indicated by
6
2
1
.82 (m, 2H, CH ), 2.49 (m, 2H, NCH ), 2.91 (m, 2H, NCH ), 3.62 TLC, the mixture was diluted with water. The resultant suspension
2 2 2
(
m, 1H, CHOH), 4.00 (s, 2H, CH ), 6.08 (d, J=8 Hz, 1H, COCH=C), was filtered and the collected solid washed with water and dried,
2
1
3
6
.89 (s, 1H, ArH),7.81 (d, J=8 Hz, 1H, PhCH=C); C NMR (100 then crude compound was recrystallized from methanol to afford
1
MHz, DMSO-d ) δ: 33.50, 49.72, 52.56, 106.77, 108.87, 109.71, compound 12 as light yellow solid with 80% yield. H NMR (400
6
1
10.38, 142.79, 144.87, 146.79, 153.60, 160.62; ESI-MS: M = 291, MHz, DMSO-d ) δ: 1.47 (m, 2H, CH ), 1.81 (m, 2H, CH ), 2.31 (s,
6 2 2
+
+
found 292.1 [M+H] ; HRMS (ESI): C H NO calcd for [M + H] : 1H, CH ), 2.45 (m, 2H, NCH ), 2.87 (m, 2H, NCH ), 3.61 (m, 1H,
1
5
17
5
3
2
2
2
92.1185, found: 292.1181.
CHOH), 3.99 (s, 2H, CH ), 6.04 (s, 1H, COCH=C), 6.94 (s, 1H,
2
1
13
Compound 9: H NMR (400 MHz, DMSO-d ) δ: 1.68 (m, 2H, CH ), ArH); C NMR (100 MHz, DMSO-d ) δ: 18.30, 33.61, 49.77, 52.88,
6
2
6
1
.92 (m, 2H, CH ), 3.09 (m, 2H, NCH ), 3.29 (m, 2H, NCH ), 3.76 107.02, 107.58, 109.18, 109.77, 142.49, 145.82, 152.80, 153.61,
2 2 2
+
(
m, 1H, CHOH), 4.44 (s, 2H, CH ), 6.27 (d, J=8 Hz, 1H, COCH=C), 160.32; ESI-MS: M = 305, found 306.1 [M+H] ; HRMS (ESI):
2
1
3
+
6
.97 (s, 1H, ArH), 8.33 (d, J=8 Hz, 1H, PhCH=C); C NMR (100 C H NO calcd for [M + H] : 306.1341, found: 306.1337.
16 19 5
MHz, DMSO-d ) δ: 30.38, 48.53, 49.61, 103.75, 110.68, 111.56, 6,7-dihydroxy-8-(pyrrolidin-1-ylmethyl)-4-methyl-2H-chromen-
6
1
12.92, 141.95, 143.54, 148.89, 150.28, 160.18; ESI-MS: M = 291, 2-one (13). According to the general procedure, 4-methyl esculetin
+
+
found 292.1 [M+H] ; HRMS (ESI): C H NO calcd for [M + H] : was reacted with aqueous formaldehyde and pyrrolidine. After
1
5
17
5
2
92.1185, found: 292.1183.
completion of the reaction as indicated by TLC, the mixture was
6
,7-dihydroxy-8-(pyrrolidin-1-ylmethyl)-2H-chromen-2-one (10) diluted with water. The resultant suspension was filtered and the
According to the general procedure, esculetin was reacted with collected solid washed with water and dried, then crude compound
aqueous formaldehyde and pyrrolidine. After completion of the was recrystallized from methanol to afford compound 13 as yellow
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5RA06070B
Page 7 of 8
Journal Name
RSC Advances
ARTICLE
1
solid with 88% yield. H NMR (400 MHz, DMSO-d ) δ: 1.84 (m, 4H, 200 μl of 50 mM Tris buffer (pH 7.4), as described in the reported
6
3
6
2
5
CH ), 2.29 (s, 3H, CH ), 2.83 (m, 4H, 2NCH ), 4.15 (s, 2H, CH ), manuscript . Controls were incubated in the absence of UDPGA,
2
3
2
2
1
3
.96 (s, 1H, COCH=C), 6.89 (s, 1H, ArH); C NMR (100 MHz, PAPS and SAM. After incubation at 37°C for 0 to 40 min, the
DMSO-d ) δ: 18.89, 23.60, 50.17, 53.33, 106.97, 108.32, 108.70, reactions were terminated by adding 200 μl of cold acetonitrile. The
6
1
2
2
8
43.65, 146.90, 154.23, 155.87, 161.07; ESI-MS: M = 275, found samples were centrifuged at 20,000 g for 20 min. The supernatants
+
+
76.1 [M+H] ; HRMS (ESI): C H NO calcd for [M + H] : (200 μl) were subjected to assay for the time-dependent esculetins
1
5
17
4
76.1236, found: 276.1230.
-((dimethylamino)methyl)-4-methyl-2H-chromen-2-one
depletion using HPLC.
(14). Cell culture and maintenance
According to the general procedure, 4-methyl esculetin was reacted The human cancer cell lines A549 (non-small cell lung carcinoma)
with aqueous formaldehyde and aqueous dimethylamine. After and B16 (mouse melanoma cell) were obtained from American Type
completion of the reaction as indicated by TLC, the mixture was Culture Collection (ATCC), which were cultivated in the Dulbecco's
diluted with water. The resultant suspension was filtered and the modified. Eagle medium (DMEM, GIBCO), containing 10% (v/v)
-
1
collected solid washed with water and dried, then crude compound heat inactivated fetal bovine serum (FBS, GIBCO), 100 IU mL
-
1
was recrystallized from methanol to afford compound 14 as yellow penicillin and 100 μg mL streptomycin (GIBCO) in culture dish in
1
o
solid with 78% yield. H NMR (400 MHz, DMSO-d ) δ: 2.30 (s, 3H, a humidified atmosphere of 5% CO at 37 C.
6
2
CH ), 2.42 (s, 6H, 2CH ), 4.00 (s, 2H, CH ), 5.99 (s, 1H, COCH=C), Antiproliferative activity assays in vitro
3
3
2
1
3
6
5
1
.92 (s, 1H, ArH); C NMR (100 MHz, DMSO-d ) δ: 18.87, 43.83, The monolayer cell culture was trypsinized and the cell count was
6
5
4.51, 106.93, 107.48, 108.82, 109.27, 143.39, 146.82, 154.21, adjusted to 0.5-1.0 x 10 cells/ml using medium containing 10% new
+
55.03, 160.98 ; ESI-MS: M = 249, found 250.0 [M+H] ; HRMS born sheep serum. To each well of the 96 well microtitre plate, 0.1ml
+
(
ESI): C H NO calcd for [M + H] : 250.1079, found: 250.1074.
of the diluted cell suspension (approximately 7,000 cells) was added.
After 24 hours, when a partial monolayer was formed, the
1
3
15
4
6
2
4
,7-dihydroxy-8-(pyrrolidin-1-ylmethyl)-4-trifluoromethyl-
H
-chromen-2-one (15). According to the general procedure, supernatant was flicked off, washed once and 100 μl of different test
-trifluoromethyl esculetin was reacted with aqueous compound concentrations were added to the cells in microtitre plates.
o
formaldehyde and pyrrolidine. After completion of the reaction The plates were then incubated at 37 C for 48 hours in 5% CO2
as indicated by TLC, the mixture was diluted with water. The incubator and microscopic examination was carried out and
resultant suspension was filtered and the collected solid washed observations recorded every 24 hours. After 48 hours, 100μl of 10%
with water and dried, then crude compound was recrystallized trichloroacetic acid was added to the wells gently such that it forms a
o
from methanol to afford compound 15 as yellow solid with 87% thin layer over the test compounds. The plates were incubated at 4 C
1
yield. H NMR (400 MHz, DMSO-d ) δ: 1.92 (m, 4H, 2CH ), for one hour. The plates were flicked and washed five times with tap
6
2
3
.17 (m, 4H, 2NCH ), 4.29 (s, 2H, CH ), 6.15 (s, 1H, water to remove traces of medium, sample and serum, and were then
2
2
1
3
COCH=C), 6.71 (s, 1H, ArH); C NMR (100 MHz, d-DMSO) air-dried. The air-dried plates were stained with 40 μL SRB and kept
δ: 23.27, 49.55, 53.45, 98.26, 102.01, 102.07, 103.10, 104.17, for 30 minutes at room temperature. The plates were then air-dried.
146.41, 151.66, 160.33, 164.84; ESI-MS: M = 329, found 330.1 100 μl of 10 mM Tris base was then added to the wells to solubilize
+
+
[
M+H] ; HRMS (ESI): C H F NO calcd for [M + H] : the dye. The plates were shaken vigorously for 5 minutes. The
15 14 3 4
3
30.0953, found: 330.0946.
absorbance was measured using microplate reader at a wavelength of
540 nm. All experiments were repeated at least three times. The data
Metabolic stability assay in vitro
To determine the effects of a combination of glucuronidation, were calculated using Graph Pad Prism version 6.0. The IC50 values
sulfation, and methylation, the 6,7-dihydroxycoumarins were were fitted using a nonlinear regression model with a sigmoidal dose
incubated with S9 fraction in the presence of three cofactors (2 mM response.
UDPGA, 0.2 mM PAPS and 0.2 mM SAM). The incubation mixture
Acknowledgements
contained pooled human S9 fraction (0.02 mg/ml protein), 5 mM
This work was supported by the National S&T Major Projects of
MgCl , 2 mM UDPGA, 0.2 mM PAPS, 0.2 mM SAM and 5 μM
2
China (2012ZX09501001, 2012ZX09506001), the National Basic
esculetin derivatives (dissolved in DMSO, final concentration 1%) in
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

View Article Online
DOI: 10.1039/C5RA06070B
RSC Advances
Page 8 of 8
ARTICLE
Journal Name
Research Program of China (2013CB531800), and NSF of China 25
Y. L. Xia, G. B. Ge, P. Wang, S. C. Liang, J. Ning, X. K. Qian, Y. li, L.
Yang, Drug Metab. Dispos., 2015, 43 (4), 553-560.
R. B. Desai, J. Org.Chem., 1961, 26, 5251-5253.
(
81402822, 81273590, and 81302793).
2
2
6
7
C A. Kontogiorgis, D J. Hadjipavlou-Litina, J. Med. Chem.,2005, 48,
Notes and references
6
401.
Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian,116023, China. E-
mail: geguangbo@dicp.ac.cn & ylingdicp@gmail.com
2
2
30
3
3
3
8
9
M. Bulut, C. Erk, Dyes Pigm., 1996, 30, 99-104.
H. V. Pechmann, Ber. Dtsch. Chem. Ges., 1884, 17, 929–936.
J. March, John Wiley & Sons, USA, 1992.
1
13
†
Electronic Supplementary Information (ESI) available: The H NMR,
C
1
2
3
M M .Garazd, Y L Garazd, V P Khilya, Chem. Nat. Compd., 2005,
NMR, HSQC, HMBC and HRMS spectra for synthesized compounds.
See DOI: 10.1039/b000000x/
4
1(3), 245-271.
J M. Timonen, R M. Nieminen and O. Sareila, Eur. J. Med. Chem.,
011, 46(9), 3845-3850.
V. N. Gupta, B. R.Sharima and R. B. Avora, J. Sci. Indust. Res.,1961,
0B, 300.
‡
These authors contributed equally to this work.
2
1
2
3
4
5
6
7
8
9
1
F. Borges, F. Roleira, N. Milhazes, L. Santana, E. Uriarte, Curr. Med.
Chem, 2005, 12, 887.
L. Wu, X. Wang, W. Xu, F. Farzaneh and R. Xu, Curr. Med. Chem,
2
3
3
4
5
M. G. Patel, S .Sethna, J. Indian Chem. Soc., 1962, 39, 595-598.
J. Zak, D. Ron and E. Riva E, Chem. –Eur. J., 2012, 18(32), 9901-
2
009, 16, 4236-4260.
9
910.
M. E. Riveiro, N. De Kimpe, A. Moglioni, R. Vazquez, F. Monczor, C.
Shayo, C. Davio, Curr. Med. Chem, 2010 17(13), 1325-1338.
M. J. Matos, L. Santana, E. Uriarte, G. Delogu, M. Corda, M. B. Fadda,
B. Era, A. Fais, Bioorg. Med. Chem. Lett, 2011, 21, 3342.
3
6
X. Wen and T. Walle, Xenobiotica, 2006, 36, 387–397.
A. Galkin, A. Fallarero, P. M. Vuorela, J. Pharm. Pharmacol, 2009, 61
(2), 177-84.
P. Petersen, J.Godtfredsen, G. Boysen, E. Andersen, B. Andersen, The
Lancet, 1989, 333(8631): 175-179.
Y. Z. Yang, A. Ranz, H. Z. Pan, Z. N. Zhang, X. B. Lin, & S. R.
Meshnick, Am. J. Trop. Med. Hyg., 1992, 46(1), 15-20.
Y. Wang, P. Li, Y. Tang, J. P. Fawcett, J. Gu, J. Pharm. Biomed. Anal,
2
007, 43 (5), 1860-3.
U. S. Weber, B. Steffen, C. P. Siegers, Res. Commun. Mol. Pathol.
Pharmacol.,1998, 99(2), 193-206.
MA. elasco-Velazquez, J. Agramonte-Hevia, D. Barrera, A. Jimenez-
Orozco, MJ. Garcia-Mondragon, N. Mendoza-Patino, Cancer Letts,
0
2
003, 198, 179-186.
1
1
1
2
I. Kostova, Anticancer Agents. Med. Chem., 2005, 5(1), 29-46.
H. Kolodziej, O. Kayser, H. J. Woerdenbag, W. van Uden, N. Pras, Z.
Naturforsch. C., 1997, 52, 240-244.
1
1
1
1
3
4
5
6
Y. Masamoto, H. Ando, Y. Murata, Y. Shimoishi, M. Tada, K, Biosci.
Biotechnol. Biochem., 2003, 67, 631-634.
C. C. Chu, Y. Y. Tsai, C. J. Wang, W. L. Lin, T. H. Tseng, Eur. J.
Pharmacol, 2001, 416 , 25-32.
C. J. Wang, Y. J. Hsieh, C. Y. Chu, Y. L. Lin, T. H. Tseng, Cancer Lett,
2
002, 183 , 163-168.
S. H. Lee, C. Park, C. Y. Jin, G. Y. Kim, S. K. Moon, J. W. Hyun, W. H.
Lee, B. T. Choi, T. K. Kwon, Y. H. Yoo, Y. H. Choi, Biomed.
Pharmacother., 2008, 62, 723-729.
1
1
7
8
C. Park, C. Y. Jin, G. Y. Kim, I. W. Choi, K. K. Taeg, T. C. Byung, J. L.
Su, H. L. Won, H. C. Yung, Toxicol. Appl. Pharmacol., 2008, 227, 219-
2
28.
H. C. Kuo, H. J. Lee, C. C. Hu, H. I. Shun, T. H. Tseng, Toxicol. Appl.
Pharmacol., 2006, 210, 55-62.
1
2
9
0
K. T. Lee, D. A. Lillard, J. Food Lipids., 1997, 4 (2), 119–127.
M. Payá, B. Halliwell, J. R. Hoult, Biochem. Pharmacol., 1992, 44,
2
05-214.
2
2
2
1
2
3
S. Tahara, N. Baba, M. Matsuo, T. Kaneko, Biosci. Biotechnol.
Biochem., 2005, 69, 620-622.
K. Matsunaga, N. Yoshimi, Y. Yamada, M. Shimizu, K. Kawabata, Y.
Ozawa, A. Hara, H. Mori, Jpn. J. Cancer Res., 1998, 89, 496-501.
S. C. Liang, G. B. Ge; H. X. Liu, Y. Y. Zhang, L. M. Wang, J. W.
Zhang, L. Yin, W. Li, Z. Z. Fang, L. Yang, Drug Metab. Dispos., 2010,
3
8, 973-980.
2
4
Y. L. Xia, S. C. Liang, L. L. Zhu, G. B. Ge, G. Y. H, J. Ning, X. Lv, X.
C. Ma, L. Yang, S. L. Yang, Drug Metab. Pharmacokinet.,2014,29 (2),
1
35-40.
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012