Synthesis and structure-activity relationship of daphnetin derivatives as potent antioxidant agents

Article abstract of DOI:10.3390/molecules23102476

In this study, daphnetin 1 was chosen as the lead compound, and C-3 or C-4-substituted daphnetins were designed and synthesized to explore the potential relationship between the antioxidant activities and the chemical structures of daphnetin derivatives. The antioxidant activities of the generated compounds were evaluated utilizing the free radical scavenging effect on 2,2-diphenyl-1-picrylhydrazyl, 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation, and the ferric reducing power assays, and were then compared with those of the standard antioxidant Trolox. The results showed that the catechol group was the key pharmacophore for the antioxidant activity of the daphnetins. The introduction of an electron-withdrawing hydrophilic group at the C-4 position of daphnetin enhanced the antioxidative capacity, but this trend was not observed for C-3 substitution. In addition, introduction of a a hydrophobic phenyl group exerted negative effects on the antioxidant activity in both the C-3 and C-4 substitutions. Among all of the derivatives tested, the most powerful antioxidant was 4-carboxymethyl daphnetin (compound 9), for which the strongest antioxidant activity was observed in all of the assays. In addition, compound 9 also displayed strong pharmaceutical properties in the form of metabolic stability. To summarize, compound 9 holds great potential to be developed as an antioxidant agent with excellent antioxidant activity and proper pharmacokinetic behavior.

Full text of DOI:10.3390/molecules23102476

molecules
Article
Synthesis and Structure-Activity Relationship of
Daphnetin Derivatives as Potent Antioxidant Agents
Yangliu Xia 1,†, Chen Chen 2,†, Yong Liu , Guangbo Ge , Tongyi Dou * and Ping Wang *
1
3
1,
3,
1
School of Life Science and Medicine, Dalian University of Technology, Panjin 124221, China;
xiayl@dlut.edu.cn (Y.X.); yliu@dlut.edu.cn (Y.L.)
College of Life Science, Dalian Minzu University, Dalian 116600, China; chenchen@dlnu.edu.cn
Institute of Interdisciplinary Medicine, Shanghai University of Traditional Chinese Medicine,
2
3
Shanghai 201203, China; gegbdicp@sohu.com
*
Correspondence: douty@dicp.ac.cn (T.D.); s200541025@126.com (P.W.);
Tel.: +86-427-263-1425 (T.D.); +86-21-513-23184 (P.W.)
These authors contributed equally to this work.
†
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 12 September 2018; Accepted: 26 September 2018; Published: 27 September 2018
Abstract: In this study, daphnetin 1 was chosen as the lead compound, and C-3 or C-4-substituted
daphnetins were designed and synthesized to explore the potential relationship between the
antioxidant activities and the chemical structures of daphnetin derivatives. The antioxidant
activities of the generated compounds were evaluated utilizing the free radical scavenging effect
0
0
on 2,2 -diphenyl-1-picrylhydrazyl, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation, and the
ferric reducing power assays, and were then compared with those of the standard antioxidant
Trolox. The results showed that the catechol group was the key pharmacophore for the antioxidant
activity of the daphnetins. The introduction of an electron-withdrawing hydrophilic group at the C-4
position of daphnetin enhanced the antioxidative capacity, but this trend was not observed for C-3
substitution. In addition, introduction of a a hydrophobic phenyl group exerted negative effects on
the antioxidant activity in both the C-3 and C-4 substitutions. Among all of the derivatives tested,
the most powerful antioxidant was 4-carboxymethyl daphnetin (compound
antioxidant activity was observed in all of the assays. In addition, compound
pharmaceutical properties in the form of metabolic stability. To summarize, compound
9
), for which the strongest
also displayed strong
holds great
9
9
potential to be developed as an antioxidant agent with excellent antioxidant activity and proper
pharmacokinetic behavior.
Keywords: daphnetin; antioxidant activity; radical scavenging effect; structure-activity relationship
1
. Introduction
Aerobic organisms form reactive oxygen species (ROS) as an unavoidable consequence of cell
metabolism. In a healthy individual, there is an equilibrium between the natural antioxidative defense
system and ROS. When the equilibrium is disrupted, ROS can induce both cellular damage responsible
for aging and various pathologies in humans, such as rheumatoid arthritis, diabetes, and cancer [
A variety of powerful natural antioxidants have been identified that act as chemopreventive agents,
which could prevent cancer through the action of antioxidants [ ]. These compounds could also exert
1–3].
4
anti-mutagenicity, anti-carcinogenicity, and anti-aging properties, which are thought to originate from
their antioxidant activities [5].
Natural compounds isolated from dietary or medicinal plants, which serve as rich sources of
antioxidant agents, have been of increasing interest in recent decades. With the goal of searching for
antioxidant components, the structure-activity relationships of some natural compounds have been
Molecules 2018, 23, 2476; doi:10.3390/molecules23102476
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2476
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extensively studied, generally focusing on the flavonoids and phenolic acids [
6–
8]. These reports
demonstrated that the free radical scavenging and antioxidant activity of these compounds primarily
depends on the type and position of the substituents on the aromatic ring of the molecules.
Coumarins are widely distributed in food and Chinese medicinal herbs, which are considered to
be good natural sources of antioxidants. With excellent safety, high solubility, and good permeability,
this family has received widespread attention [
9–13]. As a representative coumarin, daphnetin
(7,8-dihydroxycoumarin) has been approved as an adjunctive therapy for cardiovascular diseases
in China since the 1980s, as well as receiving recognition as the marker compound for the quality
control of the Zushima tablet, which has been used as a traditional Chinese medicine preparation to
treat rheumatoid arthritis [14]. Daphnetin
protecting mononuclear cells of umbilical blood from oxidative attack than resveratrol [15], and
-methyl daphnetin was found more effective in protection of human single cell DNA from
oxidative attack than curcumin and resveratrol [16]. Although daphnetin and its analogues
Figure 1) have been recognized to be good antioxidants, limited information is available on
1 and 4-methyl daphnetin 2 exhibited stronger activities in
4
2
1
(
the structure–antioxidant activity relationship of daphnetin derivatives, which may provide vital
information for the development of antioxidative agents based on daphnetin for the treatment or
adjuvant treatment of oxidative stress-related diseases.
Figure 1. The structure of daphnetin and its analogues.
Therefore, daphnetin
1 was utilized in this study as a lead compound, and different groups with
varied properties were introduced to the 3- or 4-position of daphnetin. The antioxidant activities
of the derivatives were evaluated by examining their free radical scavenging effect using the FRAP,
+
DPPH, and ABTS assays, and the potential structure-antioxidant activity relationships of daphnetins
were discussed. In addition, the pharmaceutical activities of daphnetin derivatives, especially
metabolic stability, which is the largest obstacle for the development of catechol coumarins as drugs,
were investigated. In addition, we proposed a strategy of the simultaneous optimization of metabolic
stability and pharmacological activity for these types of compounds. These findings could provide
useful guidance to design and optimize potent antioxidants and the structural modifications of
coumarin derivatives.
2
. Results and Discussion
2
.1. Chemistry
To explore the effects of different property groups and their position on daphnetin on
its antioxidant activities, a series of 3- or 4-substituted daphnetin derivatives were designed.
The substituents were selected mainly based on the electronic and lipophilic considerations defined by
0
Craig s plot [17]. In the present study, trifluoromethyl, phenyl and carboxylic ester were selected for
their electron-withdrawing and hydrophobic property; carboxymethyl and carboxyl were selected to
represent electron-withdrawing and hydrophilic substituents, while groups like tert-butyl and methyl
represented electron-donating and hydrophobic substituents; hydroxymethyl, azidomethyl, and
chloromethyl were representative of electron-donating and hydrophilic substitution. The derivatives
of daphnetin were prepared via Knoevenagel condensation and acid catalyzed Pechmann reaction.
First, the introduction of different property groups at the C-4 position of daphnetin is shown

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in Scheme 1. The compounds
2–
6
were synthesized via acid catalytic Pechmann condensation.
1
,2,3-Phenenyl triacetate was reacted with the correctly substituted β-ketoester under perchloric acid
catalysis to produce compounds . Subsequently, compound and compound were obtained from
chloride by refluxing in a mixture of dimethylformamide (DMF) and water (3:10, v:v) and treating
with sodium azide, respectively. 4-Carboxylic daphnetin and its ester 10 were obtained by treating
,2,3-phenenyl triacetate with 1,3-acetonedicarboxylic acid and dimethyl 1,3-acetonedicarboxylate,
respectively, in the present of perchloric acid.
2
–6
7
8
6
9
1
Ultimately a series of different property groups, including phenyl, cyano, carbonyl, carboxyl,
carboxylic ester and benzothiazole, were introduced to the C-3 site of daphnetin. All these compounds
were prepared from 2,3,4-trihydroxybenzaldehyde through Knoevenagel condensation (Scheme 2) [18].
The detailed experimental procedures are described in the Supporting Information. In addition,
a cyano group was introduced in the position of daphnetin 19 by an addition/elimination reaction
similar to that described in reference [19] with a brief modification. Subsequently, a cyano group
of 20 was hydrolyzed to acid 21 and amide 22, as well as being converted to tetrazol 23 via the
zinc-catalyzed [3 + 2] cycloadditon reaction (Scheme 3). Compounds 21–23 were purified using
preparative high-performance liquid chromatography.
Scheme 1. Synthesis of 4-substituted daphnetin derivatives. Reagents and conditions: (a) HClO ,
4
◦ ◦
5–60 C, 6–8 h; (b) DMF/H O (1:3), reflux, 20 h; (c) DMF, NaN , rt, 10 h; (d) HClO , 50 C, 4 h.
2 3 4
2

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Scheme 2. Synthesis of 3-substituted daphnetin derivatives. Reagents and conditions: (a) phenylacetic
◦
acid, AcONa, Ac O, 6 h, 160 C, 58%; (b) malononitrile, CH COONH , H O, rt, 3 h; HCl (2 mol/L),
2
3
4
2
◦
0
7
5 C, 1 h, 80%; (c) ethyl acetoacetate, piperidine, MeOH, reflux, 3 h, 85%; (d) Meldrum s acid,
CH COONH , H O, rt, 4 h, 83%; (e) 2-propynylamine, 1-hydroxybenzotriazole, EDCI, DMF, 5 C,
◦
h, 74%; (f) diethyl malonate, CH COONH , H O, 50 C, 5 h, 76%; (g) ethyl 2-(benzo[d]thiazol-2-yl)
3 4 2
◦
3
4
2
5
◦
acetate, piperidine, MeOH, 40 C, 5 h, 88%.
Scheme 3. Synthesis of 3-benzothiazole daphnetin derivatives. Reagents and conditions: (a) NaCN,
◦
◦
DMF, I , 6 h, 58%; (b) H SO , H O, 80 C, 3 h, 36%. (c) H SO , H O, 100 C, 8 h; 60%; (d) ZnBr, NaN ,
dioxane, 90 C, 12 h, 42%.
2
2
4
2
2
4
2
3
◦
2
.2. Antioxidant Activity Assay In Vitro
The antioxidant activity is influenced by many factors, which cannot be fully described with
a single antioxidant assay. Thus, in this study, antioxidant activities in vitro were evaluated
0
0
using the free radical scavenging effect on 2,2 -diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azinobis-
+
(3-ethylbenzthiazoline-6-sulfonate) cation (ABTS ). and the ferric reducing power (FRAP) assay,
respectively, according to the literature with a slight modification [20–24]. The Trolox (6-hydroxy-2,5,7,

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8-tetramethylchroman-2-carboxylic acid; Sigma Aldrich, (St. Louis, MO, USA) standard solution
(concentration range: 0–0.4 mmol/L) was analyzed under the same conditions as those used for
the calibration. First, the reducing power of these compounds was evaluated using the FRAP
assay by measuring the ability of an antioxidant to reduce ferric (III) ions to ferrous (II) ions.
The results of the total equivalent antioxidant capacity (TEAC) were expressed as the Trolox equivalent,
i.e., mmol equivalents of Trolox per mmol of sample (mmol Trolox/mmol sample).
As shown in Figure 2, all the daphnetin derivatives tested exhibited better reducing power
than the standard antioxidant Trolox except compound
DAP, ) exhibited a 2.5-fold higher reducing antioxidant power than that of the standard antioxidant
Trolox. A three-fold increase in reducing antioxidant power was observed when trifluoromethyl
or carboxyl group was introduced to the C-4 position of daphnetin, while other C-4 substituents,
including phenyl , methyl , tert-butyl , chloromethyl , hydroxymethyl and azidomethyl , led to
decreased activities of daphnetin but were still superior to Trolox except for the substitution of the
azidomethyl group. The markedly decreased antioxidant activity of compound is probably due to the
8 and 12 (TEACDPPH values >1). Daphnetin
(
1
5
9
3
2
4
6
7
8
8
ability of the azide group to cause the consumption of reducing agents and ultimately led to a lower
reducing antioxidant power in comparison to the other substituents. In addition, the introduction
of cyano 14 or acetyl group 15 improves the antioxidant activity of daphnetin, resulting in 3-fold
higher antioxidant activity than that of Trolox. The C-3 carboxy substituent 16 of daphnetin displayed
little effect on its antioxidant activity, and this situation did not change when the carboxyl group
was esterified 17 or amidated 18, which is quite different from the significant decrease in antioxidant
activity observed when converting the C-4 carboxy to its ester 10. In addition, after the introduction of
a strong electron-withdrawing benzothiazole to the C-3 position, benzothiazole 19 exhibited a slightly
reduced antioxidant activity. A simultaneous substitution at the C-4 position by carboxy group 21
led to a significant decrease in antioxidant activity, while this decline in activity could be alleviated
by conversion of the carboxy group to amide 22 or its isostere tetrazolium 23. However, it still had
lower antioxidant activity compared with that of compound 19. It is notable that the blocking of two
hydroxyls by an acetyl group led to a complete loss of total reducing power, indicating that catechol
moieties are key groups for the antioxidant activity of the daphnetin derivatives.
To evaluate the antioxidant activity of daphnetin derivatives in detail, the EC5 values of these
0
+
compounds were determined using the DPPH and ABTS assays as shown in Table 1. In the DPPH
assay, the EC50 value of DAP
Among these daphnetin derivatives, compound 4-carboxymethyl DAP
radical scavenging activity with EC50 values of 31.38 M (Figure 3). Similar to the results of the
1
is 46.20
µM, which is lower than that of Trolox (EC50 = 53.16
µM).
9
showed the strongest DPPH
µ
FRAP assay, a significant increase in the DPPH radical scavenging activities of the daphnetins could
be obtained when the C-4 position was substituted by strong electron-withdrawing groups, such as
an acetate group, while few changes in the EC50 value were observed when the trifluoromethyl
group was introduced to the C-4 position of daphnetin. Positive effects of the carboxyl group to the
antioxidant activity of daphnetin also appeared when the substitution site was located in the C-3
position, which could be weakened by esterification or amidation of the carboxyl group with EC50
values of 37.67 µM, 43.98 µM, and 55.65 µM for compound 16, 17, and 18, respectively. Interestingly,
negative effects of cyano and acetyl on the DPPH radical scavenging activity of daphnetins appeared
when the substitution site was switched from the C-4 to C-3 position, with the exception of an ethyl
carboxylate group, in which a weakly enhanced activity was observed. In contrast to the same increased
trend on the antioxidant activity of daphnetins when substituted by hydrophilic carboxyl, regardless
of the C-3 or C-4 substitution, the substitution of hydrophobic phenyl exerted opposite effects towards
the antioxidant activity of daphnetins depending on the substitution site. The antioxidant activity of
4
-phenyl DAP 3 was lower than that of DAP 1, while 3-phenyl DAP 13 was more effective. In addition,
the DPPH radical scavenging activity of daphnetin dramatically decreased towards the blocked
catechol groups, illustrating the important role of the catechol moieties in the depletion of DPPH.

Molecules 2018, 23, 2476
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Figure 2. Ferric reducing power of daphnetin and its derivatives. Values marked by the same alphabet
are not significantly different (p < 0.05).
0
Table 1. Antioxidant activity of daphnetin derivatives tested in 2,2 -diphenyl-1-picrylhydrazyl (DPPH)
0
+
and 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation (ABTS ) assays.DAP = daphnetin.
+
Compound
DPPH (EC /µM)
ABTS (EC /µM)
5
0
50
ab
ab
c
k
Trolox
DAP (1)
-Me DAP (2)
-Ph DAP (3)
53.16 ± 2.02
46.20 ± 1.45
85.63 ± 2.13
55.60 ± 0.78
65.50 ± 2.84
45.69 ± 1.26
46.57 ± 2.57
61.99 ± 1.43
62.17 ± 3.73
31.38 ± 0.68
50.20 ± 1.12
45.59 ± 1.48
241.33 ± 2.52
fghi
159.34 ± 4.63
ijk
4
4
224.50 ± 1.94
ab
bc
ab
ab
bc
bc
a
ijk
198.33 ± 28.18
k
4
-CCH DAP (4)
234.68 ± 2.03
3
ab
4
-CF DAP (5)
75.23 ± 1.58
3
abcde
4
4
4
-CH Cl DAP (6)
107.39 ± 6.72
2
hij
-CH OH DAP (7)
184.94 ± 2.04
2
k
-CH N DAP (8)
238.31 ± 4.47
2
3
a
4
-CH COOH DAP (9)
-CH COOMe DAP (10)
72.31 ± 2.70
2
ab
ab
f
jk
4
222.70 ± 1.84
2
ghi
Fraxetin (11)
Ac DAP (12)
173.07 ± 4.83
l
698.04 ± 5.22
4562.49 ± 50.1
ab
abcde
3
3
3
-Ph DAP (13)
-CN DAP (14)
-Ac DAP (15)
-COOH DAP (16)
-COOEt DAP (17)
-PPA DAP (18)
-BT DAP (19)
-BT-4-COOH DAP (21)
-BT-4-CONH DAP (22)
43.67 ± 2.08
47.02 ± 2.05
50.62 ± 0.61
37.67 ± 1.16
43.98 ± 0.89
55.65 ± 1.48
71.31 ± 2.40
108.01 ± 1.42
ab
ab
ab
ab
ab
bc
d
abcd
95.80 ± 4.18
abc
80.52 ± 0.61
efgh
3
145.67 ± 5.77
defg
3
138.88 ± 0.14
abcd
3
3
91.25 ± 2.29
abcd
86.57 ± 3.17
abcde
3
130.50 ± 3.54
108.33 ± 6.35
bc
bcdef
3
66.82 ± 1.38
122.34 ± 4.41
2
e
cdef
3
4
-BT-4-CN H DAP (23)
167.85 ± 11.63
121.72 ± 0.22
4
ab
abcde
-CF -3-Me DAP (24)
53.76 ± 2.25
107.21 ± 6.23
3
Values marked by the same alphabet are not significantly different (p < 0.05).

Molecules 2018, 23, 2476
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+
In the ABTS radical assay, Table 1 and Figure 3 indicated that 4-carboxymethyl DAP
9
also
+
showed strong scavenging activity on the ABTS radical, and their EC values were 72.38
to the FRAP assay and DPPH assay results, the ABTS radical scavenging ability of the daphnetins
could be significantly enhanced by the substitution of a strong electron-withdrawing carboxyl group
µM. Similar
50
+
on the C-4 position. Taken together, 4-carboxymethyl DAP
9 is the most powerful antioxidant in
these compounds and led to the best antioxidant activity observed in all the assays used in this
study. In addition, diacetyl DAP 12 displayed very low antioxidative capacity with an EC50 value of
4
562.49
µM. This provides additional evidence for the importance of the presence of two hydroxylated
groups at the ortho position for the antioxidant activity of these compounds.
Figure 3. Concentration-response curve for the radical-scavenging activity of 4-CH COOH DAP (
9
),
2
0
daphnetin (DAP) (
1
), and positive control compound Trolox in 2,2 -diphenyl-1-picrylhydrazyl (DPPH)
0
+
(
a) and 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation (ABTS ) (b) assays.
Based on these observations, a brief structure-activity relationship could be summarized.
The existence of the phenolic groups and their positions are very important for the antioxidant activity
of daphnetin. In particular, the catechol moiety plays a key role in enhancing the radical scavenging
and ferric reducing activities. The introduction of an electron withdrawing hydrophilic group,
such as a carboxyl group at the C-4 position of daphnetin, could enhance the antioxidative capacity
significantly, but this trend was not observed in the C-3 substitution. However, the introduction of
a hydrophobic phenyl group exhibited negative effects on the antioxidant activity in both the C-3
and C-4 substitutions. It is worth repeating that the carboxyl group displays more positive effects
than all the other substituents, implying that the carboxyl group could serve as a pharmacophore for
the antioxidant activity of daphnetins, and experiments to examine the antioxidant mechanism of
carboxymethyl-substituted daphnetins are merited.
Although catechol coumarins have been reported to possess a variety of therapeutic benefits,
this family suffers from low oral bioavailability due to poor metabolic stability [25,26]. The
simultaneous improvement of utility as a drug and pharmacological activity is still a challenge for
the structural optimization of lead compounds. From the view of the chemical structure, the catechol
group is not only the pharmacophore of the coumarins but also the susceptible group for the attack of
phase II metabolic enzymes. As phenolic hydroxyls, catechol coumarins primarily undergo conjugative
reactions mediated by phase II metabolizing enzymes, including uridine-5-diphosphate glucuronosyl
transferase (UGT) and catechol-O-methyltransferase (COMT) [27–29]. Considering that compound
9
exhibited the best antioxidant result in this study, the metabolic elimination t1/2 of compound
9
in the UGT and COMT incubation systems was investigated in more detail and compared with that
of Compound 1 in vitro (Supporting Information). The results demonstrated a five-fold longer t1/2
for compound
9 than that of compound 1, with t1/2 values of 96.5 min and 20.3 min, respectively
(Figure S1). These results indicated that C-4 carboxyethyl substitution improves the metabolic

Molecules 2018, 23, 2476
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stability of daphnetin. In summary, 4-carbethoxyl DAP
9
holds strong potential to be developed
as an antioxidant agent with excellent antioxidant activity and good pharmacokinetic properties.
3
. Materials and Methods
3
.1. Materials and Measurements
The H NMR and 13C NMR spectra were recorded on a Bruker ARX (Bruker, Rheinstetten,
1
Germany) 400 MHz or Bruker AMX 400 MHz or 600 MHz spectrometer in dimethyl sulfoxide
(
DMSO-d ) if not noted otherwise, and the chemical shifts were expressed as ppm using trimethylsilane
6
(TMS) as an internal reference. High-resolution mass spectral (HRMS) analyses were measured on
a TripleTOF
™ Mass Spectrometer (TripleTOF 5600, SCIEX, Foster City, CA, USA). Daphnetin and its
derivatives were analyzed using an ultra-fast liquid chromatography spectrometry system (Shimadzu,
Kyoto, Japan) equipped with two LC-20AD pumps, a DGU-20A3 vacuum degasser, a SIL-20ACHT
auto-sampler, a CTO-20AC column oven, and an SPD-M 20A diode-array detector (DAD). All the
reagents used in the synthesis were obtained commercially and used without further purification.
The reactions were monitored using thin layer chromatography (TLC) on glass packed precoated
silica gel GF254 plates. Flash column chromatography was performed using silica gel (200–300 mesh),
which was purchased from the Qingdao Ocean Chemical Co., Ltd (Qingdao, China). Daphnetin and
1
1 were purchased from Chengdu Pufei De Biotech Co., Ltd. (Sichuan, China).
3
.2. Synthesis of 4-Substituted Daphnetin Derivatives
General procedure for the synthesis of 4-substituted daphnetin derivatives (
was added drop-wise at room temperature to a mixture of 1,2,3-phenenyl triacetate (5.0 mmol) and
the correctly substituted -ketoester (10.0 mmol) and stirred for 6–8 h. After TLC analysis indicated
that the reaction was complete, the reaction mixture was slowly poured into a mixture of ice water
100 mL) with stirring. The resultant suspension was filtered, and the collected solid was washed
2–6): Perchloric acid (5.0 mL)
β
(
with water and dried; the crude compound was recrystallized from methanol to produce the desired
compound [30].
7
,8-Dihydroxy-4-methyl-2H-chromen-2-one (
was combined with ethyl acetoacetate in the presence of perchloric acid and left to react. The crude
compound was recrystallized from the methanol to produce compound as a light white solid with
a yield of 78%. 1H NMR (600 MHz, DMSO)
10.05 (s, 1H), 9.28 (s, 1H), 7.09 (d, J = 8.6 Hz, 1H),
.82 (d, J = 8.6 Hz, 1H), 6.13 (d, J = 1.1 Hz, 1H), 2.36 (s, 3H); 13C NMR (151 MHz, DMSO)
160.67,
2): Using the general procedure, 1,2,3-phenenyl triacetate
2
δ
6
δ
1
1
54.40, 149.87, 143.79, 132.62, 115.95, 113.23, 112.58, 110.65, 18.72; HRMS (ESI) for C H O , Calcd.
92.0423, found 192.0355 [M − H] .
10
8
4
−
7,8-Dihydroxy-4-phenyl-2H-chromen-2-one (
3): Using the general procedure, 1,2,3-phenenyl triacetate
was combined with ethyl benzoylacetate in the presence of perchloric acid and left to react. The crude
compound was recrystallized from the methanol to produce compound
3 as a white solid with a yield
1
of 82 %. H NMR (600 MHz, DMSO)
6
δ
10.19 (s, 1H), 9.42 (s, 1H), 7.77–7.32 (m, 5H), 6.88–6.65 (m, 2H),
160.56, 156.42, 150.11, 144.37, 135.94, 133.10, 129.94, 129.20,
28.90, 117.78, 112.81, 111.97, 110.72; HRMS (ESI) for C H O , Calcd. 254.0579, found 253.0515
.14 (s, 1H); 13C NMR (151 MHz, DMSO)
δ
1
15
10
4
−
[M − H] .
7
,8-Dihydroxy-4-tert-butyl-2H-chromen-2-one (4): Using the general procedure, 1,2,3-phenenyl triacetate
was combined with ethyl 4,4-dimethyl-3-oxovalerate in the presence of perchloric acid and left to
react. The crude compound was recrystallized from methanol and purified using flash column
1
chromatography on silica gel to produce compound
600 MHz, DMSO)
4
as a white solid with a yield of 39%. H NMR
(
δ
10.21 (s, 1H), 9.25 (s, 1H), 7.34 (d, J = 8.6 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.04 (s, 1H),
1
1
.33 (s, 9H); 13C NMR (151 MHz, DMSO)
δ
177.58, 175.25, 150.54, 147.23, 133.35, 116.88, 115.31, 114.13,
−
05.45, 36.69, 28.04. HRMS (ESI) for C H O , Calcd. 234.0892, found 233.0826 [M − H] .
13
14
4

Molecules 2018, 23, 2476
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7,8-Dihydroxy-4-trifluoromethyl-2H-chromen-2-one (
5): Using the general procedure, 1,2,3-phenenyl
triacetate was combined with ethyl 4,4,4-trifluoroacetoacetate in the presence of perchloric acid and left
to react. The crude compound was recrystallized from methanol to produce compound
5
as a yellow
1
solid with a yield of 58%. H NMR (600 MHz, DMSO)
δ
10.53 (s, 1H), 9.61 (s, 1H), 7.07 (dd, J = 8.8,
159.29, 151.03, 144.54,
1
1
2
.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.74 (s, 1H); 13C NMR (151 MHz, DMSO)
δ
40.76, 140.55, 133.40, 115.73, 113.61, 112.27, 106.45; HRMS (ESI) for C H F O , Calcd. 236.0140, found
10
5
3
4
−
45.0072 [M − H] .
7,8-Dihydroxy-4-chloromethyl-2H-chromen-2-one (6): Using the general procedure, 1,2,3-phenenyl
triacetate was combined with ethyl 4-chloroacetoacetate in the presence of perchloric acid and left to
react. The crude compound was recrystallized from methanol to produce compound
6 as a white solid
1
with a yield of 69%. H NMR (600 MHz, DMSO)
δ
10.19 (s, 1H), 9.40 (s, 1H), 7.18 (d, J = 8.7 Hz, 1H),
160.55, 151.86, 150.23,
6
.85 (d, J = 8.7 Hz, 1H), 6.42 (s, 1H), 4.94 (s, 2H); 13C NMR (151 MHz, DMSO)
δ
1
2
44.15, 132.92, 115.95, 112.79, 111.42, 110.57, 41.95; HRMS (ESI) for C H ClO , Calcd. 226.0033, found
24.9967 [M − H] .
10
7
4
−
7,8-Dihydroxy-4-hydroxymethyl-2H-chromen-2-one (7): Chloride 6 (0.5 g, 2.2 mmol) was dissolved in
a mixture of DMF (3 mL) and H O (10 mL) with stirring and refluxed for 20 h. After TLC indicated that
2
the reaction was complete, the mixture was diluted with water. The resultant suspension was filtered,
and the collected solid was washed with water and dried. The crude compound was recrystallized
1
from methanol to yield hydroxymethyl
DMSO)
Hz, 1H), 5.55 (t, J = 5.5 Hz, 1H), 4.69 (d, J = 4.2 Hz, 2H); C NMR (151 MHz, DMSO)
49.75, 143.82, 132.75, 114.75, 112.65, 110.73, 106.85, 59.56; HRMS (ESI) for C H O , Calcd. 208.0372,
7
as a light brown solid with a yield of 80%. H NMR (600 MHz,
δ
10.03 (s, 1H), 9.30 (s, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.24 (t, J = 1.4
13
δ
161.03, 157.70,
1
1
0
8
5
−
found 207.0304 [M − H] .
,8-Dihydroxy-4-azidemethyl-2H-chromen-2-one (
10 mL) and sodium azide (0.37 g, 5.5 mmol) was added and stirred overnight. After LC-MS indicated
7
(
8): Chloride 6 (0.5 g, 2.2 mmol) was dissolved in DMF
that the reaction was complete, the mixture was diluted with water. The resultant suspension was
filtered, and the collected solid was washed with water and dried. The crude compound was
1
recrystallized from methanol to yield azidemethyl
600 MHz, DMSO)
.27 (s, 1H), 4.77 (d, J = 0.8 Hz, 2H); 13C NMR (151 MHz, DMSO)
15.51, 112.82, 110.74, 109.76, 50.19; HRMS (ESI) for C H N O , Calcd. 233.0437, found 234.0513
8
as a grey solid with a yield of 85%. H NMR
(
6
δ
10.18 (s, 1H), 9.41 (s, 1H), 7.06 (d, J = 8.7 Hz, 1H), 6.83 (d, J = 8.7 Hz, 1H),
160.47, 151.05, 150.26, 144.01, 132.88,
δ
1
1
0
7
3
4
+
[
M + H] .
-(7,8-Dihydroxy-2-oxo-2H-chromen-4-yl)acetic acid (
triacetate was combined with 1,3-acetonedicarboxylic acid in the presence of perchloric acid and left
2
9): Using the general procedure, 1,2,3-phenenyl
to react. The crude compound was recrystallized from methanol to yield compound
9 as a white
1
solid with a yield of 78%. H NMR (400 MHz, DMSO)
δ
12.75 (s, 1H), 10.11 (s, 1H), 9.35 (s, 1H), 7.03
13
(d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 6.22 (s, 1H), 3.80 (s, 2H); C NMR (100 MHz, DMSO)
δ
1
71.23, 160.63, 150.99, 149.93, 143.95, 132.74, 116.03, 112.71, 112.57, 112.43, 37.88.
Methyl 2-(7,8-dihydroxy-2-oxo-2H-chromen-4-yl)acetic acid (10).
Using the general procedure,
1,2,3-phenenyl triacetate was combined with dimethyl 1,3-acetonedicarboxylate in the presence of
perchloric acid and left to react. The crude compound was recrystallized from methanol to yield
1
compound 10 as a white solid with a yield of 83%. H NMR (600 MHz, DMSO)
(
δ
10.12 (s, 1H), 9.36
13
s, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 6.24 (s, 1H), 3.92 (s, 2H), 3.65 (s, 3H); C NMR
(
3
151 MHz, DMSO)
δ
170.20, 160.52, 150.33, 150.06, 143.96, 132.78, 116.00, 112.77, 112.57, 112.40, 52.64,
−
7.27. HRMS (ESI) for C H O , Calcd. 250.0477, found 249.0413 [M − H] .
16
10
6
2-oxo-2H-Chromene-7,8-diyl diacetate (12): A solution of daphnetin 1 (0.5 g, 2.8 mmol), pyridine
(2.0 mL) and acetic anhydride (1 mL) in dichloromethane (DCM) (30 mL) was stirred overnight.
After TLC indicated that the reaction was complete, the reaction mixture was poured into water and

Molecules 2018, 23, 2476
10 of 13
acidified with 1 mol/L hydrochloric acid. The reaction mixture was extracted with DCM (50 mL
The combined organic layer was washed with water and brine, dried over anhydrous sodium sulfate,
×
3).
and concentrated by evaporation. The resultant suspension was recrystallized from methanol to yield
1
ester 12 as a grey solid with a yield of 92%. H NMR (400 MHz, CDCl )
7
δ
10.22 (s, 1H), 8.40 (s, 1H),
3
13
.59 (d, J = 8.6 Hz, 1H), 7.42–7.10 (m, 1H), 2.43 (s, 3H), 2.36 (s, 3H); C NMR (101 MHz, CDCl3)
δ
1
87.26, 167.34, 167.06, 158.66, 148.72, 148.01, 144.99, 130.57, 127.95, 121.23, 120.13, 116.85, 20.69, 20.27.
3
.3. Synthesis of 3-Substituted Daphnetin Derivatives
The synthetic routes of daphnetin derivatives 13–19 are illustrated in Scheme 2, and the details in
the synthetic procedure of the compounds are described in the Supporting Information based on our
previous study [18].
3-(Benzo[d]thiazol-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-4-carboxylic acid (21) and 3-(benzo[d]thiazol-2-yl)
-
7,8-dihydroxy-2-oxo-2H-chromene-4-carboxamide (22): NaCN (0.24 g, 5.4 mmol) was added to a solution
◦
of 19 (0.5 g, 1.6 mmol) in DMF (10 mL) and stirred for 3 h at 50 C. After the reaction was cooled
to 0 C, I (0.60 g, 2.4 mmol) was added and stirred for 5 h. After TLC indicated that the reaction
◦
2
was complete, the reaction mixture was poured into water and treated with an aqueous solution of
NaHSO . The reaction mixture was extracted with ethyl acetate. The combined organic layer was
3
washed with water and brine, dried over anhydrous sodium sulfate, and concentrated by evaporation.
The resultant suspension was recrystallized from methanol to yield compound 20 as a brown solid.
◦
Compound 20 was suspended in 60% sulfuric acid solution and heated to 100 C for 8 h. The reaction
mixture was extracted with abundant ethyl acetate and methanol. The combined organic layer was
concentrated by evaporation and purified using preparative high-performance liquid chromatography
1
to yield acid 21 with a yellowish-brown solid with a yield of 60%. H NMR (600 MHz, DMSO)
δ 14.30
(
7
s, 1H), 10.84 (s, 1H), 9.77 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.61–7.53 (m, 1H),
.52–7.46 (m, 1H), 7.05 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H); 13C NMR (151 MHz, DMSO)
δ
166.56,
1
60.36, 159.17, 152.45, 152.13, 143.79, 136.00, 132.90, 126.97, 125.92, 123.02, 122.48, 119.02, 114.52, 109.64;
−
HRMS (ESI) for C H NO S, Calcd. 355.0151, found 354.0084 [M − H] .
17
9
6
◦
Compound 20 was suspended in 60% sulfuric acid solution and heated to 80 C for 3 h.
The reaction mixture was extracted with abundant ethyl acetate and methanol. The combined
organic layer was concentrated by evaporation and purified using preparative high-performance
1
liquid chromatography to yield acid 22 with a yellowish-brown solid with a yield of 36%. H NMR
(
7
600 MHz, DMSO)
δ
10.77 (s, 1H), 9.67 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 8.06 (s, 1H), 8.00–7.90 (m, 2H),
.60–7.53 (m, 1H), 7.52–7.45 (m, 1H), 7.14 (d, J = 8.8 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H); 13C NMR (151 MHz,
DMSO)
δ 166.50, 160.63, 159.42, 152.39, 152.09, 151.09, 143.76, 135.97, 132.67, 126.75, 125.81, 123.14,
1
22.31, 119.61, 114.28, 110.49, 109.76; HRMS (ESI) for C H N O S, Calcd. 354.0310, found 353.0245
17 9 2 5
−
[M −·H] .
3
-(Benzo[d]thiazol-2-yl)-7,8-dihydroxy-4-(1H-tetrazol-5-yl)-2H-chromen-2-one (23): NaN (0.30 g 4.5 mmol)
3
was added to a solution of 19 (0.25 g, 0.8 mmol) and ZnBr (0.45 g, 2.0 mmol) in dioxan (20 mL) and
2
◦
heated to 90 C for 10 h. After TLC indicated that the reaction was complete, the reaction mixture was
extracted with abundant ethyl acetate and methanol. The combined organic layer was concentrated by
evaporation and purified using preparative high-performance liquid chromatography to yield acid
1
2
3
with a brown solid with a yield of 42%. H NMR (600 MHz, DMSO)
δ
10.88 (s, 1H), 9.84 (s, 1H),
8
.16–8.08 (m, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.48–7.36 (m, 2H), 6.90 (d, J = 8.8 Hz, 1H), 6.50 (d, J = 8.7 Hz,
13
1
H); C NMR (151 MHz, DMSO)
δ 159.54, 152.62, 151.88, 143.19, 135.88, 132.96, 126.89, 126.14, 123.07,
1
22.32, 119.23, 115.91, 114.29, 112.24, HRMS (ESI) for C H N O S, Calcd. 379.0375, found 378.0311
17
9
5
4
−
[M − H] .
7,8-Dihydroxy-3-methyl-4-(trifluoromethyl)-2H-chromen-2-one (24): Drop-wise perchloric acid (5.0 mL)
was added to a mixture of 1,2,3-phenenyl triacetate (0.5 g, 2.0 mmol) and ethyl 4,4,4-trifluoro-
-methyl-3-oxobutanoate (0.4 g, 0.2 mmol) at room temperature and stirred for 6 h. After TLC indicated
2

Molecules 2018, 23, 2476
11 of 13
that the reaction was complete, the reaction mixture was poured into water and extracted with ethyl
acetate. The combined organic layer was washed with water and brine, dried over anhydrous sodium
sulfate, and concentrated by evaporation. The residue was purified by column chromatography using
1
silica gel to obtain compound 24 as a white solid with a yield of 28%. H NMR (600 MHz, DMSO)
1
δ
13
0.28 (s, 1H), 9.48 (s, 1H), 7.14–6.97 (m, 1H), 6.87 (d, J = 9.0 Hz, 1H), 2.27 (q, J = 3.7 Hz, 3H); C NMR
(
1
151 MHz, DMSO)
δ
161.34, 149.38, 142.52, 134.88, 134.68, 132.87, 124.76, 123.30, 122.91, 115.56, 113.29,
−
07.74, 14.05. HRMS (ESI) for C H F O , Calcd. 260.0296, found 259.0231 [M − H] .
11
7
4
4
3
.4. Antioxidant Activity Assay In Vitro
3
.4.1. DPPH Assay
Each sample in an ethanol solution (0.1 mL) was added to 3.9 mL DPPH ethanol solution
0.1 mmol/L). The solution was vortexed for 10 s and incubated at room temperature for 30 min.
(
The absorbance of the resulting solution was measured at 517 nm (Hitachi U-2800 Spectrophotometer,
Tokyo, Japan). The Trolox standard solution (concentration range: 0–0.4 mmol/L) in ethanol was
analyzed under the same conditions as those used for calibration. The values of the DPPH radical
scavenging activity were determined using a calibration curve of Trolox and expressed as the Trolox
equivalent (TEACDPPH, mmol Trolox/mmol sample).
+
3
.4.2. ABTS Assay
ABTS+ solution was produced by reacting 7 mmol/L ABTS water solution (10 mL) with
2
.45 mmol/L potassium persulphate (10 mL). The mixture was incubated in the dark at room
+
temperature for 15–16 h before use. To evaluate the antioxidant activity, the ABTS solution was
diluted with ethanol until its absorbance reached 0.7 at 734 nm. Each sample solution (0.1 mL) was
added to 3.9 mL ABTS solution, and the absorbance was measured at room temperature after 3 min.
+
The Trolox standard solution (concentration range: 0–0.4 mmol/L) in ethanol was analyzed under the
same conditions as those used for the calibration. Values of the ABTS radical scavenging activity were
determined using a calibration curve of Trolox and expressed as the Trolox equivalent (TEACABTS
mmol Trolox/mmol sample).
,
3
.4.3. FRAP Assay
The FRAP (ferric reducing antioxidant power) reagent was prepared with acetate buffer
(
(
300 mmol/L, pH 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ) (10 mmol/L in HCl, 40 mmol/L) and FeCl3
20 mmol/L). The proportions were 10:1:1 (v:v:v), respectively. Each sample solution was added to the
◦
FRAP reagent (1:30, v:v) and incubated at 37 C for 5 min. The absorbance was determined at 593 nm
using a spectrophotometer (Hitachi U-2800 Spectrophotometer, Tokyo, Japan). The Trolox standard
solution (concentration range: 0–0.4 mmol/L) in ethanol was analyzed under the same conditions as
those used for the calibration. The values of the reducing power were determined using a calibration
curve of Trolox and expressed as the Trolox equivalent (TEACFRAP, mmolTrolox/mmol sample).
3
.5. Statistical Analysis
All the experiments were conducted in triplicate, and the data were analyzed using SPSS software
(
(
Version 14.0; SPSS, Chicago, IL, USA). A one-way analysis of variance and least significant difference
LSD) were used to differentiate the mean values.
4
. Conclusions
In summary, daphnetin was chosen as the lead compound and more than twenty derivatives
were designed and synthesized by structural modification on C-3 and/or C-4 position of
daphnetin. The antioxidant activities of these compounds were assayed. Meanwhile, the potential
structure–inhibition relationships of these daphnetin derivatives were well-summarized and discussed.

Molecules 2018, 23, 2476
12 of 13
The results clearly demonstrated that the catechol group of daphnetins was essential for antioxidant
activity, while introduction of an electron withdrawing hydrophilic group at the C-4 position of
daphnetin might be beneficial for antioxidant effect. Among all the tested compounds, 4-carboxymethyl
daphnetin (compound
this compound could serve as an ideal lead compound for the development of a potent antioxidant.
Further investigation on the metabolic stability demonstrated compound exerted a three-fold longer
9) exhibited the strongest antioxidant activity in all of the assays, implying that
9
t1/2 than that of compound 1 in the UGT and COMT incubation systems in vitro. All these findings are
very useful for medicinal chemists to design and develop potent antioxidants based on the structural
modifications of coumarin derivatives.
Supplementary Materials: The following are available online, Figure S1: Plot of incubation time vs. natural log
percent remaining.
Author Contributions: Conceptualization, P.W.; methodology, Y.X., C.C., and T.D.; writing-original draft
preparation, Y.X.; writing-review and editing, all authors.
Funding: This work was supported by National Key Research and Development Program of China
(
2017YFC1700200, 2017YFC1702000), National Natural Science Foundation of China (81603187, 31601517 and
3
1600641), Natural Science Foundation of Shanghai (18ZR1436500), and the Fundamental Research Funds for the
Central Universities (DUT18LK39).
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(