Novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives as topoisomerase I inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.12.035

In this study, two series of novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives containing an α,β-unsaturated lactone fragment were synthesized and screened for Topo I inhibition and antitumor activity. The topoisomerase I inhibitory activities and cytotoxicities against three human cancer cell lines (MCF-7,Hela,A549) were evaluated. The results revealed that series 2, compounds bearing an exocyclic double bond on the furanone ring, generally showed more potent activity than series 1, compounds lacking an exocyclic double bond. Several compounds of series 2 possess significant Topo I inhibitory activity and potent antiproliferative activity against cancer cell lines. Further mechanism studies of the most active compound of series 2 (B-15) indicated that synthetic compounds can not only stabilize the drug-enzyme-DNA covalent ternary complex as well as camptothecin, but also interfere with the binding between Topo I and DNA. The binding patterns of these compounds with Topo I and structure-activity relationships are discussed.

Full text of DOI:10.1016/j.ejmech.2016.12.035

Accepted Manuscript
Novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives as topoisomerase I
inhibitors
Cheng-Kang Peng, Ting Zeng, Xing-Jun Xu, Yi-Qun Chang, Wen Hou, Kuo Lu, Hui
Lin, Ping-Hua Sun, Jing Lin, Wei-Min Chen
PII:
S0223-5234(16)31042-X
10.1016/j.ejmech.2016.12.035
EJMECH 9126
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 September 2016
Revised Date: 13 December 2016
Accepted Date: 17 December 2016
Please cite this article as: C.-K. Peng, T. Zeng, X.-J. Xu, Y.-Q. Chang, W. Hou, K. Lu, H. Lin, P.-H. Sun,
J. Lin, W.-M. Chen, Novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives as topoisomerase I
inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.035.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives as
topoisomerase I inhibitors
1
1
Cheng-Kang Peng , Ting Zeng , Xing-Jun Xu, Yi-Qun Chang, Wen Hou, Kuo Lu, Hui Lin,
Ping-Hua Sun, Jing Lin*, Wei-Min Chen*
College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
These authors contributed equally to this work.
1
*Corresponding authors. Tel.: +86 20 8522 1367(J. Lin), +86 20 8522 4497 (W.-M. Chen).
Fax: +86 20 8522 4766.
E-mail address: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn(W.-M. Chen).

ACCEPTED MANUSCRIPT
Novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives as
topoisomerase I inhibitors
1
1
Cheng-Kang Peng , Ting Zeng , Xing-Jun Xu, Yi-Qun Chang, Wen Hou, Kuo Lu, Hui Lin,
Ping-Hua Sun, Jing Lin*, Wei-Min Chen*
College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
1
These authors contributed equally to this work.
*Corresponding authors. Tel.: +86 20 8522 1367(J. Lin), +86 20 8522 4497 (W.-M. Chen).
Fax: +86 20 8522 4766.
E-mail address: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn(W.-M. Chen).
ABSTRACT
In this study, two series of novel 4-(4-substituted amidobenzyl)furan-2(5H)-one
derivatives containing an α,β-unsaturated lactone fragment were synthesized and
screened for Topo I inhibition and antitumor activity. The topoisomerase I inhibitory
activities and cytotoxicities against three human cancer cell lines (MCF-7，Hela，A549)
were evaluated. The results revealed that series 2, compounds bearing an exocyclic
double bond on the furanone ring, generally showed more potent activity than series 1,
compounds lacking an exocyclic double bond. Several compounds of series 2 possess
significant Topo I inhibitory activity and potent antiproliferative activity against
cancer cell lines. Further mechanism studies of the most active compound of series 2
(B-15) indicated that synthetic compounds can not only stabilize the
drug-enzyme-DNA covalent ternary complex as well as camptothecin, but also
interfere with the binding between Topo I and DNA. The binding patterns of these
compounds with Topo I and structure-activity relationships are discussed.
Keywords: Furan-2(5H)-one; α, β-unsaturated lactones; Topoisomerase I; Antitumor
1

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1
. Introduction
Cancer is one of the leading global health problems worldwide with an
increasing number of patients every year [1]. The current oncotherapy methods
include radiotherapy, immunotherapy, surgery and chemotherapy, and the design and
synthesis of new drugs for the treatment of cancer is a crucial area of research in
medicinal chemistry [2].
Topoisomerase I (Topo I) is a ubiquitous nuclear enzyme that catalyzes
transient cleavage and reconnection in DNA, events which are associated with DNA
replication, transcription, recombination and chromatin remodeling [3,4]. Evidence
suggests that DNA topoisomerase I is an effective target for the discovery of
antitumor drugs as a result of its high over-expression in cancer cells [5-8].
Compounds bearing α,β-unsaturated lactones or 2-furanone (Fig. 1) have been
reported to have a broad spectrum of biological roles, including antitumor,
antibacterial, and anti-inflammatory activities [9], which suggests that the
α,β-unsaturated lactone is a promising pharmacophore for drug design. Other studies
have revealed that compounds bearing an α,β-unsaturated lactone fragment, such as
some cardiac glycosides, acetogenins and securinines could serve as Topo I inhibitors
[
7,10,11]. Meanwhile, several reports have revealed that exocyclic double bonds are
an essential structural feature which increases antitumor activity [12,13]. Many
clinical trials have been conducted of anticancer drugs, such as Eribulin [14],
calicheamicin [15], all of which contain an exocyclic double bond. In this study, two
series of 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives bearing an
α,β-unsaturated lactone fragment (Fig. 1, Series 1: without exocyclic double bond;
Series 2: with exocyclic double bond) have been designed, synthesized and evaluated
as Topo I inhibitors.
The topoisomerase I inhibitory activity and the cytotoxicity against three human
cancer cell lines (MCF-7, Hela, A549) of the synthesized compounds was evaluated
and several compounds that show both excellent Topo I inhibitory activity and
effective potent antiproliferative activity against cancer cell lines were identified. The
novel dual action mechanism on Topo
I
of these 4-(4-substituted
2

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amidobenzyl)furan-2(5H)-one derivatives was illuminated by three different
electrophoresis experiments and docking studies. In addition, the structure-activity
relationships of these compounds are discussed.
2
. Chemistry
The design strategy for 4-(4-substituted amidobenzyl)furan-2(5H)-one
derivatives is shown in Fig. 1. Securinine bearing an α,β-unsaturated lactone,
extracted from securinega [16], shows potent antiproliferative activity as a Topo I
inhibitor [7,17,18] suggesting that securinine may be used as a lead compound for the
development of topoisomerase I inhibitors as antitumor agents. An attempt was made
to simplify the structure of securinine and design target compounds whose structure
can be easily synthesized. First, the bond between C2 and C9 of securinine was
opened, producing structure 1. A molecular docking study using Sybyl-8.1 was
performed to investigate the interaction between structure 1 and Topo I (PDB: 1K4T).
Guided by the docking results (shown in Supporting Information, SI), compounds in
series 1 were designed by scaffold hopping from structure 1. Rings B and C of
structure 1 were replaced with a phenyl ring and an amido bond. The amide is
connected to the phenyl ring by a variable length linker. In series 1, C4 of the
furanone ring is a linear hydrocarbon, while in series 2, C4 of the furanone ring was
modified to produce an exocyclic double bond to assess whether such an exocyclic
double bond is related to antitumor activity. The synthesis of the designed compounds
in Fig. 1 is outlined in Scheme 1 (series 1: A-1~A-7) and Scheme 2 (series 2:
B-1~B-15).
[
Figure 1]
2
.1. Synthesis of the target compounds, series 1
-Phenylpropanal was reacted with glyoxylic acid monohydrate and potassium
3
hydroxide in dichloromethane at room temperature affording (Z)-3-benzyl-4-oxobut-
o
2
-enoic acid (1-1) [19]. Compound 1-1, treated with nitrosonitric acid at -10 C
afforded the p-nitrophenyl compound 1-2, which was reduced to the hydroxyl
3

ACCEPTED MANUSCRIPT
o
compound (1-3) with sodium borohydride in methanol at 0 C [20]. Compound 1-3
with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) in dry
dichloromethane at room temperature afforded 4-(4-nitrobenzyl)furan-2(5H)-one
(1-4), which was reduced to 4-(4-aminobenzyl)-furan-2(5H)-one (1-5) with iron and
o
ammonium chloride in ethanol at 80 C [21]. This compound (1-5), treated with acyl
o
chloride and dry pyridine in tetrahydrofuran at 0 C afforded the amides A-1~A-7
[
22].
[
Scheme 1]
Synthesis of the target compounds series 2
Reaction of 1-(bromomethyl)-4-nitrobenzene with 2,4-pentanedione in methanol
o
at 75 C gave 4-(4-nitrophenyl)butan-2-one (2-1) which, treated with glyoxylic acid
o
monohydrate in phosphoric acid at 85 C afforded compound 2-2. This was refluxed
with
p-toluenesulfonic
acid
in
toluene
to
afford
5-methylene-4-
(4-nitrobenzyl)furan-2(5H)-one (2-3) [23]. This was refluxed with iron and
ammonium chloride in ethanol to obtain the amine 2-4 [21] which, with the
o
appropriate acyl chloride and dry pyridine in tetrahydrofuran at 0 C afforded the
target compounds B-1~B-15 [22].
[
Scheme 2]
1
13
All the compounds were fully analyzed and characterized by H NMR, C NMR,
mass spectrometry (MS) and high resolution mass spectrometry (HRMS).
3
. Results and Discussion
3
.1. Topo I inhibitory activity
It has been reported that Topo I is one of the targets of compounds bearing a
-furanone with an α,β-unsaturated lactone [9]. Consequently, a DNA relaxation
2
assay was used to investigate the inhibition of Topo I by 4-(4-substituted
amidobenzyl)furan-2(5H)-one derivatives. As shown in Fig. 2A, the Topo I inhibitory
activity is directly related to the conversion of supercoiled DNA to its relaxed form
4

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[
24]. Camptothecin (CPT), a well-known Topo I inhibitor, was chosen as a positive
control. As shown in Fig. 2A, when treated with 100 µM of various furanone
derivatives, inhibition of Topo I between 11-94%, revealed by gray scale value
analysis, shown in Table 1, is observed. Notably, the most active compound is B-15
which showed an inhibition rate of 94.04% at 100 µM, significantly higher than that
of the positive control CPT (79.50% at 100 µM). In addition, compounds B-3, B-5,
B-8, B-12, B-13 exhibited considerable activity, with inhibition rates of 47.94%,
4
1.93%, 43.32%, 48.64%, and 44.62% respectively. However, compounds of series 1
that lack the exocyclic double bond generally showed poor inhibitory activity, with
inhibition rates <20%. These results show that introduction of exocyclic double bond
improves the Topo I inhibitory activity of 4-(4-substituted amido-benzyl)-
furan-2(5H)-one derivatives. We then applied the most active compound (B-15) to a
concentration gradient experiment, shown in Fig. 3 and found that the inhibition rate
of B-15 decreased linearly in a concentration-dependent manner. Based on the results
of Topo I inhibitory activity, the structure-activity relationship of the furanone
derivatives can be summarized as follows: (1) introduction of exocyclic double bond
dramatically improves the Topo I inhibitory activity; (2) the longer the carbon chain in
the furanone derivatives, the higher the inhibitory activity; (3) substitution of both
electron withdrawing groups or electron donating groups at the para-positon of the
phenyl ring can improve the Topo-I inhibitory activity.
[
Table 1]
[
[
Figure 2]
Figure 3]
To determine if the exocyclic double bond is essential to the Topo I inhibitory
activity, a molecular docking study was performed with Sybyl-8.1 [25] to clarify the
possible interaction mode between the furanone derivatives and Topo I (PDB:
1
K4T).The complexes were validated by molecular dynamics (MD) simulation
(RMSD plots were shown in Figure S2) and the free binding energy (∆G) was
calculated afterward (Table S1). As shown in Fig. 4, the furanone rings of the active
5

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compounds (B-13, B-14, B-15) were selected as representatives of the compounds in
series 2. These compounds could interact with the active pocket of Topo I by
π-π stacking or the π-alkyl stacking. However, no π-π stacking interactions or π-alkyl
stacking interactions could be observed between compounds in series 1 and the active
pocket of Topo I (data not shown), which probably because removing the double-bond
broken the cyclic conjugate structure. These results show that the exocyclic double
bond could effectively enhance the interaction between the compounds and Topo I by
π-π stacking interactions, the π-lone pair stacking interaction and π-alkyl stacking
interaction, most likely through strengthening the conjugative effect of the furanone
ring of series 2. The docking results suggest that introduction of a double bond into
the furanone ring may be a feasible strategy for the further development Topo I
inhibitors.
[
Figure 4]
3
.2. Cytotoxicity
The MTT assay was used to evaluate antitumor activity by testing the
cytotoxicity against three human cancer cells lines, MCF-7 (human breast
adenocarcinoma cell line), HeLa (human cervix tumor cell line), A549
(adenocarcinomic human alveolar basal epithelial cell line). Camptothecin (CPT) was
again used as a positive control. The cell growth inhibition activities (IC ) of the
50
tested compounds are shown in Table 2 which shows that the results of cytotoxicity
are generally consistent with the inhibitory activity. Compounds B-3, B-5, B-8, B-12,
B-13, B-15 exhibit higher cytotoxicity than CPT against MCF-7 cells and Hela cells.
Furthermore, B-15 with the highest antiproliferative activity, is the most active against
all three human cancer cell lines. Consistent with the Topo I inhibitory results,
compounds of series 1, lacking the exocyclic double bond, also generally showed
poor inhibitory activity. These results demonstrate that 4-(4-substituted amidobenzyl)-
furan-2(5H)-one derivatives exhibit positive antitumor activity, probably due, in part
to their Topo I inhibitory activity.
[
Table 2]
6

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3
.3. DNA-insertion assay
Since the results of the Topo I inhibition assay clearly indicate that Topo I is a
target of the 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives, the
mechanism of action of these derivatives was investigated. According to the catalytic
process of Topo I (Fig. 5), the mechanism of inhibition of Topo I possibly consists of
insertion into DNA, forming a drug-enzyme-DNA ternary complex, covalent or
noncovalent, and retarding the combination between enzyme and DNA [7,26]. Thus,
the DNA-insertion assay was performed to confirm whether these compounds can
insert into DNA. Ethidium bromide (EB), a DNA intercalating agent, was used as a
positive control [27] and CPT, which inhibits Topo I by forming the
drug-enzyme-DNA covalent ternary complex, was used as a negative control. As
shown in Fig. 6, as a DNA-inserting agent, EB can successfully inhibit the conversion
of the supercoiled form (lower bands) to the relaxed form (upper bands) in the
presence of a large excess of Topo I. However, all of our compounds showed no
unwinding effect on DNA even at levels up to 100 µM, and thus are similar to the
negative control CPT. This result demonstrates that these furanone derivatives fail to
exert Topo I inhibitory activity through DNA insertion.
[
[
Figure 5]
Figure 6]
3
.4. Topo I-mediated DNA cleavage assay
The Topo I-mediated DNA cleavage assay was conducted to investigate whether
B-15, the most active compound towards Topo I, can stabilize the Topo I-DNA
cleavage complex through formation of a drug-enzyme-DNA covalent ternary
complex. CPT, which has been shown to stabilize Topo I-cleavable complexes [28],
was used as a positive control and the percentage of nicked DNA is shown in Fig. 7B.
In this assay, Topo I was hydrolyzed by proteinase K, and a secondary electrophoresis
was applied to separate the relaxed DNA from nicked DNA. As presented in Fig. 7,
when treated with 25 µM and 50 µM CPT, the amount of linear DNA was distinctly
7

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increased, as expected. When Topo I was incubated with 25, 50, 100, 200, 400 µM of
B-15, the percentage of nicked DNA was increased linearly in a concentration-
dependent manner. These results indicate that similar to CPT, one of the Topo I
inhibitory compounds stabilizes the Topo I-DNA cleavage complex.
[
Figure 7]
3
.5. Inhibition of the combination between DNA and Topo I
In order to investigate whether B-15 can also interfere with the binding between
Topo I and DNA, an electrophoretic mobility shift assay (EMSA) was performed [29].
In this assay, CPT, which does not inhibit of the binding of Topo I to DNA, was used
as a negative control. The proportion of Topo I-DNA complex formed was used to
explain the result of the electrophoretic mobility shift assay (Fig. 8B). As shown in
Fig. 8, when treated with 50 and 100 µM of CPT, the amount of Topo I-DNA complex
(
the middle bands) was generated at a similar level as in the control (Lane B).
However, when treated with 50, 100, 200, 300, 400 µM of B-15, the percentage of
Topo I-DNA complex (the middle bands) diminished linearly in
a
concentration-dependent manner. These EMSA results clearly indicate that another
Topo I inhibitory mechanism of our compound is interfering with the binding of Topo
I to DNA.
[
Figure 8]
3
.6. Molecular docking
The mechanism studies above indicated that our compounds not only can
stabilize the drug-enzyme-DNA covalent ternary complex, but also interfere with the
binding between Topo I and DNA. Molecular docking studies were carried out in an
attempt to confirm this dual action mechanism. The average minimized complex of
representative compound B-15 with Topo I (validated by MD simulation for 20 ns,
Figure S2), whose maximum free energy (∆G) calculated by MM-PBSA were -29.7
kcal/mol, was used for binding mode analysis. Firstly, as shown in Fig. 9, B-15 could
fit well into the Topo I-DNA covalent complex by interacting not only with amino
8

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acid residues, but also with DNA bases in the active pocket of Topo I. Specifically, the
O of amido bond and the lactonic O of furanone ring of B-15 binds well with residue
ARG364 and ASN722 respectively by forming hydrogen bonds (shown as green
dashed lines). Meanwhile, the N-H of amido bond of B-15 forms hydrogen bonds
with the DNA base DA113. In addition, the fluorine atom in the para-position can
form a strong hydrogen-halogen bond of -5.5 kcal • mol-1 with ASN722, while such
interaction cannot be observed in the complex of compounds B-13 and B-14 (nearly
identical with B-15 except for the substituted fluorine atom attached to the different
position on the terminal phenyl ring). As shown in Figure 10, lack of this
hydrogen-halogen change the whole binding mode, of which the furan ring is bending
over, which can provide one possible explanation for the significant potency of B-15
superior to both B-13 and B-14. As shown in Fig. 9B, the π-π stacking interactions
(shown as red dashed lines) can be observed between the aromatic ring of B-15 and
amino acid residue TGP11 as well as the DNA bases DA113, DC112 and DT10.
Moreover, the furanone ring of B-15 forms with the amino acid residue LYS425 by
the π-alkyl stacking interaction (blue dashed lines). These effective interactions
between B-15 and the Topo I-DNA covalent complex are evidence for the mechanism
of
stabilization
of
the
drug-enzyme-DNA
covalent
4-(4-substituted
amidobenzyl)furan-2(5H)-one derivatives.
Since the mechanism studies also reveal that 4-(4-substituted amidobenzyl)furan-
(5H)-one derivatives apparently interfere with the binding of Topo I to DNA, we
2
further docked B-15 into the 3-dimensional crystal structure of Topo I (PDB: 1K4T),
from which the DNA was removed. As shown in Fig. 9C, B-15 also interacts well
with the active pocket of DNA-free Topo I by forming hydrogen bonds (showed as
yellow dashed lines) with different amino acid residues (HIS367, LYS425, LYS493,
ALA499). The results provide evidence for the second mechanism with which these
compounds interfere with the binding between Topo I and DNA.
[
Figure 9]
[
Figure 10]
9

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4
. Conclusion
In this study, two series of novel 4-(4-substituted amidobenzyl)furan-2(5H)-one
derivatives bearing an α,β-unsaturated lactone moiety, a pharmacophore with
potential antitumor activity [9], were designed, synthesized and evaluated for Topo I
inhibitory activity and cytotoxicity against three human cancer cell lines. Among the
synthetic 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives, series 2 with an
exocyclic double bond, exhibited promising Topo I inhibitory activity and cytotoxicity
against cancer cell lines. This suggests that introduction of the exocyclic double bond
into the α,β-unsaturated lactones 2-furanone moiety is essential to develop
4
-(4-substituted amidobenzyl)furan-2(5H)-one derivatives as Topo I-based antitumor
agents. In series 2, several compounds showing promising activity as Topo I inhibitors
and cytotoxicity, especially B-15, were identified. Some design principles derived
from the binding patterns of this type of compound with Topo I as well as the
structure-activity relationships have been summarized, and may be useful in the future
to guide the design and modification of new furanone derivatives as Topo I inhibitors.
A series of mechanism studies indicate that our compounds can not only stabilize the
drug-enzyme-DNA covalent ternary complex but can also interfere with the binding
of Topo I to DNA. In the future, we will attempt to develop more potent Topo I
inhibitors.
5
. Experimental section
5
.1. Chemistry
All chemicals were purchased from Alfa Aesar or Sigma Aldrich. The synthetic
routes to two series of 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives are
shown in Scheme 1 and Scheme 2. All the compounds were fully identified through
1
13
H and C-nuclear magnetic resonance (NMR), mass spectrometry (MS) and high
resolution mass spectrometry (ESI-HRMS). Melting points were determined with a
digital melting point apparatus and are uncorrected.
5
.1.1. Intermediate compounds 1-1~1-4 and 2-1~2-4
5
.1.1.1.
1
0

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(E)-3-Benzyl-4-oxobut-2-enoic acid (1-1). Glyoxylic acid monohydrate (0.037
mol, 2.76 g) was dissolved in 50 mL of MeOH, and KOH (0.037 mol, 2.08 g) was
o
added to the solution at 0 C under nitrogen followed by dropwise addition of
phenylpropyl aldehyde (0.037 mol, 5 g). The mixture kept overnight at room
temperature. The solvent and excess phenylpropyl aldehyde were then removed under
reduced pressure, and the residue was diluted with 30 mL of H O, and the mixture
2
was extracted with EtOAc (3 × 30 mL). The aqueous phase was acidified with 10%
HCl to pH 3 and then extracted with EtOAc (3 × 50 mL). The organic layer was dried
over anhydrous Na SO , filtered concentrated to dryness and purified through a silica
2
4
gel column (EA: PE: AcOH = 5: 1: 5%) to give (Z)-3-benzyl-4-oxobut-2-enoic acid
o
1
(
1-1) as a white solid, mp 149.3-150.0 C, yield 65%; H NMR (300 MHz, CDCl ) δ
3
9
.59 (s, 1H, -CHO), 7.32 – 7.16 (m, 5H, Ar-H), 6.61 (s, 1H, -C=CH), 4.14 (s, 2H,
1
3
benzylic CH2); C NMR (75 MHz, CDCl ) δ 193.97 (-CHO), 170.85 (-COOH),
3
1
54.32, 137.33, 134.18, 129.18, 128.65, 126.79, 30.46 (benzylic CH ); ESI-HRMS
2
+
m/z: 189.0559 [M+H] , calcd for C H O 189.0557.
11
9
3
5
.1.1.2.
E)-3-Formyl-4-(4-nitrophenyl)but-2-enoic acid (1-2). (Z)-3-benzyl-4-oxobut-2-
(
enoic acid (1-1, 1.29 mmol, 193 mg) was added portion-wise into stirred nitrosonitric
o
acid (1 mL, 23.80 mmol) at -10 C. After 4 h, 20 mL of ice was added into the
solution and the mixture was stirred for 30 min. The mixture was extracted with
EtOAc (3 × 20 mL). The organic layer was dried over anhydrous Na SO , filtered and
2
4
concentrated to dryness, then purified through silica gel column (EA: PE: AcOH = 2:
1
1
8
4
: 5%) to give (E)-3-formyl-4-(4-nitrophenyl)but-2-enoic acid as a yellow solid, mp
o
1
76.6-177.6 C, yield 56%; H NMR (300 MHz, acetone -d ) δ 9.69 (s, 1H, aldehydic),
6
.12 (d, J = 8.7 Hz, 2H, Ar-H), 7.54 (d, J = 8.7 Hz, 2H, Ar-H), 6.94 (s, 1H, -C=CH),
13
.18 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 194.52 (-CHO), 165.93
2
6
(-COOH), 150.55, 146.61, 146.38, 137.38, 129.93, 123.27, 29.64 (benzylic CH2);
-
ESI-HRMS m/z: 234.0410 [M-H] , calcd for C H NO 234.0408.
11
8
5
5
.1.1.3.
1
1

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(E)-4-Hydroxy-3-(4-nitrobenzyl)but-2-enoic acid (1-3). Compound 1-2 (0.51
mmol, 120 mg) was added into a flask, and 5 mL MeOH was added to the flask at
room temperature. Sodium borohydride (2.04 mmol, 80 mg) was added portion-wise
to the rapidly stirred reaction mixture. After 2 h, 20 mL of 10 % HCl was added into
the solution. The mixture was extracted with EtOAc (3 × 20 mL). The organic layer
was dried over anhydrous Na SO , filtered and concentrated to dryness, which was
2
4
purified through silica gel column (EA: PE: AcOH = 2: 1: 5%) to give
o
(
E)-4-hydroxy-3-(4- nitrobenzyl)but-2-enoic acid as a white solid, mp 165.4-166.3 C,
1
yield 78%; H NMR (300 MHz, DMSO-d ) δ 8.60 (d, J = 8.7 Hz, 2H, Ar-H), 8.01 (d,
6
J = 8.7 Hz, 2H, Ar-H), 6.69 (s, 1H, -C=CH), 4.61 (s, 2H, -CH -OH), 4.57 (d, J = 1.6
2
1
3
Hz, 2H, benzylic CH ); C NMR (75 MHz, DMSO-d ) δ 167.51 (-COOH), 159.57,
2
6
1
47.71, 130.24, 123.83, 115.29, 64.39 (-CH -OH), 34.30 (benzylic CH ); ESI-HRMS
2 2
-
m/z: 236.056 [M-H] , calcd for C H NO 236.0564.
11
9
5
5
.1.1.4.
-(4-Nitrobenzyl)furan-2(5H)-one (1-4). Compound 1-3 (0.61mmol, 145 mg)
4
and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.92 mmol, 176
mg) were placed in a dry double-neck flask, and 5 ml of dry DCM and 0.17 mL of
triethylamine (1.23 mmol) were added to the flask under nitrogen separately. The
mixture was reacted for 2 h at room temperature then was washed with saturated
sodium bicarbonate solution. The organic layer dried over anhydrous Na SO , filtered
2
4
and concentrated to dryness, and then purified through a silica gel column (EA: PE =
o
5
: 1) to give 4-(4-nitrobenzyl)furan-2(5H)-one as a white solid, mp 140.0-140.9 C,
1
yield 72%; H NMR (300 MHz, DMSO-d ) δ 8.19 (d, J = 8.7 Hz, 2H, Ar-H), 7.57 (d,
6
J = 8.7 Hz, 2H, Ar-H), 5.82 – 5.78 (m, 1H, -C=CH), 4.87 (d, J = 1.7 Hz, 2H, -O-CH2),
13
3
.96 (s, 2H, benzylic CH2); C NMR (75 MHz, DMSO-d ) δ 173.28 (carbonyl),
6
1
69.88, 146.55, 144.67, 130.35, 123.80, 115.57, 72.80 (-O-CH ), 33.77 (benzylic
2
+
CH ); ESI-HRMS m/z: 220.0612 [M+H] , calcd for C H NO 220.0604.
2
11 10
4
5
.1.1.5.
4-(4-Nitrophenyl)butan-2-one (2-1). 4-Nitrobenzyl bromide (0.2 mol, 42.8 g) and
2
,4-pentanedione (0.2 mol, 20 g) were placed a double-neck flask, and 300 mL of
1
2

ACCEPTED MANUSCRIPT
MeOH was added to the flask. When these were completely dissolved, 27.6 g of
potassium carbonate was added and the mixture was refluxed for 4 h. When the
reaction solution temperature cooled to room temperature, the solvent was removed
under reduced pressure, and then 200 mL of H O was added. The mixture was
2
extracted with EtOAc (3 × 200 mL), and then the organic layer was dried over
anhydrous Na SO , filtered and concentrated to dryness, and purified through silica
2
4
gel column (EA: PE = 10: 1) to give 4-(4-nitrophenyl)butan-2-one as white crystals,
o
1
mp 56.1-57.0 C, yield 66%; H NMR (300 MHz, CDCl ) δ 8.08 (d, J = 8.7 Hz, 2H,
3
Ar-H), 7.32 (d, J = 8.7 Hz, 2H, Ar-H), 2.96 (t, J = 7.3 Hz, 2H, benzylic CH ), 2.79 (t,
2
13
J = 7.2 Hz, 2H, -CO-CH ), 2.13 (s, 3H, -CO-CH ); C NMR (75 MHz, CDCl ) δ
2
3
3
2
06.83 (carbonyl), 149.09, 146.45, 129.31, 123.71, 44.14 (benzylic CH ), 30.06
2
+
(
-CO-CH ), 29.33 (-CO-CH ); ESI-HRMS m/z: 194.0814 [M+H] , calcd for
2
3
C H NO 194.0812.
10
12
3
5
.1.1.6.
E)-3-(4-Nitrobenzyl)-4-oxopent-2-enoic acid (2-2). Compound 2-1 (128.20
(
mmol, 25 g) and glyoxylic acid monohydrate (270.27 mmol, 20 g) were added into a
double-neck flask, and 100 mL of phosphoric acid was added to the flask. The
mixture was refluxed for 4 h. When the reaction solution temperature cooled to room
temperature, 200 mL of H O was added. The mixture was extracted with EtOAc (3 ×
2
2
00 mL), and the organic layer was extracted with 30% K CO (3 × 150 mL). The
2 3
aqueous phase was acidified with 10% hydrochloric acid to pH 3 and then extracted
with EtOAc (3 × 100 mL). The organic layer was dried over anhydrous Na SO ,
2
4
filtered and concentrated to dryness, then purified through a silica gel column
CH Cl : MeOH = 200: 1) to give (E)-3-(4-nitrobenzyl)-4-oxopent-2-enoic acid as a
(
2
2
o
1
yellow solid, mp 177.6-178.5 C, yield 72%; H NMR (300 MHz, CDCl ) δ 8.10 (d, J
3
=
8.7 Hz, 2H, Ar-H), 7.40 (d, J = 8.7 Hz, 2H, Ar-H), 6.78 (s, 1H, -C=CH), 4.27 (s, 2H,
13
benzylic CH ), 2.40 (s, 3H, -CO -CH ); C NMR (75 MHz, CDCl ) δ 198.78
2
3
3
(carbonyl), 170.30 (-COOH), 153.31, 146.71, 145.99, 129.91, 127.30, 123.78, 31.85
-
(
benzylic CH ), 26.68 (-CO-CH ); ESI-HRMS m/z: 248.0561 [M-H] , calcd for
2
3
C H NO 248.0564.
1
2
10
5
1
3

ACCEPTED MANUSCRIPT
5
.1.1.7.
-Methylene-4-(4-nitrobenzyl)furan-2(5H)-one (2-3). Compound 2-2 (0.28 mol,
0 g) and p-toluenesulfonic acid (0.84 mol, 159.6 g) was added into a double-neck
5
7
flask, and 400 mL of toluene was added to the flask. The mixture was refluxed for 6 h.
When the reaction solution temperature cooled to room temperature, the solvent was
removed under reduced pressure, and then 400 mL of H O was added. The mixture
2
was extracted with EtOAc (3 × 200 mL). The organic layer was dried over anhydrous
Na SO , filtered and concentrated to dryness, and purified through a silica gel column
2
4
(EA: PE = 5: 1) to give 5-methylene-4-(4-nitrobenzyl)furan-2(5H)-one as a yellow
o
1
solid, mp 136.7-137.6 C, yield 45%; H NMR (300 MHz, CDCl ) δ 8.23 (d, J = 8.7
3
Hz, 2H, Ar-H), 7.40 (d, J = 8.7 Hz, 2H, Ar-H), 5.87 (d, J = 0.9 Hz, 1H, -C=CH), 5.24
(dd, J = 3.1, 1.8 Hz, 1H, exocyclic -C=CH), 4.93 (dd, J = 3.1, 0.7 Hz, 1H, exocyclic
1
3
-
C=CH), 3.94 (s, 2H, benzylic CH ); C NMR (75 MHz, CDCl ) δ 168.20 (-CO),
2
3
1
3
2
5
55.88, 155.23, 147.45, 143.37, 129.83, 124.40, 119.25, 95.55 (exocyclic -C=CH),
+
2.44 (benzylic CH ); ESI-HRMS m/z: 232.0612 [M+H] , calcd for C H NO
2
12 10
4
32.0604.
.1.1.8.
4-(4-Aminobenzyl)-5-methylenefuran-2(5H)-one (2-4). Compound 2-3 (10 mmol,
2
.5 g), ammonium chloride (10 mmol, 532 mg) and Fe (30 mmol, 1.67 g) were placed
in a double-neck flask, and 40 mL of THF and 8 mL of H O were added to the flask.
2
The mixture was refluxed under nitrogen for 4 h. When the reaction solution
temperature cooled to room temperature, 20 mL of H O was added. The excess of Fe
2
was filtered, and the mixture was extracted with EtOAc (3 × 20 mL). The organic
layer was dried over anhydrous Na SO , filtered and concentrated to dryness, then
2
4
was purified through silica gel column (EA: PE = 5: 1) to give
1
4
-(4-aminobenzyl)-5-methylene- furan-2(5H)-one as a yellow oil, yield 67%; H
NMR (300 MHz, CDCl ) δ 6.95 (d, J = 8.4 Hz, 2H, Ar-H), 6.63 (d, J = 8.4 Hz, 2H,
3
Ar-H), 5.80 (d, J = 0.9 Hz, 1H, -C=CH), 5.14 (dd, J = 2.8, 1.8 Hz, 1H, exocyclic
-C=CH), 4.93 (dd, J = 2.9, 0.6 Hz, 1H, exocyclic -C=CH), 3.66 (d, J = 1.1 Hz, 2H,
1
3
benzylic CH ); C NMR (75 MHz, CDCl ) δ 169.01 (-CO), 159.03, 155.53, 145.67,
2
3
1
4

ACCEPTED MANUSCRIPT
1
29.61, 125.41, 117.99, 115.44, 94.96 (exocyclic -C=CH), 31.83 (benzylic CH2);
+
ESI-HRMS m/z: 202.0854 [M+H] , calcd for C H NO 202.0863.
1
2
12
3
5
.1.2. General procedure of the preparation of A-1~A-7
Compound 1-4 (10 mmol, 2.5 g), ammonium chloride (10 mmol, 532 mg) and Fe
30 mmol, 1.67 g) were placed in a double-neck flask, and 40 mL of THF and 8 mL of
(
H O were added to the flask. The mixture was refluxed under nitrogen for 4 h. When
2
the reaction solution temperature cooled to room temperature, 20 mL of H O was
2
added. The excess of Fe was filtered, and the mixture was extracted with EtOAc (3 ×
2
0 mL). The organic layer was dried over anhydrous Na SO , filtered and
2 4
concentrated to obtain the intermediate aminofuranone, which was used in the next
reaction without purification. The carboxylic acid (0.75 mmol) was added into a dry
double-neck flask, and 5 mL of dry THF was added to the flask.
N,N-Dimethylformamide (1.50 mmol, 0.15 mL) was added into the solution under
o
nitrogen at 0 C. When these were completely dissolved, oxalyl chloride (3.75 mmol)
was added slowly. After 30 min, the solvent and excess oxalyl chloride were then
removed under reduced pressure to obtain an acyl chloride. The acyl chloride was
kept dry. 100 mg of the intermediate amino furanone was added into a dry
double-neck flask, and 5 mL of dry THF was added to the flask. When these were
completely dissolved, dry pyridine (1.06 mmol, 0.01 mL) was added under nitrogen at
o
0
C. The THF solution of an acyl chloride (0.793 mmol) was added into the mixture
slowly. After 30 min, 20 mL of saturated ammonium chloride solution was slowly
added into the mixture, and the mixture was extracted three times with EtOAc (3 × 20
mL). The organic layer was dried over anhydrous Na SO , filtered and concentrated
2
4
to dryness, which was purified through silica gel column (EA: PE = 2: 1) to give
A-1~A-7.
5
.1.2.1.
N-(4-((5-Oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(p-tolyl)acetamide (A-1)
o
1
White solid, mp 105.5-106.4 C, yield 78%; H NMR (300 MHz, Acetone-d ) δ 9.31
6
(s, 1H, N-H), 7.63 (d, J = 8.4 Hz, 2H, Ar-H), 7.23 (dd, J = 15.3, 8.1 Hz, 4H, Ar-H),
7
.12 (d, J = 8.4 Hz, 2H, Ar-H), 5.73 (s, 1H, -C=CH), 4.79 (s, 2H, -O-CH ), 3.78 (s,
2
1
5

ACCEPTED MANUSCRIPT
13
2
H, benzylic CH ), 3.63 (s, 2H, benzylic CH ), 2.28 (s, 3H, Ar-CH ); C NMR (75
2 2 3
MHz, Acetone-d ) δ 173.99 (-CO), 171.59 (-CO), 170.03, 139.32, 136.87, 133.76,
6
1
32.37, 130.06, 129.90, 129.81, 120.39, 116.15, 73.34 (-O-CH ), 44.34 (benzylic
2
+
CH ), 34.76 (benzylic CH ), 21.02 (Ar-CH ); ESI-MS m/z: 322.1 [M+H] ;
2
2
3
+
ESI-HRMS m/z: 322.1450 [M+H] , calcd for C H NO 322.1438.
2
0
20
3
5
.1.2.2.
N-(4-((5-Oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(2-(trifluoromethyl)phenyl
o
1
)
acetamide (A-2). White solid, mp 127.8-128.3 C, yield 55%; H NMR (300 MHz,
Acetone-d ) δ 9.44 (s, 1H, N-H), 7.73 (d, J = 7.8 Hz, 1H, Ar-H), 7.68 – 7.56 (m, 4H,
6
Ar-H), 7.49 (t, J = 7.5 Hz, 1H, Ar-H), 7.24 (d, J = 8.4 Hz, 2H, Ar-H), 5.78 – 5.73 (m,
1
2
1
1
H, -C=CH), 4.81 (d, J = 1.6 Hz, 2H, -O-CH ), 3.98 (s, 2H, benzylic CH ), 3.81 (s,
2 2
1
3
H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 173.17 (-CO), 170.71 (-CO),
2
6
67.81, 138.31, 134.05, 133.30, 132.04, 131.65, 129.25, 127.22, 126.56, 125.65,
22.49, 119.64, 115.32, 72.50 (-O-CH ), 40.03 (benzylic CH ), 33.90 (benzylic CH );
2
2
2
+
+
ESI-MS m/z: 376.3 [M+H] ; ESI-HRMS m/z: 376.1160 [M+H] , calcd for
C H FNO 376.1155.
20
17
3
5
.1.2.3.
N-(4-((5-Oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(3-(trifluoromethyl)phenyl
o
1
)
acetamide (A-3). White solid, mp 126.6-127.2 C, yield 51%; H NMR (300 MHz,
Acetone-d ) δ 9.47 (s, 1H, N-H), 7.73 (s, 1H, Ar-H), 7.70 – 7.51 (m, 5H, Ar-H), 7.22
6
(d, J = 8.5 Hz, 2H, Ar-H), 5.75 – 5.66 (m, 1H, -C=CH), 4.79 (d, J = 1.7 Hz, 2H,
13
-
O-CH ), 3.84 (s, 2H, benzylic CH ), 3.79 (s, 2H, benzylic CH ); C NMR (75 MHz,
2
2
2
Acetone-d ) δ 174.00 (-CO), 171.54 (-CO), 169.17, 139.13, 138.19, 134.15, 132.63,
6
1
4
30.84, 130.14, 130.02, 126.84, 125.36, 124.28, 120.51, 116.21, 73.37 (-O-CH2),
+
4.00 (benzylic CH ), 34.78 (benzylic CH ); ESI-MS m/z: 376.2 [M+H] ; ESI-HRMS
2
2
+
m/z: 376.1158 [M+H] , calcd for C H FNO 376.1155.
2
0
17
3
5
.1.2.4.
N-(4-((5-Oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(4-(trifluoromethyl)phenyl
o
1
)
acetamide (A-4). White solid, mp 139.8-140.7 C, yield 66%; H NMR (300 MHz,
Acetone-d ) δ 9.46 (s, 1H, N-H), 7.75 – 7.51 (m, 6H, Ar-H), 7.22 (d, J = 8.4 Hz, 2H,
6
1
6

ACCEPTED MANUSCRIPT
Ar-H), 5.79 – 5.64 (m, 1H, -C=CH), 4.79 (d, J = 1.7 Hz, 2H, -O-CH ), 3.83 (s, 2H,
2
1
3
benzylic CH ), 3.79 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 174.00
2
2
6
(-CO), 171.54 (-CO), 169.05, 141.50, 139.12, 135.27, 132.65, 130.92, 130.13, 125.98,
1
25.46, 120.53, 116.21, 73.37 (-O-CH ), 44.20 (benzylic CH ), 34.78 (benzylic CH );
2
2
2
+
+
ESI-MS m/z: 376.2 [M+H] ; ESI-HRMS m/z: 376.1146 [M+H] , calcd for
C H FNO 376.1155.
20
17
3
5
.1.2.5.
E)-3-(2-Fluorophenyl)-N-(4-((5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)acryl
(
o
1
-
amide (A-5). Yellow solid, mp 154.0-155.0 C, yield 65%; H NMR (300 MHz,
Acetone-d ) δ 9.57 (s, 1H, N-H), 7.83 – 7.75 (m, 3H, Ar-H, -C=CH), 7.67 (dd, J =
6
1
0.8, 4.6 Hz, 1H, Ar-H), 7.49 – 7.39 (m, 1H, Ar-H), 7.31 – 7.15 (m, 4H, Ar-H), 6.96
(d, J = 15.8 Hz, 1H, -C=CH), 5.85 – 5.65 (m, 1H, -C=CH), 4.82 (d, J = 1.1 Hz, 2H,
1
3
-
O-CH ), 3.82 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 174.05 (-CO),
2 2 6
1
1
71.57, 164.29 (-CO), 162.04, 139.27, 139.14, 134.10, 132.72, 132.28, 130.19,
25.67, 125.62, 123.70, 120.63, 117.00, 116.86, 73.40 (-O-CH ), 34.82 (benzylic
2
+
+
CH ); ESI-MS m/z: 338.1 [M+H] ; ESI-HRMS m/z: 338.1191 [M+H] , calcd for
2
C H FNO 338.1187.
20
17
3
5
.1.2.6.
E)-3-(3-Fluorophenyl)-N-(4-((5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)acryl
(
o
1
-
amide (A-6). Yellow solid, mp 155.6-156.3 C, yield 61%; H NMR (300 MHz,
Acetone-d ) δ 9.55 (s, 1H, N-H), 7.77 (d, J = 8.5 Hz, 2H, Ar-H), 7.67 (d, J = 15.6 Hz,
6
1
H, -C=CH), 7.44 (m, 2H, Ar-H), 7.37 (d, J = 10.5 Hz, 1H, Ar-H), 7.26 (d, J = 8.5 Hz,
H, Ar-H), 7.20 – 7.10 (m, 1H, Ar-H), 6.90 (d, J = 15.6 Hz, 1H, -C=CH), 5.79 – 5.73
2
13
(
m, 1H, -C=CH), 4.82 (d, J = 1.3 Hz, 2H, -O-CH ), 3.81 (s, 2H, benzylic CH ); C
2 2
NMR (75 MHz, Acetone-d ) δ 174.10 (-CO), 171.60, 164.20 (-CO), 163.85, 140.27,
6
1
1
39.20, 138.52, 132.72, 131.63, 130.18, 124.84, 124.44, 120.65, 117.11, 116.20,
+
14.69, 73.41 (-O-CH ), 34.80 (benzylic CH ); ESI-MS m/z: 338.4 [M+H] ;
2
2
+
ESI-HRMS m/z: 338.1200 [M+H] , calcd for C H FNO 338.1187.
2
0
17
3
5
.1.2.7.
1
7

ACCEPTED MANUSCRIPT
(E)-3-(4-Fluorophenyl)-N-(4-((5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)acry-
o
1
lamide (A-7). White solid, mp 182.1-183.0 C, yield 71%; H NMR (300 MHz,
Acetone-d ) δ 9.45 (s, 1H, N-H), 7.76 (d, J = 8.5 Hz, 2H, Ar-H), 7.70-7.64 (m, 3H,
6
Ar-H, -C=CH), 7.26 (d, J = 8.5 Hz, 2H, Ar-H), 7.19 (t, J = 8.8 Hz, 2H, Ar-H), 6.80 (d,
J = 15.6 Hz, 1H, -C=CH), 5.77 – 5.74 (m, 1H, -C=CH), 4.82 (d, J = 1.7 Hz, 2H,
1
3
-
O-CH ), 3.83 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 173.12 (-CO),
2 2 6
1
1
3
5
70.70, 165.11 (-CO), 163.47, 139.51, 138.49, 131.71, 131.65, 129.87, 129.32,
21.93, 119.66, 115.80, 115.34, 72.50 (-O-CH ), 33.95 (benzylic CH ); ESI-MS m/z:
2
2
+
+
38.2 [M+H] ; ESI-HRMS m/z: 338.1199 [M+H] , calcd for C H FNO 338.1187.
2
0
17
3
.1.3. General procedure for the preparation of B-1~B-15
A carboxylic acid (0.75 mmol) was added into a dry double-neck flask, and 5 mL
of dry THF was added to the flask. N,N-dimethylformamide (1.50 mmol, 0.15 mL)
o
was added into the solution under nitrogen at 0 C. When the solids were completely
dissolved, oxalyl chloride (3.75 mmol) was added slowly. After 30 min, the solvent
and excess oxalyl chloride were removed under reduced pressure to obtain an acyl
chloride which was kept dry. 100 mg of compound 2-4 (0.53 mmol) was added into a
dry double-neck flask, and 5 mL of dry THF was added to the flask. When these were
completely dissolved, dry pyridine (1.06 mmol, 0.01 mL) was added under nitrogen at
o
0
C. The THF solution of an acyl chloride (0.793 mmol) was added into the mixture
slowly. After 30 min, 20 mL of saturated ammonium chloride solution was slowly
added into the mixture, and the mixture was extracted with EtOAc (3 × 20 mL). The
organic layer was dried over anhydrous Na SO4, filtered and concentrated to dryness,
2
then purified through silica gel column (EA: PE = 2: 1) to give B-1~B-15.
5
.1.3.1.
N-(4-((2-Methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)benzamide
B-1).
(
1
Yellow oil, yield 67%; H NMR (300 MHz, Acetone-d ) δ 9.59 (s, 1H, N-H), 7.99 (d,
6
J = 8.5 Hz, 2H, Ar-H), 7.85 (d, J = 8.5 Hz, 2H, Ar-H), 7.47 – 7.56 (m, 3H, Ar-H),
7
.32 (d, J = 8.5 Hz, 2H, Ar-H), 6.02 (s, 1H, -C=CH), 5.16 - 5.19 (m, 2H, exocyclic
1
3
-
C=CH ), 3.94 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 168.23
2 2 6
1
8

ACCEPTED MANUSCRIPT
(-CO), 165.50 (-CO), 158.71, 155.68, 138.38, 135.36, 132.19, 131.49, 129.16, 128.41,
1
27.45, 120.48, 117.85, 94.44 (exocyclic -C=CH ), 31.44 (benzylic CH ); ESI-MS
2 2
+
+
m/z: 306.3 [M+H] ; ESI-HRMS m/z: 306.1142 [M+H] , calcd for C H NO
3
19
16
3
06.1125.
5
.1.3.2.
N-(4-((2-Methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-phenylacet-a
1
mide (B-2). Yellow oil, yield 71%; H NMR (300 MHz, Acetone-d ) δ 9.38 (s, 1H,
6
N-H), 7.64 (d, J = 8.5 Hz, 2H, Ar-H), 7.37 (d, J = 7.4 Hz, 2H, Ar-H), 7.33 (s, 1H,
Ar-H), 7.30 (d, J = 7.4 Hz, 2H, Ar-H), 7.24 (d, J = 8.5 Hz, 2H, Ar-H), 5.98 (s, 1H,
-
C=CH), 5.19 – 5.10 (m, 2H, exocyclic -C=CH ), 3.89 (s, 2H, benzylic CH ), 3.68 (s,
2 2
1
3
2
H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 168.97 (-CO), 168.18 (-CO),
2 6
1
58.71, 155.65, 138.41, 135.98, 131.77, 129.53, 129.16, 128.32, 126.61, 119.52,
17.79, 94.37 (exocyclic -C=CH ), 43.84 (benzylic CH ), 31.36 (benzylic CH );
1
2
2
2
+
+
ESI-MS m/z: 320.0 [M+H] ; ESI-HRMS m/z: 320.1295 [M+H] , calcd for C H NO
3
2
0
18
3
20.1281.
5
.1.3.3.
N-(4-((2-Methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(p-tolyl)acet-
o
1
amide (B-3). White solid, mp 119.8-120.7 C, yield 71%; H NMR (300 MHz,
Acetone-d ) δ 9.36 (s, 1H, N-H), 7.64 (d, J = 8.5 Hz, 2H, Ar-H), 7.25 (dd, J = 8.1, 4.9
6
Hz, 4H, Ar-H), 7.13 (d, J = 8.5 Hz, 2H, Ar-H), 6.00 (s, 1H, -C=CH), 5.13 – 5.18 (m,
2
H, exocyclic -C=CH ), 3.90 (s, 2H, benzylic CH ), 3.64 (s, 2H, benzylic CH ), 2.29
2 2 2
1
3
(
s, 3H, Ar-CH ); C NMR (75 MHz, Acetone-d ) δ 169.20 (-CO), 168.19 (-CO),
3
6
1
58.72, 155.66, 138.44, 136.00, 132.89, 131.73, 129.13, 129.04, 128.95, 119.49,
1
17.73, 94.36 (exocyclic -C=CH ), 43.47 (benzylic CH ), 31.36 (benzylic CH ), 20.17
2 2 2
+
+
(
Ar-CH ); ESI-MS m/z: 334.1 [M+H] ; ESI-HRMS m/z: 334.1451 [M+H] , calcd for
3
C H NO 334.1438.
21
20
3
5
.1.3.4.
N-(4-((2-Methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(2-(trifluoro-
o
1
methyl)phenyl)acetamide (B-4). White solid, mp 124.6-125.5 C, yield 56%; H NMR
(
300 MHz, CDCl ) δ 7.68 (d, J = 7.7 Hz, 1H, Ar-H), 7.55-7.50 (m, 2H, Ar-H), 7.44 (d,
3
1
9

ACCEPTED MANUSCRIPT
J = 8.3 Hz, 2H, Ar-H), 7.40 (d, J = 8.4 Hz, 1H, Ar-H), 7.10 (d, J = 8.3 Hz, 2H, Ar-H),
5
.78 (s, 1H, -C=CH), 5.17 – 5.14 (m, 1H, exocyclic -C=CH), 4.92 (d, J = 2.8 Hz, 1H,
1
3
exocyclic -C=CH), 3.88 (s, 2H, benzylic CH ), 3.74 (s, 2H, benzylic CH ); C NMR
2
2
(
75 MHz, CDCl ) δ 168.94 (-CO), 168.24 (-CO), 158.13, 155.48, 137.03, 132.80,
3
1
9
3
5
32.67, 132.43, 131.96, 129.36, 129.12, 127.82, 126.39, 122.62, 120.65, 118.53,
5.30 (exocyclic -C=CH ), 41.16 (benzylic CH ), 32.05 (benzylic CH ); ESI-MS m/z:
2
2
2
+
+
88.4 [M+H] ; ESI-HRMS m/z: 388.1148 [M+H] , calcd for C H F NO 388.1155.
2
1
17
3
3
.1.3.5.
N-(4-((2-Methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(3-(trifluoro-
o
1
methyl)phenyl)acetamide (B-5). White solid, mp 115.6-116.3 C, yield 59%; H NMR
(
300 MHz, Acetone-d ) δ 9.52 (s, 1H, N-H), δ 7.74 (s, 1H, Ar-H), 7.69 (d, J = 7.4, 1H,
6
Ar-H), 7.65 (d, J = 8.5 Hz, 2H, Ar-H), 7.60 (d, J = 3.3, 1H, Ar-H), 7.56 (d, J = 7.4 Hz,
H, Ar-H). 7.25 (d, J = 8.5 Hz, 2H, Ar-H), 5.99 (s, 1H, -C=CH), 5.18 – 5.14 (m, 2H,
1
13
exocyclic -C=CH ), 3.90 (s, 2H, benzylic CH ), 3.84 (s, 2H, benzylic CH ); C NMR
2
2
2
(
75 MHz, Acetone-d ) δ 168.29 (-CO), 168.18 (-CO), 158.67, 155.66, 138.24, 137.31,
6
1
9
3
5
33.29, 131.96, 129.94, 129.20, 129.14, 128.08, 125.96, 123.40, 119.59, 117.80,
4.35 (exocyclic -C=CH ), 43.11 (benzylic CH ), 31.36 (benzylic CH ); ESI-MS m/z:
2
2
2
-
+
86.2 [M-H] ; ESI-HRMS m/z: 388.1147 [M+H] , calcd for C H F NO 388.1155.
2
1
17
3
3
.1.3.6.
N-(4-((2-Methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)-2-(4-(tri-fluoro
o
1
methyl)phenyl)acetamide (B-6). White solid, mp 145.3-146.3 C, yield 70%; H NMR
300 MHz, Acetone-d ) δ 9.49 (s, 1H, N-H), 7.69 – 7.58 (m, 6H, Ar-H), 7.26 (d, J =
(
6
8
.5 Hz, 2H, Ar-H), 5.99 (s, 1H, -C=CH), 5.18 – 5.12 (m, 2H, exocyclic -C=CH ), 3.90
2
1
3
(
s, 2H, benzylic CH ), 3.82 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ
2 2 6
1
1
68.17 (-CO), 168.16 (-CO), 158.67, 155.65, 140.58, 138.23, 131.98, 131.35, 130.05,
29.20, 125.10, 122.32, 119.59, 117.80, 94.36 (exocyclic -C=CH ), 43.31 (benzylic
2
+
CH ), 31.36 (benzylic CH ); ESI-MS m/z: 388.4 [M+H] ; ESI-HRMS m/z: 388.1150
2
2
+
[
M+H] , calcd for C H F NO 388.1155.
21 17 3 3
5
.1.3.7.
2
0

ACCEPTED MANUSCRIPT
2-(2-Fluorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)ph-e
1
nyl)acetamide (B-7). Yellow oil, yield 61%; H NMR (300 MHz, Acetone-d ) δ 9.42 (s,
6
1
H, N-H), 7.65 (d, J = 8.5 Hz, 2H, Ar-H), 7.44 – 7.38 (m, 1H, Ar-H), 7.35-7.29 (m,
H, Ar-H), 7.27 (d, J = 8.5 Hz, 2H, Ar-H), 7.18 – 7.05 (m, 2H, Ar-H), 5.99 (s, 1H,
1
-
C=CH), 5.19 – 5.11 (m, 2H, exocyclic -C=CH ), 3.89 (s, 2H, benzylic CH ), 3.77 (s,
2 2
1
3
2
H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 168.19 (-CO), 167.89 (-CO),
2 6
1
61.18, 158.70, 155.66, 138.33, 131.94, 131.84, 129.18, 128.78, 124.13, 122.88,
19.57, 117.80, 115.08, 94.44 (exocyclic -C=CH ), 36.59 (benzylic CH ), 31.37
1
2
2
+
+
(
benzylic CH ); ESI-MS m/z: 338.4 [M+H] ; ESI-HRMS m/z: 338.1193 [M+H] ,
2
calcd for C H FNO 338.1187.
2
0
17
3
5
.1.3.8.
-(4-Fluorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)ph-e
2
o
1
nyl)acetamide (B-8). White solid, mp 135.1-136.0 C, yield 75%; H NMR (300 MHz,
Acetone-d ) δ 9.38 (s, 1H, N-H), 7.63 (d, J = 8.4 Hz, 2H, Ar-H), 7.40 (dd, J = 8.2, 5.7
6
Hz, 2H, Ar-H), 7.24 (d, J = 8.4 Hz, 2H, Ar-H), 7.07 (t, J = 8.8 Hz, 2H, Ar-H), 5.98 (s,
1
3
H, -C=CH), 5.20 – 5.07 (m, 2H, exocyclic -C=CH ), 3.89 (s, 2H, benzylic CH ),
2 2
13
.69 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 168.87 (-CO), 168.19
2
6
(-CO), 161.79, 158.69, 155.65, 138.33, 132.01, 131.86, 131.02, 129.18, 119.58,
1
17.79, , 114.91, 94.37 (exocyclic -C=CH ), 42.77 (benzylic CH ), 31.37 (benzylic
2
2
+
+
CH ); ESI-MS m/z: 338.2 [M+H] ; ESI-HRMS m/z: 338.1196 [M+H] , calcd for
2
C H FNO 338.1187.
20
17
3
5
.1.3.9.
-(2-Chlorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)ph-
2
o
1
enyl)acetamide (B-9). White solid, mp 122.5-130.5 C, yield 65%; H NMR (300
MHz, Acetone-d ) δ 9.41 (s, 1H, N-H), 7.65 (d, J = 8.4 Hz, 2H, Ar-H), 7.47 – 7.39 (m,
6
2
H, Ar-H), 7.31 – 7.27 (m, 2H, Ar-H), 7.25 (d, J = 8.4, 2H, Ar-H), 6.00 (s, 1H,
-
C=CH), 5.18 – 5.11 (m, 2H, exocyclic -C=CH ), 3.89 (s, 2H, benzylic CH ), 3.87 (s,
2
2
1
3
2
1
1
H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 169.04 (-CO), 168.59 (-CO),
2
6
59.54, 156.50, 139.18, 135.05, 134.81, 132.94, 132.67, 130.03, 129.96, 129.37,
27.82, 120.43, 118.64, 95.23 (exocyclic -C=CH ), 42.03 (benzylic CH ), 32.22
2
2
2
1

ACCEPTED MANUSCRIPT
+
+
(
benzylic CH ); ESI-MS m/z: 354.3 [M+H] ; ESI-HRMS m/z: 354.0897 [M+H] ,
2
calcd for C H ClNO 354.0891.
2
0
17
3
5
.1.3.10.
2-(3-Chlorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)ph-
o
1
enyl)acetamide (B-10). Yellow solid, mp 128.8-129.5 C, yield 68%; H NMR (300
MHz, Acetone-d ) δ 9.44 (s, 1H, N-H), 7.63 (d, J = 8.4 Hz, 2H, Ar-H), 7.43 (s, 1H,
6
Ar-H), 7.35 – 7.27 (m, 3H, Ar-H), 7.25 (d, J = 8.4 Hz, 2H, Ar-H), 5.98 (s, 1H,
-
C=CH), 5.18 – 5.10 (m, 2H, exocyclic -C=CH ), 3.89 (s, 2H, benzylic CH ), 3.72 (s,
2 2
1
3
2
1
1
H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 169.21 (-CO), 169.03 (-CO),
2
6
59.53, 156.51, 139.14, 139.11, 134.38, 132.78, 130.78, 130.11, 130.04, 128.67,
27.53, 120.44, 118.65, 95.22 (exocyclic -C=CH ), 44.01 (benzylic CH ), 32.22
2
2
+
+
(
benzylic CH ); ESI-MS m/z: 354.3 [M+H] ; ESI-HRMS m/z: 354.0892 [M+H] ,
2
calcd for C H ClNO 354.0891.
2
0
17
3
5
.1.3.11.
-(4-Chlorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)ph-
2
o
1
enyl)acetamide (B-11). White solid, mp 118.7-119.6 C, yield 72%; H NMR (300
MHz, Acetone-d ) δ 9.41 (s, 1H, N-H), 7.63 (d, J = 8.4 Hz, 2H, Ar-H), 7.41 – 7.31 (m,
6
4
H, Ar-H), 7.25 (d, J = 8.4 Hz, 2H, Ar-H), 5.98 (s, 1H, -C=CH), 5.19 – 5.11 (m, 2H,
13
exocyclic -C=CH ), 3.90 (s, 2H, benzylic CH ), 3.70 (s, 2H, benzylic CH ); C NMR
2
2
2
(
75 MHz, Acetone-d ) δ 169.40 (-CO), 169.03 (-CO), 159.54, 156.52, 139.17, 135.74,
6
1
32.90, 132.75, 131.84, 130.03, 129.14, 120.43, 118.66, 95.21 (exocyclic -C=CH2),
+
4
3.76 (benzylic CH ), 32.23 (benzylic CH ); ESI-MS m/z: 376.3 [M+Na] ;
2
2
+
ESI-HRMS m/z: 354.0895 [M+H] , calcd for C H ClNO 354.0891.
2
0
17
3
5
.1.3.12.
N-(4-((2-Methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)cinnamamide
o
1
(
B-12). White solid, mp 139.3-140.0 C, yield 75%; H NMR (300 MHz, Acetone-d )
6
δ 9.46 (s, 1H, N-H), 7.77 (d, J = 8.4 Hz, 2H, Ar-H), 7.68 (d, J = 15.6 Hz, 1H, -C=CH),
7
.63 – 7.60 (m, 2H, Ar-H), 7.46 – 7.28 (m, 3H, Ar-H), 7.30 (d, J = 8.4 Hz, 2H, Ar-H),
.86 (d, J = 15.6 Hz, 1H, -C=CH), 6.02 (s, 1H, -C=CH), 5.20 – 5.14 (m, 2H,
6
2
2

ACCEPTED MANUSCRIPT
1
3
exocyclic -C=CH ), 3.93 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ
2
2
6
1
1
69.10 (-CO), 164.46 (-CO), 159.57, 156.57, 141.65, 139.52, 136.06, 132.78, 130.55,
30.12, 129.79, 128.63, 122.84, 120.41, 118.58, 95.31 (exocyclic -C=CH ), 32.09
2
+
+
(
benzylic CH ); ESI-MS m/z: 332.0 [M+H] ; ESI-HRMS m/z: 332.1274 [M+H] ,
2
calcd for C H NO 332.1281.
2
1
18
3
5
.1.3.13.
E)-3-(2-Fluorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)
(
o
1
phenyl)acrylamide (B-13). White solid, mp 119.7-120.5 C, yield 73%; H NMR (300
MHz, Acetone-d ) δ 9.57 (s, 1H, N-H), 7.81 – 7.76 (m, 3H, Ar-H, -C=CH), 7.72 –
6
7
7
5
.65 (m, 1H, Ar-H), 7.48 – 7.40 (m, 1H, Ar-H), 7.30 (d, J = 8.5 Hz, 2H, Ar-H), 7.27 –
.17 (m, 2H, Ar-H), 6.96 (d, J = 15.6 Hz, 1H, -C=CH), 6.02 (s, 1H, -C=CH), 5.20 –
13
.14 (m, 2H, exocyclic -C=CH ), 3.93 (s, 2H, benzylic CH ); C NMR (75 MHz,
2
2
Acetone-d ) δ169.06 (-CO), 164.25 (-CO), 162.03, 159.54, 156.55, 139.32, 134.07,
6
1
32.92, 132.28, 130.13, 125.71, 123.71, 120.58, 118.70, 117.01, 116.71, 95.24
+
(
exocyclic -C=CH ), 32.27 (benzylic CH ); ESI-MS m/z: 372.3 [M+Na] ; ESI-HRMS
2
2
+
m/z: 350.1198 [M+H] , calcd for C H FNO 350.1187.
2
1
17
3
5
.1.3.14.
E)-3-(3-Fluorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)
(
o
1
phenyl)acrylamide (B-14). White solid, mp 122.9-123.7 C, yield 77%; H NMR (300
MHz, Acetone-d ) δ 9.49 (s, 1H, N-H), 7.77 (d, J = 8.4 Hz, 2H, Ar-H), 7.67 (d, J =
6
1
5.6 Hz, 1H, -C=CH), 7.50 – 7.44 (m, 2H, Ar-H), 7.39 (d, J = 10.5 Hz, 1H, Ar-H),
.30 (d, J = 8.4 Hz, 2H, Ar-H), 7.20 – 7.13 (m, 1H, Ar-H), 6.89 (d, J = 15.6 Hz, 1H,
7
-
C=CH), 6.02 (s, 1H, -C=CH), 5.20 – 5.14 (m, 2H, exocyclic -C=CH ), 3.93 (s, 2H,
2
13
benzylic CH2); C NMR (75 MHz, Acetone-d ) δ 169.05 (-CO), 165.66 (-CO),
6
1
1
64.08, 159.54, 156.55, 140.26, 139.29, 138.56, 132.94, 131.65, 130.14, 124.84,
24.44, 120.56, 118.70, 117.13, 114.70, 95.24 (exocyclic -C=CH ), 32.27 (benzylic
2
-
+
CH ); ESI-MS m/z: 348.3 [M-H] ; ESI-HRMS m/z: 350.1199 [M+H] , calcd for
2
C H FNO 350.1187.
21
17
3
5
.1.3.15.
2
3

ACCEPTED MANUSCRIPT
(E)-3-(4-Fluorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)
o
1
phenyl)acrylamide (B-15). White solid, mp 100.9-101.9 C, yield 71%; H NMR (300
MHz, Acetone-d ) δ 9.50 (s, 1H, N-H), 7.77 (d, J = 8.4 Hz, 2H, Ar-H), 7.70 – 7.63 (m,
6
3
H, -C=CH, Ar-H), 7.29 (d, J = 8.4 Hz, 2H, Ar-H), 7.18 (t, J = 8.7 Hz, 2H, Ar-H),
.81 (d, J = 15.6 Hz, 1H, -C=CH), 6.01 (s, 1H, -C=CH), 5.17 (s, 2H, exocyclic
6
1
3
-
C=CH ), 3.92 (s, 2H, benzylic CH ); C NMR (75 MHz, Acetone-d ) δ 169.02
2 2 6
(
-CO), 165.95 (-CO), 164.38, 159.56, 156.54, 140.38, 139.29, 132.80, 132.52, 130.74,
30.11, 122.80, 120.54, 118.68, 116.65, 95.25 (exocyclic -C=CH ), 32.15 (benzylic
1
2
+
+
CH ); ESI-MS m/z: 372.3 [M+Na] ; ESI-HRMS m/z: 350.1185 [M+H] , calcd for
2
C H FNO 350.1187.
21
17
3
5
.2.1. DNA Topo I Mediated Relaxation Assay in vitro
DNA Topo I inhibitory assay was conducted using a previously reported method
[
7]. The tested compounds were previously dissolved in DMSO at a concentration of
00 mM as stock solutions. CPT was used as a positive control. A mixture of 0.1 µg
1
pBR322 DNA (TaKaRa, Kyoto, Japan) and 0.2 units of recombinant human Topo I
was incubated in the presence or absence of the prepared compounds at 37 °C for 30
min in the Topo I buffer (20 mL) which was provided by the supplier (TaKaRa, Kyoto,
Japan). The reaction was terminated by the addition of a dye solution containing
0
.25% bromophenol blue, 0.25% xylene cyanol ff and 40% glycerol. Then samples
were electrophoresed on a 1% agarose gel at 100 V for 30 min in a running buffer of 1
x TAE buffer (40 mM Tris-acetate, 2 mM EDTA, 19.9 mM HOAc). Gels were stained
for 30 min in 200 mL 1 x TAE buffer containing ethidium bromide (0.5 µg/mL) and
destained with 200 mL double distilled water for 20 min. DNA bands were visualized
by transillumination with UV light and quantitated using an Alpha Imager (Alpha
Innotech Corporation).
5
.2.2. Anticancer activity in vitro by MTT
To measure the antiproliferative activity of the compounds in various cancer cell
lines, cell viability was assessed by a classical MTT assay [30]. Three human cancer
cell lines, MCF-7 (human breast adenocarcinoma cell line), HeLa (human cervix
tumor cell line), A549 (adenocarcinomic human alveolar basal epithelial cell line)
2
4

ACCEPTED MANUSCRIPT
were used in this study. The cells were seeded in 96-well plates at a density of 3.5
3
×
10 per well and incubated overnight in 100 µL of a medium containing 10% Fetal
Bovine Serum (Gibco, USA). On the following day, the culture medium in each well
was replaced by fresh medium containing a range of concentrations of the
4
-(4-substituted amidobenzyl)furan-2(5H)-one derivatives for a cultivation lasting 48
h. Then 20 µL of 5 mg/mL MTT solution were added into each well with a subsequent
o
incubation for 4 h at 37 C. Culture medium was then removed, and 200 mL DMSO
was added to dissolve the formazan dye. The absorption was determined by a
microplate-reader (Bio-Tek) at 570 nm. Parallel triplicate repetition for each dose was
made.
5
.2.3. DNA-insertion assay
The DNA-insertion assay was performed in 20 µL of Topo I buffer (50 mM
Tris-HCl, pH 7.5, 10 mM MgCl , 50 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, and 30
2
mg/mL bovine serum albumin) containing 0.1 µg supercoiled pBR322DNA and
excess Topo I (4 units). The DNA was pre-incubated with different compounds at 25
o
o
C for 10 min. Then Topo I was added to initiate the reaction for 30 min at 37 C.
Afterwards 4 µL of 6 x DNA loading buffer (0.25% bromophenol blue, xylene cyanol
ff 0.25% and 40% glycerol) were added into the samples and electrophoresed for 40
min in a 1% agarose gel in 1 x TAE buffer (40 mM Tris-acetate, 2 mM EDTA, 19.9
mM HOAc). Finally the gels were stained for 30 min in 200 mL 1 x TAE buffer
containing EB (0.5 µg /mL), and destained for 20 min with 200 mL double distilled
water. DNA bands were visualized by transillumination with UV light and
photographed by an Alpha Innotech digital imaging system.
5
.2.4. Topo I-mediated DNA cleavage assay
DNA cleavage assay was carried as follows. 0.125 µg supercoiled pBR322DNA
was incubated with 7.5 units Topo I in 10 µL of Topo I buffer (50 mM Tris-HCl, pH
7
.5, 50 mM KCl, 10 mM MgCl , 0.5 mM DTT, 0.1 mM EDTA, and 30 mg/mL bovine
2
o
serum albumin) at 37 C for 30 min in the presence of tested compounds. After 30
min, the reaction was stopped by adding 1% SDS and 0.8 mg/mL proteinase K. An
o
additional incubation for 10 min at 50 C to digest the Topo I was necessary before
2
5

ACCEPTED MANUSCRIPT
samples were mixed with 2 µL of 6 x loading buffer (0.25% bromophenol blue,
xylene cyanol ff 0.25% and 40% glycerol), and electrophoresed in 1% agarose gel in
1
x TAE buffer (40 mM tris-acetate, 2 mM EDTA, 19.9 mM HOAc) for 60 min at 60
V after. Then gels were stained by 200 mL 1 x TAE containing 0.5 µg/mL EB for 30
min and a secondary electrophoresis of 30 min was significant to separate the relaxed
DNA and nicked DNA. DNA bands were visualized by transillumination with UV
light, photographed by an Alpha Innotech digital imaging system.
5
.2.5. EMSA assay
The DNA mobility shift assay was performed as described in a paper with minor
modifications [26]. First Topo I (4 units) was added into 10 µL of Topo I buffer (50
mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl , 0.5 mM DTT, 0.1 mM EDTA, and
2
3
0 mg/mL bovine serum albumin), which was incubated in the absence or in the
o
presence of compounds at 25 C for 20 min. Then 0.1 µg of pBR322 DNA was added
as another incubation for 3 min. The samples were immediately analyzed using a 1%
agarose gel with 1% EB at 1 V/cm for 8 h on ice. Finally, DNA bands were visualized
by transillumination with UV light, photographed by an Alpha Innotech digital
imaging system.
5
.3. Molecular docking
Sybyl-8.1 surflex-dock module (Tripos, Inc. St. Louis, MO) was used in molecular
docking, and the structure of Topo I was downloaded from the Protein Data Bank
PDB: 1K4T). The water and small molecule ligands were removed with other
(
necessary modification in order to create a simulated physiological environment. A
threshold parameter of 0.5 and a bloat parameter of 0 Å were set to generate the
protomol, which is a computational representation of a ligand that makes every
potential interaction with the binding site, and the Kollman all-charges were added.
We then conducted the staged minimization with 100 steps at every stage and applied
GeomX model mode to the docking study. Sequentially all of our compounds were
docked into the binding site. Minimum RSMD between final poses and other
parameters were respectively set as 0.5 Å to distinguish conformations and default.
2
6

ACCEPTED MANUSCRIPT
After docking study, the binding conformation with highest score was subjected to
MD simulations in AMBER molecular dynamics package [31]. The total MD time
was 20 ns in explicit solvent. We used the Antechamber tool to generate the partial
atomic charges of each ligand and the AMBER force field ff12SB [32] were loaded.
The complexes were then immersed in a truncated octahedral water box by the TIP3P
water model before minimization and MD. Analysis was performed with the ptraj
analysis tool based on the last 2ns trajectories of the simulation. The binding free
energies of complexes were calculated using the MM-PBSA.
Acknowledges
We sincerely thank the Natural Science Foundation of China (81273362) for financial
support of this study.
2
7

ACCEPTED MANUSCRIPT
Reference
[
(
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