Natural tanshinone-like heterocyclic-fused ortho-quinones from regioselective Diels-Alder reaction: Synthesis and cytotoxicity evaluation

Article abstract of DOI:10.1016/j.ejmech.2009.04.016

A series of new natural tanshinone-like oxoheterocyclic-fused ortho-quinone derivatives were synthesized from readily available benzofuranol and N-substituted dienes via IBX oxidation-cycloaddition-aromatization procedure. The regiospecific Diels-Alder cycloaddition reactions of N-dienes were achieved efficiently with a variety of dienophiles. It is found that the amide moiety in the molecular could be preserved or eliminated by control of the aromatization conditions. Selected oxoheterocyclic-fused ortho-quinones as well as several thioheterocyclic-fused ortho-quinones we obtained before were evaluated for their cytotoxicities on different cancer cell lines and the Structure-Activity Relationship (SAR) was discussed.

Full text of DOI:10.1016/j.ejmech.2009.04.016

European Journal of Medicinal Chemistry 44 (2009) 3915–3921
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Natural tanshinone-like heterocyclic-fused ortho-quinones
from regioselective Diels–Alder reaction: Synthesis
and cytotoxicity evaluation
,
,
a
Yu-Dong Shen b 1, Yuan-Xin Tian c, Xian-Zhang Bu a , Lian-Quan Gu
*
a School of Pharmaceutical Sciences, Sun Yat-Sen University, Xin-Gang West Road 135, Guangzhou 510275, China
b College of Food Science, South China Agricultural University, Guangzhou 510642, China
c School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new natural tanshinone-like oxoheterocyclic-fused ortho-quinone derivatives were synthe-
sized from readily available benzofuranol and N-substituted dienes via IBX oxidation–cycloaddition–
aromatization procedure. The regiospecific Diels–Alder cycloaddition reactions of N-dienes were ach-
ieved efficiently with a variety of dienophiles. It is found that the amide moiety in the molecular could be
preserved or eliminated by control of the aromatization conditions. Selected oxoheterocyclic-fused
ortho-quinones as well as several thioheterocyclic-fused ortho-quinones we obtained before were
evaluated for their cytotoxicities on different cancer cell lines and the Structure–Activity Relationship
(SAR) was discussed.
Received 28 October 2008
Received in revised form
26 March 2009
Accepted 3 April 2009
Available online 14 April 2009
Keywords:
Naphthofuranoquinone
IBX
Ó 2009 Elsevier Masson SAS. All rights reserved.
Regioselective
Diels–Alder reaction
Cytotoxicity
1. Introduction
Other than the reported procedures [19–23], we present herein
our results concerning an alternative strategy based on IBX oxida-
It’s well known that many natural and synthetic heterocyclic
quinones possess a variety of bioactivities. For example, naturally
occurring tanshinones from Salvia miltiorrhiza Bunge exhibits
interesting physiologic properties including effects on cardiac
function [1–4], antioxidant activity [5–7], cytotoxicity [8–12],
apoptosis induction [13,14], etc., and have attracted particular
attention from medicinal chemists and clinicians. We have engaged
in a program directed at searching for new bioactive ortho-quinoid
compounds for several years [15–17]. Recently, we described
regioselective cycloaddition reaction of o-quinones with electron-
rich N-dienes for synthesis of novel thioheterocyclic-fused ortho-
quinones [18]. As an extension of our previous works, the synthesis
of oxoheterocyclic-fused ortho-quinones I as novel analogues of
natural tanshinones (Fig. 1) was planned.
tion–regiospecific cycloaddition–aromatization (DDQ and silica
gel) sequence which we developed previously [18]. Cycloaddition
reaction was adopted to construct the functionalized polycyclic
systems, the N-substituted dienes were believed to be able to
provide a rapid and powerful strategy for many complex natural
products and synthetic alkaloids with high regioselectivity [24],
therefore, N-substituted diene derivatives II–IV (Fig. 1) were used
in this paper for the efficient construction of functional oxoheter-
ocyclic-fused ortho-quinone derivatives with general structure I
(Fig. 1). To the best of our knowledge, there are few reports on the
cycloaddition of ortho-benzofuranoquinones with N-substituted
dienes.
2. Chemistry
These key steps of our strategy involved intramolecular cycli-
zation, IBX oxidation, cycloaddition and subsequent aromatization,
starting from readily available benzofuran-5-ol 5a–e and 13
(Schemes 1 and 2).
* Corresponding author. Tel./fax: þ86 20 39943054.
E-mail address: phsbxzh@mail.sysu.edu.cn (X.-Z. Bu).
Tel./fax: þ86 20 85288279. sydscau@yahoo.cn.
1
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.016

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Y.-D. Shen et al. / European Journal of Medicinal Chemistry 44 (2009) 3915–3921
O
Diels–Alder reaction–aromatization sequence. As anticipated, IBX
O
R2
O
also worked very well in these reactions of regiospecifically and
almost quantitatively transforming benzofuan-5-ol 5a–e and 13
into o-benzofuranoquinones 6a–e and 14, as shown in Scheme 3.
On the other hand, the isolation of the o-benzofuranoquinone
intermediates was not pursued for their volatility and high reac-
tivity. However, we found that these o-benzofuranoquinone inter-
mediates could remain stable in dry benzene solution for several
weeks.
O
R1
R1 = Me or H
R2 = Ar or H
R3
R4
O
O
O
R
3 = amide groups or H
R5
R4 = R5 = Me or H
Tanshione IIA
I
O
N
O
N
N
H
2.3. Synthesis of ortho-naphthofuranoquinone derivatives 8a–e,
9a–e, 16, 17, 20 and 21
O
IV
II
III
Our first plan is to finish the construction of the naphthofur-
anoquinone skeleton using cycloaddition between ortho-benzo-
furanoquinones and N-diene II as key step. However, because of the
low stability of the intermediate ortho-benzofuran-4,5-diones,
these compounds were not isolated from the reaction mixture, and
a one-pot reaction was first designed. After the IBX oxidation
process was completed at room temperature, the N-diene II was
directly added to the reaction mixture, and the mixture was heated
to 45 ꢀC for a given time, after which DDQ was added in for the
aromatization at the same temperature. However, the reaction gave
unpredictable and complicated products. We then adopted a multi-
step strategy. After the completion of IBX oxidation process, the
reaction mixture was first diluted with water, and the organic
content was then extracted into benzene. After the benzene solu-
tion containing benzofuran-4,5-diones was dried with anhydrous
sodium sulfate, N-diene II was added for cycloaddition. The reac-
tion was carried out at 45 ꢀC for 16 h and monitored by TLC. The
products of the reaction were subsequently aromatized into the
final ortho-naphthofuranoquinones at 45 ꢀC by DDQ or silica gel.
After evaporation of the solvent and purification by chromatog-
raphy on silica gel (CHCl3/CH3OH), two types of compounds 8a–e,
16 and 9a–e, 17 were obtained in high yields. The cycloaddition
reactions of N-dienes of types III and IV with ortho-benzofur-
anoquinones 6a and 6b were also carried out (Scheme 4). Inter-
estingly, the reaction rates of these dienes were much slower than
Fig. 1.
2.1. Synthesis of substituted benzofuran-5-ol (5a–e, 13)
Compounds 5a–e were obtained in high overall yield (5a 81%, 5b
70%, 5c 82%, 5d 69%, 5e 78%) through three steps (condensation,
cyclization [25] and deprotection) by starting from the inexpensive
and readily available compounds 1 and 2a–e as outlined in
Scheme 1. Compound 13 was synthesized in high yield in four steps
(condensation in 98% yield, rearrangement [26] in 96% yield,
cyclization [27] in 88% yield and deprotection in 86% yield) using
the readily available compounds 1 and 9 as the starting materials as
shown in Scheme 2.
2.2. Synthesis of substituted benzofuran-4,5-dione (6a–e, 14)
As part of our ongoing program dedicated to the construction of
new bioactive molecules, we reported our strategy in 2005 for the
synthesis of thioheterocyclic-fused o-quinones based on IBX
oxidation and Diels–Alder reactions [18]. Herein our first effort was
focused on extending this strategy to the synthesis of oxaheter-
ocyclic-fused o-quinones by o-Iodoxybenzoic acid (IBX) oxidation–
O
O
O
O
R
i
ii
O
OH
Br
+
R
1
2a-e
3a e
-
R
R
2a 3a 4a 5a
,
,
,
: R=Ph
HO
O
2b,3b,4b,5b: R=4-ClPh
iii
2c 3c 4c 5c
,
,
,
: R=4-FPh
O
O
2d,3d,4d,5d: R=4-MePh
2e,3e,4e,5e: R=3,4-2Me-Ph
5a e
-
4a-e
Scheme 1. Conditions: (i) K2CO3, KI, Acetone, r.t., 16 h; (ii) PPA or Amberlyst 15, Toluene, r.t., 2–5 h; (iii) Ac2O–HBr (48%), 140 ꢀC, 4 h.
O
HO
ii
i
O
OH
+
Br
O
O
9
1
10
11
O
O
iv
iii
HO
O
12
13
Scheme 2. Conditions: (i) K2CO3, KI, Acetone, r.t., 10 h; (ii) N2, r.t., 2 h; (iii) PdCl2 (2 mol%), Cu(OAc)2, LiCl, DMF–H2O, r.t., 5 h; (iv) Ac2O–HBr (48%), 140 ꢀC, 1.5 h.

Y.-D. Shen et al. / European Journal of Medicinal Chemistry 44 (2009) 3915–3921
3917
O
6a: R1=H, R2=Ph
structure isomers 2,7,9-trimethylnaphtho[1,2-b]furan-4,5-dione
(17), 7,9-dimethyl-3-p-tolylnaphtho[1,2-b]furan-4,5-dione (9d)
and 7,9-dimethyl-3-(3,4-dimethyl)naphtho[1,2-b]furan-4,5-dione
(9e) formed (Scheme 5, H-6 and C-5 show correlation in HMBC).
Furthermore, we obtained the single crystal of 9a and b, the X-ray
data show clearly the structures as 7,9-dimethyl-3-phenyl-
naphtho[1,2-b]furan-4,5-dione (9a) and 7,9-dimethyl-3-(4-
chlorophenyl)naphtho[1,2-b]furan-4,5-dione (9b) (Fig. 2). The
structure of 16, 8a and b, 8d and e could therefore be confirmed.
Regioisomers 8c, 9c and 21 were judged by deduction.
R2
6b: R1=H, R2=4-ClPh
O
6c
: R1=H, R2=4-FPh
6d
i
R1
5a-e,13
: R1=H, R2=4-MePh
O
6e: R1=H, R2=3, 4-MePh
14: R1=Me, R2=H
6a e 14
- ,
Scheme 3. Conditions: (i) IBX, DMF, r.t., 16 h.
that of N-diene II even at elevated temperatures. TLC analysis of the
reaction mixture showed that the reaction could not be completed
even after refluxing for 24 h. 1.5 equiv of DDQ was added under
reflux for aromatizing the intermediates 18 and 19, and the
aromatization procedure was completed within 2 h for 18 and 6 h
for 19 respectively, giving compound 21 in 50% yield, and 20 in 30%
yield. For the reaction of diene III, the amide group was eliminated
from 18 during the reaction process, giving 20 as the only product.
The possible reason for this might be the effect of steric hindrance
of larger isopropyl which caused the easy removal of amide group.
For diene IV, two carbonyls connected to N atom significantly
decreased the electron donating capability, and the reactivity of
diene IV was also remarkably decreased. Furthermore, ortho-ben-
zofuranoquinones tended to decompose when it was heated for
a long time, this should be the possible cause for the low yield of
compound 20.
3. Results and discussion
3.1. Regioselectivity and reactivity of the cycloaddition reactions
N-Dienes II–IV were unsymmetrical dienes, and the cycloaddi-
tion reaction could provide a mixture of regioisomers. However,
structural analysis of the aromatized products showed that the
aromatization reaction gave only compounds 8a–e, 16, 21 and 9a–e,
17 which should be originated from the single regioisomers 7a–e,15,
18, and 19 (Schemes 4 and 5 and Table 1) [28]; and their structural
isomers 80a–e, 160 and 90a–e, 170 were not detected. These results
indicated that the cycloaddition reactions were essentially regio-
specific. These observations on regioselectivity and different reac-
tivities of the different N-dienes could be explained by the electronic
effects of the diene induced by the carbonyl (from II–IV) and/or
hydrogen bonding (II) (Scheme 5). The intermolecular hydrogen
bond-between the amide group (NH) of the type II N-diene and the
carbonyl oxygen of ortho-quinones, made the transition structure in
EA, HRMS, 1H NMR, 13C NMR were used for final products’
characterization. For the regioisomer identification, the HMBC of 17,
9d and e was recorded, in each case, the HMBC spectra show
apparent correlation of an aromatic H (dH: 7.75, 7.62 and 7.77
referring to 17, 9d and e respectively) with a carbonyl C (dC: 181.48,
180.06 and 181.43 referring to 17, 9d and e respectively), indicating
O
O
R1
O
R1
R1
O
O
O
ii
i
III IV
+ N-diene or
R2
R2
O
O
O
20
:R1= Ph
R2=H
R2=succinimido
18:R1= Ph
19:R1= 4-ClPh
R2=N-isopropylacetamido
R2=succinimido
5a
:R1 = Ph
5b:R1 =4-Cl Ph
21:R1= 4-ClPh
Scheme 4. Conditions: (i) Benzene, r.t., 24 h; (ii) DDQ, benzene, r.t., 24 h.
O
O
O
R2
R2
R2
3
4
5
O
O
O
H
N
R1
R1
2
iior iii
H
N
R1
+
6
O
O
O
8
O
O
7
O
δ−
R2
8a-e, 16
7a-e, 15
9a e 17
- ,
O
δ−
H
N
i
O
R1
δ−
δ+
δ+
+
δ+
O
O
O
R2
δ−
R2
O
R2
3
4
O
5
O
O
II
6a-e, 14
R1
O
R1
R1
6
2
O
iior iii
+
O
8
NH
7
NH
O
O
8'a-e, 16'
7'a-e, 15'
9'a-e, 17'
6a,7a,8a,9a
6b,7b,8b,9b
: R1 = H, R2 = Ph
: R1 = H, R2 = 4-ClPh
6c,7c,8c,9c
6d,7d,8d,9d
: R1 = H, R2 = 4-FPh
6e,7e,8e,9e: R1 = H, R2 = 3, 4-MePh
: R1 = H, R2 = 4-MePh
14,15,16,17: R1 = Me, R2=H
Scheme 5. Conditions: (i) benzene, 45 ꢀC, 16 h; (ii) DDQ, benzene, 45 ꢀC, 16 h; (iii) silica gel, 45 ꢀC, 16 h.

3918
Y.-D. Shen et al. / European Journal of Medicinal Chemistry 44 (2009) 3915–3921
9a
9b
Fig. 2. X-ray crystal structure of 9a (CCDC No. 254246) and 9b (CCDC No. 260179).
a more ordered state and been easier to form. This was proved to be
true from the NMR signal assignments of the aromatized products
and the single-crystal structure analysis.
3.3. Cytotoxicity evaluation and SAR
To investigate the bioactivities of obtained analogues, the oxo-
heterocyclic-fused ortho-quinones (8a–e, 9a–e, 17, 21) obtained in
the current work, as well as 5 thioheterocyclic-fused ortho-
quinones (s1–s5, Fig. 3), which were prepared in our previous work
[18], were chosen for the primary cytotoxicity evaluation on human
lung adenocarcinoma cells GLC-82 and human nasopharyngeal
carcinoma cell Line CNE2 using MTT (3-(4,5-dimethylthiazol-2-yl)-
3.2. Facile control of the aromatization procedure
Noticeably, our experimental results (Table 1, Schemes 4 and 5)
showed that different method of aromatization had great effect on
the ratio of products. These results made it possible to fully control
the structures of the final products. During the aromatization
process, there were two competitive reactions at the same time:
one would be the thermal elimination of amide groups, and the
other would be the dehydrogenation of the cycloaddition inter-
mediate by oxidant DDQ or oxygen species from the air. When DDQ
is used as dehydrogenation reagent in solvent, the reaction
happens in homogeneous phase. Thus, dehydrogenation procedure
with DDQ finished smoothly and quickly, leaving the amide groups
intact. However, when the cycloaddition intermediates were
loaded on silica gel, the reaction proceeded heterogeneously. In this
case it was not easy for the cycloaddition products to react with
oxygen from the air. As a result, the dehydrogenation proceeded
slowly, and the thermal elimination reaction was the predominant
one. In addition, the hydrogen bonding interaction between amide
groups and the Si–OH groups on the surface of silica gel could
accelerate the elimination of amide groups. Therefore, when
aromatization was carried out on silica gel, the products with amide
groups eliminated were the major products.
2,5-diphenyl-2H-tetrazolium
bromide)
assay.
10-Hydrox-
ycamptothecin (HCPT), a clinically used antitumor agent, was also
evaluated for positive control; unfortunately, most of compounds
without the 6-amido substitution (9b and c, 9e, 17) and 8e were
found to be insoluble and the data cannot be obtained; the results
are summarized in Table 2.
The obtained data (8a–d, 9a, 9d, 21, s1–s5) reveal a general SAR
for the cytotoxicity of tested compounds. Comparing the cytotox-
icities of 8a–d with that of s1–s4 respectively, it seems that the
replacement of O with S in the heterocyclic moiety can elevate the
activity slightly. On the other hand, though s2 has slight higher IC50
than other thioheterocyclic-fused ortho-quinones s1, s3 and s4, no
evidence shows that the different substitution on the 3-phenyl has
apparent effects on their cytotoxicity to both cancer cell lines.
Interestingly, 9a, 9d and s5, which without the 6-acetamido
substitution, have much lower cytotoxicity to both cancer cell lines
than others especially,
a 6-N-succinimidyl moiety bearing
compound 21 has much stronger toxicity than others, and has
closer IC50 (2.0 and 1.6 mmol/L refer to CNE2 and GLC-82
Table 1
Synthesis from substituted benzofuran-5-ol to ortho-naphthofuranoquinones.d
Entry
1
Benzofuranoquinones
N-Diene
Naphthofuranoquinones
Total yields (%)
Ratioa
5a
II
8a þ 9a
85b
82c
91b
78c
90b
90c
85b
92c
88b
94c
89b
90c
30b
50b
92/8
23/77
88/12
35/65
100/0
27/73
94/6
26/74
92/8
42/58
86/14
20/80
–
2
3
4
5
6
5b
5c
5d
5e
13
II
II
II
II
II
8b þ 9b
8c þ 9c
8d þ 9d
8e þ 9e
16 þ 17
7
8
5a
5b
IV
III
20
21
–
a
Ratios ¼ (products with amide group)/(products without amide group).
DDQ aromatization method.
Silica catalyzed aromatization method.
b
c
d
All isolated yields were based on substituted benzofuran-5-ol.

Y.-D. Shen et al. / European Journal of Medicinal Chemistry 44 (2009) 3915–3921
3919
O
glass plate coated with silica gel with fluorescent indicator (GF254).
Flash chromatography was performed on silica gel H. 1H NMR, 13
R4
O
C
: R =acetamido, R =R =Me, R =Ph
s1
: R1=acetamido, R2=R3=Me, R4=4-Cl-Ph
NMR spectra were measured on a Varian UNITY INOVA 500 MHz or
300 MHz spectrometer using TMS as an internal standard. The
electrospray (ESI) MS analysis was performed on a Finnigan LCQ
Deca XP ion trap mass spectrometer. FAB-MS was measured on
a VG ZAB-HS analytical spectrometer. HRMS were measured on
a Thermo MAT 95XP; N-dienes II–IV were obtained by using the
reported method [24,29,30]; Analyses indicated by the symbols of
the element functions were within 0.4% of the theoretical values for
the final products 8a–e, 9a–e, 16, 17, 20 and 21.
s2
: R1=acetamido, R2=R3=Me, R4=4-F-Ph
R1
R2
S
s3
: R1=acetamido, R2=R3=Me, R4=2-Me-Ph
s4
: R1=R =R =H, R =Ph3
2
4
4
s5
R3
1
2
3
Fig. 3.
Table 2
Effect of compounds on the growth of cancer cell lines.
a
Compound
IC50
(
m
mol/L)
GLC-82
Compound
IC50
(
m
mol/L)
GLC-82
5.2. General procedure for 2-(4-methoxyphenoxy)
-1-arylethanone (3a–e)
CNE2
CNE2
8a
8b
8c
8d
8e
9a
9b
9c
9d
4.8
9.2
nd
5.6
na
>25
na
na
7.2
6.0
8.9
9.5
na
>25
na
na
9e
17
21
s1
s2
s5
s3
s4
HCPT
na
na
na
na
Potassium carbonate (0.05 mol) and potassium iodide
(0.005 mol) were added to a mixture of 2-bromo-1-arylethanone
(0.05 mol), 4-methoxyphenol (0.05 mol), and dry acetone (60 mL).
The reaction mixture was covered with black cloth and was stirred
at room temperature for 16 h under nitrogen atmosphere, and then
was diluted with 120 mL of CH2Cl2. The solid salts were filtered off
and the organic phase was washed with water, dried over Na2SO4.
Evaporation gave crude compounds 3a–e which were directly used
for the next reactions without further purification.
2.0
4.1
6.8
nd
4.6
4.2
0.37
1.6
5.0
7.5
20.1
3.9
nd
23.3
12.0
1.09
nd, not determined; na, data cannot be obtained owning to the poor solubility of the
sample.
a
The IC50 represents the compound concentration required for the reduction of
the mean cell viability to 50%.
5.3. General procedure for 5-methoxy-3-arylbenzofuran (4a–e)
respectively) to that of clinical drug 10-hydroxycamptothecin
Method A. A mixture of 2-(4-methoxyphenoxy)-1-aryletha-
none (3a–e) (about 0.01 mol), polyphosphoric acid (20 g), and
toluene (60 mL) was refluxed for 5–6 h. The reaction mixture was
cooled to room temperature, poured into water, and extracted with
diethyl ether. The organic extracts were dried over Na2SO4. Evap-
oration gave crude compounds 4a–e which were directly used for
the next reactions without further purification.
Method B. A mixture of 2-(4-methoxyphenoxy)-1-arylethanone
(3a–e) (0.01 mol), dry Amberlyst 15 resin (1 g), and toluene (25 mL)
was refluxed for 2–6 h with azeotropic removal of water. The
catalyst was removed by filtration, and the filtrate was washed with
ethyl ether. Evaporation of solvent gave compounds 4a–e which
were directly used for the next reactions without further
purification.
(HCPT, IC50 ¼ 0.37 and 1.09
mmol/L refer to CNE2 and GLC-82
respectively), indicating that the amide moiety substitution on the
benzo-ring plays an important role in cancer cell growth inhibition,
and further group alteration on benzo-ring would be the direction
of more potent diversity generation in future work.
4. Conclusions
In this work, we successfully synthesized 14 new natural
tanshinone-like ortho-naphthofuranoquinones via IBX oxidation–
cycloaddition–aromatization
sequences.
The
regiospecific
cycloaddition reactions of N-dienes were achieved efficiently with
a variety of dienophiles. By selection of different aromatization
conditions, it is possible to control the amide moiety to be
preserved or eliminated; the introduction of substitution on the
5.4. General procedure for substituted benzofuran-5-ol (5a–e)
aromatic ring provided us with
a good chance to produce
compounds with a variety of functional groups, and made it
possible for us to establish a library of highly functionalized
tanshinone analogues. Cytotoxicity evaluation of selected oxohe-
terocyclic-fused ortho-quinones as well as several thioheterocyclic-
fused ortho-quinones indicates that most of the substrates have
moderate to good cytotoxicity to cancer cell lines CNE2 and GLC-82,
the SAR analysis reveals that 6-amide moiety substitution plays an
important role in cancer cell growth inhibition; especially, the 6-N-
succinimidyl moiety bearing compound 21 was found to have
strongest cytotoxicity to tested cell lines, and might serve as an
effective lead compound for further antitumor agent development.
Further exploration focuses on more potent diversity generation
mainly by the group alteration on the benzo-ring, SAR analysis and
detail mechanism is under active exploration.
Unpurified 3-aryl-5-methoxybenzofuran (about 2 mmol) was
added to mixed solution (12 mL) of 48% HBr and acetic anhydride
(1:1, v/v). The mixture was refluxed at 140 ꢀC for 0.5–4 h. The
reaction mixture was cooled to room temperature, neutralized
with saturated sodium bicarbonate solution and extracted with
diethyl ether. The organic extracts were washed with saturated
sodium chloride solution and dried over Na2SO4. Evaporation and
purification by chromatography on silica gel (Hexanes/EtOAc) gave
(5a–e) in overall yields of 69–82%.
3-Phenylbenzofuran-5-ol (5a). ESI-MS m/z: 209 ([M ꢁ H]ꢁ). 1H
NMR (500 MHz, CDCl3,
d ppm): 4.91 (br, s,1H), 6.86 (dd, 1H,
J ¼ 2.5 Hz, J ¼ 9.0 Hz), 7.24 (d, 1H, J ¼ 2.5 Hz), 7.34–7.37 (m, 1H), 7.39
(d, 1H, J ¼ 9.0 Hz), 7.43–7.47 (m, 2H), 7.58–7.60 (m, 2H), 7.75 (s, 1H).
13C NMR (75 MHz, CDCl3,
d ppm): 151.31,150.57, 142.10, 131.66,
128.67, 127.14, 126.99, 121.87, 113.22, 112.04, 105.53.
5. Experimental
3-(4-Chlorophenyl)benzofuran-5-ol (5b). ESI-MS m/z: 243
([M ꢁ H]ꢁ). 1H NMR (300 MHz, d6-acetone,
d ppm): 6.93 (dd, 1H,
5.1. General
J ¼ 2.4, J ¼ 9.0), 7.28 (d, 1H, J ¼ 2.4), 7.40 (d, 1H, J ¼ 9.0), 7.48 (d, 2H,
J ¼ 8.4), 7.69 (d, 2H, J ¼ 8.4), 8.05 (s, 1H). 13C NMR (75 MHz, d6-
All reagents were commercially available. Solvents were treated
using standard techniques. Reactions were monitored by TLC on
acetone, d ppm): 154.11, 150.34, 143.17, 132.62, 131.29, 129.21,
128.79, 126.76, 120.85, 113.84, 112.16, 104.90.

3920
Y.-D. Shen et al. / European Journal of Medicinal Chemistry 44 (2009) 3915–3921
3-(4-Fluorophenyl)benzofuran-5-ol (5c). ESI-MS m/z: 227
129.52, 129.65, 129.74, 135.51, 136.57, 137.02, 139.42, 140.18, 140.68,
163.33, 175.05, 181.43. Anal. (C22H18O3) C, H.
7,9-Dimethyl-3-p-tolylnaphtho[1,2-b]furan-4,5-dione (9d) (Red
solid). ESI-MS m/z: 317 ([M þ H]þ). HRMS-EI m/z: calcd for
C21H16O3, 316.1094, found 316.1087. 1H NMR (500 MHz, d6-DMSO,
([M ꢁ H]ꢁ). 1H NMR (300 MHz, CDCl3,
d
ppm): 7.56 (s, 1H), 7.28
(dd, 2H, J ¼ 5.4 Hz, J ¼ 8.7 Hz), 7.25 (d, H, J ¼ 8.7 Hz), 7.11 (d, 1H,
J ¼ 2.7 Hz), 6.91 (t, 2H, J ¼ 8.7 Hz), 6.84 (dd, 1H, J ¼ 2.7 Hz,
J ¼ 8.7 Hz), 6.73 (br, s,1H). 13C NMR (75 MHz, CDCl3,
d ppm): 161.75
(d), 151.08, 150.54, 141.99, 128.44 (d), 127.54, 126.95, 120.96, 115.54
(d), 113.30, 112.14, 105.36.
d
ppm): 2.32 (s, 3H), 2.35 (s, 3H), 2.61 (s, 3H), 7.23 (d, 2H, J ¼ 8.0 Hz),
7.38 (s, 1H), 7.59 (d, 2H, J ¼ 8.0 Hz), 7.62 (s, 1H), 8.16 (s, 1H). 13C NMR
3-p-Tolylphenylbenzofuran-5-ol (5d). ESI-MS m/z: 223
(500 MHz, d6-DMSO, d ppm): 20.44, 20.72, 20.93, 118.40, 123.65,
([M ꢁ H]ꢁ). 1H NMR (300 MHz, d6-acetone,
d
ppm): 2.35 (s, 3H),
125.89, 126.48, 128.13,128.20, 128.63, 129.70, 134.62,137.29, 138.65,
139.53, 141.93, 162.04, 174.37, 180.06. Anal. (C21H16O3) C, H.
7,9-Dimethyl-3-p-tolyl-6-acetamidonaphtho[1,2-b]furan-4,5-dione
(8d) (Red solid). ESI-MS m/z: 372 ([M ꢁ H]ꢁ). HRMS-EI m/z: calcd for
C23H19NO4, 373.1309, found 373.1310. 1H NMR (500 MHz, d6-DMSO,
6.93 (dd, 1H, J ¼ 2.4 Hz, J ¼ 9.0 Hz), 7.26 (d, 2H, J ¼ 7.8 Hz), 7.31
(d, 1H, J ¼ 2.4 Hz), 7.38 (d, 1H, J ¼ 9.0 Hz), 7.55 (d, 2H, J ¼ 7.8 Hz),
7.95 (s, 1H). 13C NMR (75 MHz, d6-acetone,
d ppm): 153.92, 150.37,
142.44, 137.03, 129.77, 129.49, 127.26, 127.16, 121.95, 113.62, 112.04,
105.15, 20.76.
d
ppm): 2.08 (s, 3H), 2.18 (s, 3H), 2.34 (s, 3H), 2.64 (s, 3H), 7.23 (d, 2H,
J ¼ 7.5), 7.48 (s, 1H), 7.60 (d, 2H, J ¼ 7.5), 8.22 (s, 1H), 9.65 (br, s,1H).
13C NMR (500 MHz, d6-DMSO,
ppm): 17.90, 20.72, 21.28, 23.03,
3-(3,4-Dimethylphenyl)benzofuran-5-ol (5e). ESI-MS m/z: 257
([M ꢁ H]ꢁ). 1H NMR (300 MHz, CDCl3,
d
ppm): 7.63 (s, 1H), 7.05–
d
7.29 (m, 5H), 6.80 (dd, 1H, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 2.22 (s, 3H), 2.20
117.85, 124.03, 124.68, 125.54, 126.31, 128.01, 128.70, 132.62, 137.40,
137.57, 138.28, 140.02, 141.29, 162.32, 168.24, 174.96, 182.35. Anal.
(C23H19NO4) C, H, N.
7,9-Dimethyl-3-phenyl-6-acetamidonaphtho[1,2-b]furan-4,5-
dione (8a) (Red solid). ESI-MS m/z: 358 ([M ꢁ H]ꢁ). HRMS-EI m/z:
calcd for C22H17NO4, 359.1152, found 359.1154. 1H NMR (300 MHz,
(s, 3H). 13C NMR (75 MHz, CDCl3,
d ppm): 151.17, 150.59, 141.82,
136.94, 135.66, 129.95, 129.13, 128.25, 127.27, 124.46, 121.88, 113.01,
111.96, 105.59, 19.78, 19.50.
2-Methylbenzofuran-5-ol (13). ESI-MS m/z: 147 ([[M ꢁ H]ꢁ). 1H
NMR (300 MHz, CDCl3,
d
ppm): 7.15 (d, 1H, J ¼ 8.7 Hz), 6.86 (d, 1H,
J ¼ 2.1 Hz), 6.69 (dd, 1H, J ¼ 2.1 Hz, J ¼ 8.7 Hz), 6.11 (s,1H), 2.32
CDCl3, d ppm): 2.23 (s, 3H), 2.24 (s, 3H), 2.63 (s, 3H), 7.29 (s, 1H),
(s, 3H). 13C NMR (75 MHz, CDCl3,
d
ppm): 156.24, 150.61, 149.55,
7.34–7.41 (m, 3H), 7.61 (s, 1H), 7.64 (d, 2H, J ¼ 7.2), 9.83 (br, s,1H). 13C
129.89, 111.31, 110.76, 105.48, 102.48, 14.02.
NMR (300 MHz, CDCl3, d ppm): 19.44, 21.85, 24.19, 118.30, 121.16,
125.09, 127.06, 128.33, 128.45, 128.50, 128.97, 133.18, 138.33, 140.01,
141.31, 142.10, 163.19, 168.98, 174.51, 184.68. Anal. (C22H17NO4 with
1H2O) C, H, N.
5.5. General procedure for substituted naphtho[1,2-b]furan-4,5-
dione (8a–e, 9a–e, 16, 17, 20, 21)
7,9-Dimethyl-3-phenylnaphtho[1,2-b]furan-4,5-dione (9a) (Red
solid). ESI-MS: m/z 303 ([M þ H]þ). HRMS-EI m/z: calcd for
C20H14O3, 302.0937, found 302.0925. 1H NMR (500 MHz, CDCl3,
Substituted benzofuran-5-ol (5a–e, 13) (1 mmol) and IBX
(1.2 mmol) were added to dry DMF (5 mL) and the reaction mixture
was stirred at room temperature for 4–5 h. The reaction mixture
was diluted with 120 mL water, extracted with benzene, washed
with saturated sodium chloride solution, dried over Na2SO4, and
concentrated to 50 mL. N-Dienes (1 mmol) were added and the
mixture was stirred at 45 ꢀC for 16 h for N-diene II and refluxed for
36 h for N-dienes II, IV. The cycloaddition products were then
aromatized using the following two different methods.
Method A. To one half of the above solution was added DDQ
(0.75 mmol) and the mixture was refluxed for 16 h. Evaporation
and purification by chromatography on silica gel (CHCl3/CH3OH)
gave the target compounds 8a–e and 16 in 75–86% yield, 21 in 20%
yield, 9a–e and 17 in 6–12% yield and 20 in 50% yield.
Method B. To the other half of solution was added silica gel
(10 g, 80–120 mesh) and the solvent was removed in vacuo at 45 ꢀC.
The resulted dry silica gel was then stirred at 45 ꢀC for 24 h. Puri-
fication by chromatography on silica gel (CHCl3/CH3OH) gave target
compounds 8a–e, 16 in 19–25% yield and 9a–e, 17 in 55–65% yield.
7,9-Dimethyl-3-(3,4-dimethylphenyl)-6-acetamidonaphtho [1,2-
b] furan-4,5-dione (8e) (Red solid). ESI-MS m/z: 388 ([M þ H]þ),
HRMS-EI m/z: calcd for C24H21NO4, 387.1465, found 387.1459. 1H
d
ppm): 2.35 (s, 3H), 2.67 (s, 3H), 7.23 (s, 1H), 7.35–7.42 (m, 3H), 7.59
(s, 1H), 7.64 (d, 1H, J ¼ 6.6 Hz), 7.75 (s, 1H). 13C NMR (500 MHz,
CDCl3,
d ppm): 21.16, 21.68, 118.71, 124.14, 127.19, 128.30, 128.34,
129.09, 129.46, 129.63, 135.47, 139.33, 140.19, 140.81, 163.30, 174.84,
181.10. Anal. (C20H14O3) C, H.
2,7,9-Trimethyl-6-acetamidonaphtho[1,2-b]furan-4,5-dione (16)
(Red solid). ESI-MS m/z: 296 ([M ꢁ H]ꢁ). HRMS-EI m/z: calcd for
C17H15NO4, 297.0996, found 297.0991. 1H NMR (500 MHz, CDCl3,
d
ppm): 2.23 (s, 3H), 2.24 (s, 3H), 2.40 (d, J ¼ 1 Hz, 3H), 2.59 (s, 3H),
6.40 (q, J ¼ 1 Hz, 1H), 7.26 (s, 1H), 9.86 (br, s,1H). 13C NMR (500 MHz,
CDCl3,
d ppm): 13.58, 19.30, 21.50, 24.11, 103.78, 121.00, 121.94,
125.25, 132.62, 137.87, 140.26, 141.88, 155.52, 161.21, 168.97, 173.98,
184.71. Anal. (C17H15NO4) C, H, N.
2,7,9-Trimethylnaphtho[1,2-b]furan-4,5-dione (17) (Red solid).
ESI-MS m/z: 241 ([M þ H]þ). 1H NMR (500 MHz, CDCl3,
d ppm): 2.34
(s, 3H), 2.41 (d, J ¼ 1 Hz, 3H), 2.62 (s, 3H), 6.44 (q, J ¼ 1 Hz, 1H), 7.20
(s, 1H), 7.75 (s, 1H). 13C NMR (500 MHz, CDCl3,
d ppm): 13.60, 20.99,
21.22, 104.03, 122.60, 124.42, 129.71, 129.79, 134.92, 139.17, 139.69,
154.97, 161.53, 174.80, 181.48. Anal. (C15H12O3) C, H.
7,9-Dimethyl-3-(4-chlorophenyl)-6-acetamidonaphtho[1,2-b]furan-
4,5-dione (8b) (Red solid). ESI-MS m/z: 391 ([M ꢁ H]ꢁ). HRMS-EI m/z:
calcd for C22H16ClNO4, 393.0762, found 393.0764. 1H NMR (500 MHz,
NMR (500 MHz, CDCl3,
d ppm): 2.25 (s, 3H), 2.26 (s, 3H), 2.29 (s,
3H), 2.31 (s, 3H), 2.67 (s, 3H), 7.18 (d,1H, J ¼ 7.5 Hz), 7.33 (s, 1H), 7.39
(dd, 1H, J ¼ 7.5 Hz, J ¼ 1.5 Hz), 7.45 (s, 1H), 7.61 (s, 1H), 9.91 (s, br,
CDCl3,
(d, J ¼ 8.0 Hz, 2H), 7.63 (d, J ¼ 8.0 Hz, 2H), 7.66 (s, 1H), 9.91 (br, s,1H).
13C NMR (500 MHz, CDCl3,
ppm): 19.50, 21.86, 24.21, 118.17, 121.27,
d ppm): 2.26 (s, 3H), 2.27 (s, 3H), 2.69 (s, 3H), 7.36 (s, 1H), 7.40
1H). 13C NMR (500 MHz, CDCl3,
d ppm): 19.47, 19.58, 19.76, 21.89,
24.23, 118.43, 121.16, 125.24, 125.76, 126.37, 127.14, 129.52, 129.74,
133.14, 136.67, 137.18, 138.19, 139.98, 141.05, 142.12, 163.07, 169.02,
174.56, 184.89. Anal. (C24H21NO4) C, H, N.
7,9-Dimethyl-3-(3,4-dimethyl)naphtho[1,2-b]furan-4,5-dione (9e)
(Red solid). ESI-MS m/z: 331 ([M þ H]þ). HRMS-EI m/z: calcd for
C22H18O3, 330.1250, found 330.1251. 1H NMR (500 MHz, CDCl3,
d
124.99, 126.15, 127.52, 128.74, 129.70, 133.32, 134.57, 138.62, 140.21,
141.32, 142.19, 163.45, 169.02, 174.62, 184.62. Anal. (C22H16ClNO4) C,
H, N.
7,9-Dimethyl-3-(4-chlorophenyl)naphtho[1,2-b]furan-4,5-dione
(9b) (Red solid). ESI-MS m/z: 336 ([M þ H]þ). HRMS-EI m/z: calcd for
C20H13ClO3, 336.0548, found 336.0545. 1H NMR (300 MHz, CDCl3,
d
ppm): 2.29 (s, 3H), 2.30 (s, 3H), 2.35 (s, 3H), 2.67 (s, 3H), 7.17 (d,
1H, J ¼ 7.5 Hz), 7.24 (s, 1H), 7.37 (dd, 1H, J ¼ 7.5 Hz, J ¼ 2.0 Hz), 7.45
(s, 1H), 7.57 (s, 1H), 7.77 (s, 1H). 13C NMR (500 MHz, CDCl3,
ppm):
19.57, 19.75, 21.06, 21.58, 118.91, 124.36, 125.81, 126.62, 127.33,
d
ppm): 2.36 (s, 3H), 2.66 (s, 3H), 7.24 (s, 1H), 7.35 (d, J ¼ 8.4 Hz, 2H),
d
7.58 (d, J ¼ 8.4 Hz, 2H), 7.59 (s, 1H), 7.76 (s, 1H). 13C NMR (300 MHz,
CDCl3, d ppm): 180.91, 174.82, 163.50, 140.85, 140.40, 139.39, 135.58,

Y.-D. Shen et al. / European Journal of Medicinal Chemistry 44 (2009) 3915–3921
3921
134.25, 129.62, 129.55, 128.49, 127.58, 126.14, 123.97, 118.45, 21.70,
21.21. Anal. (C20H13ClO3) C, H.
7,9-Dimethyl-3-(4-flurophenyl)-6-acetamidonaphtho[1,2-b]furan-
4,5-dione (8c) (Red solid). ESI-MS m/z: 376 ([M ꢁ H]ꢁ). HRMS-EI m/z:
calcd for C22H16FNO4, 377.1058, found 377.1055. 1H NMR (300 MHz,
a microplate reader (Bio-Rad). Cytoviability of the control cells was
taken as 100%. Tests were conducted in quadruplicate. For each dose,
the mean cell viability was expressed as a percentage of the control.
The IC50 represents the compound concentration required for the
reduction of the mean cell viability to 50%.
CDCl3,
(s, 1H), 7.61 (s, 1H), 7.64 (dd, 2H, J ¼ 5.4 Hz, J ¼ 8.5 Hz), 9.88 (br,
s,1H). 13C NMR (500 MHz, CDCl3,
ppm): 184.69, 174.65, 169.08,
d
ppm): 2.26 (s, 6H), 2.67 (s, 3H), 7.09 (t, 2H, J ¼ 8.5 Hz), 7.33
Acknowledgments
d
163.33, 162.95 (d), 142.19, 141.11, 140.11, 138.53, 133.31, 130.23 (d),
126.22, 125.04, 121.25, 118.23, 115.51 (d), 24.23, 21.89, 19.50. Anal.
(C22H16FNO4) C, H, N.
7,9-Dimethyl-3-(4-flurophenyl)naphtho[1,2-b]furan-4,5-dione (9c)
(Red solid). ESI-MS m/z: 321 ([M þ H]þ). HRMS-EI m/z: calcd for
C20H13FO3, 320.0843, found 320.0842. 1H NMR (300 MHz, CDCl3,
We are indebted to the National High-tech R&D 863 Program
(2008AA02Z304), Science and Technology Key Project of The
Education Ministry of PR China (105133), and the Guangdong
Provincial Science Foundation (5001773) for financial support of
this study.
d
ppm): 2.37 (s, 3H), 2.68 (s, 3H), 7.08 (t, 2H, J ¼ 8.7 Hz), 7.25 (s, 1H),
References
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(500 MHz, CDCl3,
d ppm): 181.26, 175.12, 163.59, 162.88 (d), 140.73,
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d ppm): 180.20, 174.28, 161.26, 141.35, 135.33, 130.18, 130.12,
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dione (21). FAB-MS m/z: 406 ([M þ H]þ). 1H NMR (300 MHz, d6-
DMSO,
d
ppm): 2.86–2.96 (m, 4H, 2CH2), 7.41 (d, 1H, J ¼ 8.0 Hz), 7.51
(d, 2H, J ¼ 8.5 Hz), 7.78 (d, 2H, J ¼ 8.5 Hz), 7.92–7.98 (m, 2H), 8.38 (s,
1H). 13C NMR (300 MHz, d6-DMSO,
d
ppm): 178.23, 176.09, 172.78,
159.62,143.41,135.90, 134.39, 132.70,132.92, 129.70,129.58,128.20,
128.06, 125.24, 123.74, 123.25, 118.48, 28.74. Anal. (C22H12ClNO5) C,
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structure assignments of these aromatized products.
5.7. Cell growth inhibition assay
The growth inhibitory effects of selected compounds on different
cancer cell lines were evaluated by means of the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay.
The cells were plated at a density of 1 ꢂ104 per well in 96-well
microplates and allowed to incubate overnight. The tested
compounds were added to the wells at increasing concentrations (0–
100
solution and the cells were further incubated at 37 ꢀC for 4 h. At the
end of incubation, the untransformed MTT was removed and 150
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mL
DMSO added. The microplates were well shaken to dissolve the
formazan dye and the absorbance at 570 nm was measured using