Synthesis and anticancer activity of 2,4-disubstituted furo[3,2-b] indole derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.06.012

We synthesized and evaluated a series of 2,4-disubstituted furo[3,2-b]indole derivatives for anticancer activity and established the structure - activity relationships (SARs) of these compounds. Among all tested compounds, we found (5-((2-(hydroxymethyl)-4H-furo[3,2-b]indol-4-yl)methyl) furan-2-yl) methanol (10a) to be the most promising agent. In screening against NCI-60 human tumor cell lines, 10a exhibited highly selective anticancer activity and significant inhibitory activity against A498 renal cancer cells. Our COMPARE analysis results suggest that the 10a fingerprint is similar to that of NSC-754549, which is an isostere of YC-1. We further con firmed the significant antitumor activity of compound 10a with tests in the A498 xenograft nude mice model. Therefore, compound 10a should be further developed as a new drug candidate for treating renal cancer.

Full text of DOI:10.1016/j.ejmech.2013.06.012

European Journal of Medicinal Chemistry 66 (2013) 466e479
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity of 2,4-disubstituted furo[3,2-b]
indole derivatives
Shi-Hong Zhuang a, Yi-Chien Lin a, Li-Chen Chou a, Mei-Hua Hsu a, Hui-Yi Lin a,
,
Chi-Hung Huang b, Jin-Cherng Lien a, Sheng-Chu Kuo a, Li-Jiau Huang a
*
a Graduate Institute of Pharmaceutical Chemistry, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan ROC
b Taiwan Advance Biopharm, Inc., 12F, No. 25, Lane 169, Kangning Street, Xizhi District, New Taipei City 221, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized and evaluated a series of 2,4-disubstituted furo[3,2-b]indole derivatives for anticancer
activity and established the structureeactivity relationships (SARs) of these compounds. Among all
tested compounds, we found (5-((2-(hydroxymethyl)-4H-furo[3,2-b]indol-4-yl)methyl)furan-2-yl)
methanol (10a) to be the most promising agent. In screening against NCI-60 human tumor cell lines, 10a
exhibited highly selective anticancer activity and significant inhibitory activity against A498 renal cancer
cells. Our COMPARE analysis results suggest that the 10a fingerprint is similar to that of NSC-754549,
which is an isostere of YC-1. We further confirmed the significant antitumor activity of compound 10a
with tests in the A498 xenograft nude mice model. Therefore, compound 10a should be further devel-
oped as a new drug candidate for treating renal cancer.
Received 8 February 2013
Received in revised form
23 May 2013
Accepted 7 June 2013
Available online 18 June 2013
Keywords:
Furo[3,2-b]indole derivatives
Structureeactivity relationships (SARs)
Anticancer activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
A498 renal cancer cells
1. Introduction
anticancer activity. Although the exact role of these two moieties is
unclear, from a molecular biology viewpoint, we continued modi-
The 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) is a
highly potent anticancer drug candidate with multiple biological
activities, including anti-angiogenesis [1,2], anti-inflammation [3],
and apoptosis induction [4,5] activities as well as the ability to
inhibit matrix metalloproteinases (MMPs) [6]. Studies on YC-1 in
various antitumor animal models, such as non-small cell lung
cancer [2], renal cancer [7], and breast cancer [8], have all
confirmed its significant anticancer activity.
The core skeleton of YC-1 is an indazole. We previously applied
the concept of bioisosterism to replace the indazole ring of YC-1
with furo[3,2-c]pyrazole (A) [9], thieno[3,2-c]pyrazole (B) [10],
and selenolo[3,2-c]pyrazole (C) [11] in an effort to discover YC-1
analogs with potential anticancer activity. We also synthesized a
series of these analogs with different core skeletons and evaluated
their anticancer activity to establish the structureeactivity re-
lationships (SARs) of YC-1 analogs (Chart 1).
fying the structure of YC-1 analogs, by fusing its furan ring with the
indole ring to form a new core structure consisting of expanded
tricyclic furo[3,2-b]indole (D). By adding a fourth type of YC-1 an-
alogs with a novel core structure, we attempted to establish
enhanced SARs, leading to the development of a more profound
anticancer drug candidate. We separately introduced similar
functional groups used previously, namely, the CH2OH-bearing
group and the CH2eAr group, onto the 2-position and 4-position of
the new skeleton. We synthesized and evaluated the analogs of this
new core skeleton for anticancer activity.
2. Results and discussion
2.1. Chemistry
Scheme 1 schematically shows the synthetic routes to the target
compounds (1e3, 6aec, 7aeo and 10aec). Starting compound 1
[12] was reacted with various alkyl halides (4a, 4b), substituted
benzyl halides (4ceo), and heteroarylmethyl halides (8aec) [13] in
the presence of NaH to yield the corresponding methyl 4-
substituted furo[3,2-b]indole-2-carboxylates (5, 9). The ester
compounds 5aeo and 9aec were either hydrolyzed with 10% NaOH
to the corresponding carboxylic acids (3, 6aec) or reduced with Ca
Our findings showed that all A-, B-, and C-type compounds
exhibited similar SARs, and those with 3-furanylcarbinol and 1-
methylaryl moieties demonstrated significant and selective
* Corresponding author. Tel.: þ886 4 22053366x5609; fax: þ886 4 22030760.
E-mail address: ljhuang@mail.cmu.edu.tw (L.-J. Huang).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.012

S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
467
Chart 1. Structures of YC-1 analogs.
Scheme 1. Reagents and conditions: (a) NaH/DMF; (b) Ca(BH4)2/THF; (c) (1) 10% NaOH (2) dilute HCl.

468
S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
(BH4)2 to afford the corresponding carbinols 7aeo and 10aec. The
chemical structures of the target compounds were determined
based on their HRMS and NMR spectra.
exhibited the greatest inhibitory activity among compounds 1e3
M). Replace-
ment of the CH2OH group (R2) of compound 2 with a COOCH3 group
(1) or with a COOH group (3) resulted in reduced inhibitory activity
against the A498 renal cancer cell line (IC50 ¼ 21.8
m
against the A498 cell line (IC50 > 50 mM). In contrast, replacement
of the H (R4) of compound 2 with a CH3 group (7a), a C2H5 group
(7b), or a benzyl group (7c) increased inhibitory activity against the
2.2. Screening tests of compounds 1e3, 5aeo, 6aec, 7aeo, 9aec
and 10aec
A498 cell line in the order of compound
2
(R4
M) < 7b (R4 ¼ C2H5,
M). Larger sub-
¼
H,
We evaluated the newly synthesized target compounds (1e3,
5aeo, 6aec, 7aeo, 9aec and 10aec) for growtheinhibitory activity
against five human cancer cell lines: HL-60 leukemia, Hep 3B
hepatoma, H460 non-small cell lung cancer, A498 renal cancer, and
COLO 205 colon cancer lines. We simultaneously tested Detroit 551
normal human fetal skin fibroblast cells with the target compounds
for comparison. The results are summarized in Table 1. Compound 2
IC50 ¼ 21.8
m
M) < 7a (R4 ¼ CH3, IC50 ¼ 4.6
m
IC50 ¼ 3.7
mM) < 7c (R4 ¼ benzyl, IC50 ¼ 0.24
m
stituents at R4 improved inhibitory activity against the A498 renal
cancer cell line, which improved by as much as 100-fold over
compound 2. Among the five cell lines tested, these compounds
showed selective inhibitory activity against the A498 renal cancer
Table 1
Cytotoxicity of compounds 1e3, 5aeo, 6aec, 7aeo, 9aec and 10aec.
R2
O
R2
O
R2
O
N
N
X
N
R4
R2'
R4'
R2'
R3'
(1-3, 5a-b, 6a-b, 7a-b)
(5c-o, 6c, 7c-o)
(9a-c, 10a-c)
Compd no.
X
R2
R4
R2’
R3’
R4’
IC50 (m
M)a
HL-60
Hep 3B
H460
A498
COLO 205
Detroit 551
1
2
3
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
O
S
COOCH3
CH2OH
COOH
H
H
H
CH3
C2H5
e
e
e
e
e
e
e
>50
>50
>50
>50*
>50
>50*
>50*
>50*
>50*
>50*
>50*
>50*
25.1
>50
>50
>50
16.5
>50*
>50
>50
>50
>50
>50
>50
>50
25
>50
16.8
14.7
38.9
13.3
35.4
54.5
>50
>50
>50
50*
>50
>50
>50
>50*
>50
>50*
>50*
>50*
>50*
>50*
>50*
>50*
50*
>50
58.6
>50
50
>50*
>50
>50
>50
>50
>50
50
>50
>50
>50
50
>50
21.8
>50
>50*
>50
>50*
>50*
>50*
>50*
>50*
>50*
>50*
>50*
>50
>50
>50
>50
>50*
>50
>50
>50
4.6
>50
>50
>50
>50*
>50
>50*
>50*
>50*
>50*
>25
>50*
>50*
32.9
>50
>50
>50
>50*
>50*
>50
>50
>50
>50
>50
30.6
40.9
>50
35.9
26.7
26.4
8.1
>50
>50
>50
>50*
>50
>50*
>50*
>50*
>50*
>50*
>50*
>50*
>50
>50
>50
>50
>50*
>50*
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
44.4
60.5
>50
>50
>50
>50
>50
>50*
>50*
>25*
>50
>50
>50
e
e
e
e
5a
5b
5c
5d
5e
5f
5g
5h
5i
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOH
e
e
e
e
e
>50
>50*
>50*
>50*
>50*
>50*
>50*
>50*
>50*
>50
>50
>50
>50
>50*
>50
>50
>50
50
H
F
H
H
Cl
H
H
OCH3
H
H
H
H
H
e
H
H
F
H
H
Cl
H
H
H
H
H
F
H
H
Cl
H
H
e
e
e
e
e
e
5j
5k
5l
e
e
OCH3
H
eOCH2Oe
COOCH3
H
e
e
OCH3
5m
5n
5o
6a
6b
6c
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
9a
9b
9c
10a
10b
10c
YC-1
e
e
H
e
COOCH3
e
CH3
C2H5
e
COOH
COOH
e
e
e
H
e
H
e
H
e
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
COOCH3
COOCH3
COOCH3
CH2OH
CH2OH
CH2OH
CH3
C2H5
e
e
e
e
50
3.7
H
F
H
H
Cl
H
H
OCH3
H
H
H
H
H
COOCH3
COOCH3
COOCH3
CH2OH
CH2OH
CH2OH
H
H
F
H
H
Cl
H
H
H
H
H
F
H
H
Cl
H
H
>50
>50
50
36.7
30.5
50
31.3
30.8
52.0
42.9
>50
>50
>50
>50*
>50*
>25*
74.1
>50
>50
0.24
0.43
0.59
0.69
5.5
1.1
0.79
4.3
0.33
0.36
0.27
0.37
0.47
>50*
>50*
>50*
0.21
0.69
0.23
0.37
e
50
e
44.0
44.2
43.1
29.8
36.2
24.6
31.9
45.9
>50
50
>50
>50*
>50*
>25*
>50
>50
>50
e
e
e
e
e
28.3
31.9
29.8
>50
>50
>50
>50*
>50*
>50*
31.1
>50
>50
e
OCH3
H
eOCH2Oe
CH2OH
H
e
e
e
e
e
e
e
OCH3
e
e
H
e
CH2OH
e
e
e
e
e
e
e
e
>50*
>25*
>50
>50
>50
25.27
Se
O
S
e
e
e
Se
e
a
Human tumor cells were treated with different concentrations of samples for 48 h. Data are presented as IC50 (mM, the concentration of 50% proliferation-inhibitory effect).

S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
469
cell line, and exhibiting low cytotoxicity against the Detroit 551
normal human fetal skin fibroblast cells (IC50 > 50 M).
In a different approach, replacement of the CH2OH group (R2) of
compounds 7a, 7b, and 7c with a COOCH3 group (5a, 5b and 5c) or
with a COOH group (6a, 6b and 6c) led to reduced inhibitory ac-
tivity. This finding indicates that the CH2OH group (R2) of com-
In summary, we learned from the previous SARs that the
CH2OH-bearing group on the 2-position and the CH2eAr group on
the 4-position of the new core skeleton played important roles in
boosting the anticancer activity of YC-1 analogs. The compounds
7c,10a, and 10c exhibited selective inhibitory activity against the A-
498 renal cancer cell line and showed improved activity approxi-
mately 1.5-fold above that of YC-1. These three compounds warrant
further investigation.
m
pounds 7a, 7b, and 7c is
a functional group essential for
maintaining high inhibitory activity against cancer cells.
Replacement of the benzyl group at the 4-position of com-
pound 7c with benzyl groups mono-substituted with moieties
such as F, Cl, OCH3, eOCH2Oe, or CH2OH yielded compounds 7de
o that also demonstrated selective inhibitory activity toward the
2.3. Growth inhibitory activity of 10a against a panel of human
cancer cell lines
A498 renal cell line (IC50 0.27e5.5
mM). Replacement of the
We evaluated the inhibitory activity of 10a against the NCI-60
human tumor cell lines [14,15], and the results are shown in Fig. 1.
The mean graph midpoint (MID) values for log GI50, log TGI, and log
LC50 were ꢀ4.57, ꢀ4.21, and ꢀ4.04, respectively, indicating its
relatively low cytotoxicity toward NCI-60 human tumor cell lines.
However, we found compound 10a to demonstrate selective inhi-
bition against NCI-H226 lung cancer, UACC-257 melanoma, OVCAR-
5 ovarian cancer, A498 and TK-10 renal cancer and MDA-MB-468
breast cancer cells. In comparison, the log GI50, log TGI, and log
LC50 values of 10a against A498 renal cancer cells were ꢀ6.85, ꢀ6.52,
and ꢀ6.18, respectively, indicating an approximate 100-fold greater
benzyl group at the 4-position of compound 7c with a 5-
(hydroxymethylfuran-2-yl)methyl (10a), 5-(hydroxymethylth-
iophen-2-yl)methyl (10b) or 5-(hydroxymethylselenophen-2-yl)
methyl group (10c) resulted in only minor changes in inhibitory
activity against the A498 cell line, with IC50 values of 0.21
mM
(10a), 0.69 M (10b) and 0.24 M (10c), respectively. Alternatively,
m
m
replacement of the CH2OH group of compounds 7aeo and 10aec
with a COOCH3 group (5aeo and 9aec) significantly attenuated
the inhibitory activity of these compounds against the A498
cancer cell line.
Fig. 1. Differential activity patterns for compound 10a against 60 human cancer cell lines. MG-MID: mean of log X values (X ¼ GI50, TGI, and LC50). Delta: logarithm of the difference
between the MG-MID and the log X of the most sensitive cell line. Range: logarithm of the difference between the log X of the most resistant cell line and the log X of the most
sensitive cell line.

470
S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
inhibitory potency than that reflected by MID values. Because of its
peculiar anticancer selectivity, compound 10a could serve as a
promising lead compound for further investigation.
We then analyzed the differential activity patterns (Fingerprint)
of 10a by using
a pattern recognition computer program
(COMPARE), which contains a database covering fingerprints from
over 175 known anticancer agents with various modes of action.
The results of COMPARE analysis at the GI50 level for compound 10a
in Table 2 showed a poor correlation (r < 0.5) with various known
anticancer agents, suggesting that the mode of action of compound
10a might differ significantly from that of most of the 175 known
anticancer agents in the NCI database. However, we observed good
similarities (r ¼ 0.796) when comparing the anticancer screening
fingerprint of 10a with that of NSC-754549, a YC-1 isostere recently
screened by the NCI.
Fig. 2. Effects of 10a on A498 cell viability. A498 cells were exposed to different
concentrations of 10a for 48 h. Cell viability was assessed using the MTT assay. The data
are presented as the mean ꢁ SEM of three independent experiments. Cells without
treatment served as a control. *P < 0.001 vs. control.
2.4. Mechanism of action of compound 10a
2.4.1. Toxicity in A498 cells induced by 10a
During apoptosis, the mitochondrial membrane potential (Djm)
Exposure of A498 cells to 10a for 48 h, followed by MTT meta-
bolism assays, confirmed the effects of 10a on cell viability. The IC50
decreased. Cells treatment with 0.5 mM of 10a for 12 h, 24 h, 36 h, or
48 h, followed by staining with JC-1, confirmed apoptosis as the
cause of decreased Djm. As shown in Fig. 4, in healthy cells with
high mitochondrial Djm, JC-1 spontaneously forms complexes
known as the JC-1 polymer (C1 and C2) with intense red fluores-
cence (0 h). A significant increase occurs in cells with reduced red
fluorescence (C3 and C4), indicative of a change in Djm, in the
population in which apoptosis is induced (12e48 h). These data
demonstrate that 10a induces cell apoptosis in A498 cells.
value was 0.21
dependent manner. Exposure of A498 cells to 0.1
0.5 M, or 1
m
M, and 10a reduced A498 cell viability in a dose-
m
M, 0.25 M,
m
m
m
M 10a reduced survival to 82.8 ꢁ 2.8%, 39.5 ꢁ 2.9%,
37.5 ꢁ 1.2%, and 19.4 ꢁ 1.8% of the control (0.1% DMSO), respectively
(Fig. 2).
2.4.2. Morphological changes and apoptosis in A498 cells induced
by 10a
Morphological analysis confirmed the cytotoxic effects of 10a. As
shown in Fig. 3A, the apoptotic morphological changes included cell
2.4.3. Effects of 10a on the cell cycle in A498 cells
We treated A498 cells with 0.5 mM of 10a for 0 h, 24 h, 36 h, or
rounding and shrinkage after a 24 h incubation with 0.5
Annexin V-FITC/PI double-labeling was used to detect phos-
phatidylserine (PS) externalization, hallmark of the early
apoptosis phase (Fig. 3B). Cells incubated in the absence of 10a for
12 h, 24 h, 36 h, or 48 h were undamaged and were negative for
both annexin V-FITC and PI staining (Q3). After incubation with
mM of 10a.
48 h, followed by flow cytometric analysis to determine the cell-
cycle distribution of treated cells. We also investigated whether
10a disrupts the cell cycle, so that we could provide further insights
into the apoptotic effects of this compound. As shown in Fig. 5, 10a
a
induced
a time-dependent accumulation of G2/M cells and
apoptotic (sub-G1) cells.
0.5 mM of 10a for 24e48 h, the number of advanced apoptotic cells
To investigate whether G2/M arrest induced by 10a was involved
stained positive by annexin V-FITC and negative with PI (Q4)
increased significantly with the incubation time. The number of
advanced apoptotic cells stained positive by annexin V-FITC and PI
(Q2) also increased significantly with the incubation time.
in activating cyclin B1 and CDK1, we exposed A498 cells to 0.5 mM
of 10a for 6 h, 12 h, 24 h, 36 h, or 48 h. We then determined the
activities of cyclin B1 and CDK1 by using western blot analysis,
which revealed the activation of cyclin B1 and CDK1 within 6 h of
10a treatment (Fig. 6).
Table 2
2.4.4. Apoptosis induced by 10a through the activation of
mitochondrial signaling pathways in A498 cells
Following our observation that 10a caused apoptosis in A498
cells, we determined the levels of selected proteins associated with
apoptosis. To investigate whether apoptosis induced by 10a was
involved in activating caspase cascades, we exposed A498 cells to
COMPARE correlation at the GI50 level for compound 10a.
Compound (NCI number)
ra
Mechanism of action
O6-Methylguanine (NSC 37364)
4-Ipomeanol (NSC 349438)
Bruceantin (NSC 165563)
0.316
0.291
0.197
Alkylating agent [20]
Alkylating agent [21]
Inhibition of protein
synthesis [22]
Inhibition of DNA
replication [23]
Inhibited the synthesis
of RNA [24]
Inhibitor of activation
of AKT [25]
0.5 mM of 10a for 6 h, 12 h, 24 h, 36 h, or 48 h. The activities of
Pibenzimol
hydrochloride (NSC 322921)
Mitramycin (NSC 24559)
0.183
0.16
caspase-3 and caspase-9 were then determined using western blot
analysis, which revealed the activation of caspase-3 and caspase-9
within 24 h of 10a treatment. Exposure to 10a also increased the
levels of Endo G, AIF, Apaf-1, and cytochrome c (Fig. 7). These results
suggest that the mitochondrial signaling pathways of A498 cells
mediate 10a-induced apoptosis.
Triciribine
0.147
phosphate (NSC 280594)
Cyanomorpholino-ADR (NSC 357704)
Aclacinomycin A (NSC 208734)
0.143
0.139
Alkylating agent [26]
Stabilizes topoisomerase I
cleavage [27]
Chloroquinoxaline
sulfonamide (NSC 339004)
Echinomycin (NSC 526417)
0.134
0.133
0.796
Topoisomerase IIa/b
poison [28]
Inhibitor of RNA
synthesis [29]
2.5. In vivo antitumor activity of compound 10a
We evaluated the target compound 10a in an A498 xenograft
nude mouse model. This compound was delivered intraperitoneally
(ip) with doses of 30 mg/kg/d or 60 mg/kg/d. As shown in Fig. 8,
compound 10a significantly suppressed tumor growth in a dose-
1-Benzyl-3-(5-hydroxymethyl-2-furyl)
selenolo[3,2-c]pyrazole (NSC754549)
YC-1 analog [10]
a
r: correlation coefficient.

Fig. 3. Morphological changes and apoptosis induced by 10a in A498 cells. (A) Morphological changes. A498 cells were treated with 0.5
bar ¼ 50 m. (B) Annexin V/PI staining. A498 cells were treated with 10a for different periods of time and apoptosis was assessed using annexin V/PI staining and flow cytometry. The fraction of annexin V-positive A498 cells was 3.9%
prior to treatment and 5.0, 12.7, 21.5 and 41.0% after treatment with A498 cells for 12, 24, 36, or 48 h, respectively.
mM 10a for 12 h, 24 h, 36 h, or 48 h. Cells without treatment served as a control (0 h). Scale
m

472
S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
Fig. 4. Effects of 10a on mitochondrial membrane potential in A498 cells. Cells (1 ꢂ 106 cells/mL) were untreated or treated with 10a (0.5
mM, 12e48 h) to induce apoptosis. Cells
were stained with JC-1 according to the protocol on a BDÔ MitoScreen as described in the section Methods for staining cells with JC-1 and analyzing by flow cytometry.
Fig. 5. Effects of 10a on the cell cycle in A498 cells. A498 cells were incubated with 0.5
mM of 10a for 0 h, 24 h, 36 h, or 48 h. They were then harvested and analyzed using flow
cytometry.
and time-dependent manner. The tumor size was found to decrease
by 80% after dosing at 60 mg/kg/d. During the course of the anti-
tumor evaluation, no significant body weight changes were
detected in either the tested or the control mice.
754549), a YC-1 isostere. We found that 10a exhibited greater cyto-
toxicity against A498 cells compared to YC-1. From the medicinal
chemistry viewpoint, compound 10a presents new possibilities for
optimizing YC-1 analogs and warrants further investigation.
4. Experimental
3. Conclusion
4.1. Material and physical measurements
We designed, synthesized, and evaluated a series of 2,4-
disubstituted furo[3,2-b]indoles in vitro for anticancer activity and
investigated the SARs of the new YC-1 analogs. Compound 10a,
which demonstrated the best anticancer activity among the tested
compounds, was submitted to the NCI for evaluation against the NCI-
60 panel of human tumor cell lines. Although compound 10a con-
tains the furoindole core skeleton, which differs from the selenolo-
pyrazole core skeleton of NSC 754549, the NCI results indicate that
the anticanceractivityand the fingerprint of 10a resemble those of 1-
benzyl-3-(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole (NSC
All solvents and reagents were obtained commercially and used
without further purification. The progress of all reactions was
monitored by TLC on 2 cm ꢂ 6 cm pre-coated silica gel 60 F254
plates, with 0.25 mm thickness (Merck). The chromatograms were
visualized under UV light at 254ꢀ366 nm. The following adsorbent
was used for column chromatography: silica gel 60 (Merck, particle
size 0.040e0.063 mm). Melting points (mp) were determined using
a Yanaco MP-500D melting point apparatus and were uncorrected.
The IR spectra were recorded on Shimadzu IR-Prestige-21 spec-
trophotometers as KBr pellets. The NMR spectra were recorded on
Bruker Avance DPX-200, DPX-400, Bruker Avance III 500 FT-NMR
spectrometers and a Varian Unity Inova-600 spectrometer at
room temperature, and chemical shifts were reported in parts per
million (d). The following abbreviations were used: s, singlet; d,
doublet; t, triplet; q, quartet; dd, double doublet; td, triple doublet;
and m, multiplet. Low- and high-resolution mass spectra were
performed using Finnigan/Thermo Qust MAT95XL at National
Chung Hsing University, Taichung, Taiwan.
4.2. Chemistry
Fig. 6. Treatment with 10a increased G2/M phase checkpoint protein expression. A498
4.2.1. General procedure for synthesizing 4-substituted methyl 4H-
furo[3,2-b]indole-2-carboxylate (5aeo, 9aec)
A mixture of 4H-furo[3,2-b]indole-2-carboxylate (1 equiv) and
NaH (3 equiv, 60%) in anhydrous DMF (5 mL) was stirred for 5 min
cells were treated with 0.5
extraction. Protein samples (20
and subjected to immunoblotting with antibodies specific to cyclin B1, CDK1, and
actin (n ¼ 3 independent experiments). -actin was used as a loading control.
mM 10a for the indicated time periods and lysed for protein
m
g protein/lane) were separated using 10% SDS-PAGE
b-
b

S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
473
(d, J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
d
(ppm): 14.91,
52.27, 106.73, 111.39, 112.52, 117.79, 120.01, 124.26, 131.75, 141.56,
143.40,145.76,159.46; MS (EI, 70 eV) m/z: 243.1 (Mþ); HRMS (EI) m/
z: calc. for C14H13NO3: 243.0895; found: 243.0891.
4.2.1.3. Methyl 4-benzyl-4H-furo[3,2-b]indole-2-carboxylate (5c).
Yield: 36%; white cubic crystals; mp: 168e170 ꢃC; IR (KBr) (cmꢀ1):
n
1718 (C]O); 1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.86 (s, 3H),
5.53 (s, 2H), 7.19 (t, J ¼ 7.6 Hz, 1H), 7.27e7.35 (m, 6H), 7.61 (s, 1H),
7.71 (d, J ¼ 8.4 Hz, 1H), 7.83 (d, J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz,
DMSO-d6)
d (ppm): 48.24, 106.67, 111.87, 112.79, 117.84, 120.36,
124.47, 127.99, 129.10, 132.34, 137.76, 141.90, 145.83, 159.38; MS (EI,
70 eV) m/z: 305.2 (Mþ); HRMS (EI) m/z: calc. for C19H15NO3:
305.1052; found: 305.1050.
4.2.1.4. Methyl 4-(2-fluorobenzyl)-4H-furo[3,2-b]indole-2-carboxylate
(5d). Yield: 38%; pale yellow flocculence crystals; mp: 136e138 ꢃC;
IR (KBr)
n
(cmꢀ1): 1708 (C]O); 1H NMR (500 MHz, DMSO-d6)
d
(ppm): 3.87 (s, 3H), 5.60 (s, 2H), 7.15 (td, J ¼ 7.4, 1.1 Hz, 1H), 7.19e
7.24 (m, 2H), 7.28 (td, J ¼ 7.7, 1.4 Hz, 1H), 7.32e7.38 (m, 2H), 7.54 (s,
1H), 7.70 (d, J ¼ 8.5 Hz, 1H), 7.83 (d, J ¼ 8.0 Hz, 1H); 13C NMR
(125 MHz, DMSO-d6)
d (ppm): 42.61, 52.33, 106.62, 111.76, 112.84,
116.05 (d, J ¼ 21.3 Hz), 117.88, 120.50, 124.44 (d, J ¼ 26.3 Hz), 124.46,
125.18 (d, J ¼ 3.8 Hz), 130.59 (d, J ¼ 8.8 Hz), 130.65, 132.25, 141.86,
143.48, 145.87, 159.36, 160.72 (d, J ¼ 243.8 Hz); MS (EI, 70 eV) m/z:
323.1 (Mþ); HRMS (EI) m/z: calc. for C19H14FNO3: 323.0958; found:
323.0952.
4.2.1.5. Methyl 4-(3-fluorobenzyl)-4H-furo[3,2-b]indole-2-carboxylate
(5e). Yield: 46%; white flocculence crystals; mp: 160e162 ꢃC; IR
(KBr) n d (ppm):
(cmꢀ1): 1708 (C]O); 1H NMR (500 MHz, DMSO-d6)
3.87 (s, 3H), 5.57 (s, 2H), 7.09e7.16 (m, 3H), 7.20 (t, J ¼ 7.0 Hz, 1H),
7.33e7.38 (m, 2H), 7.71 (s, 1H), 7.72 (d, J ¼ 6.0 Hz, 1H), 7.84 (d,
J ¼ 8.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6)
d (ppm): 47.68, 52.32,
106.67, 111.85, 112.93, 114.66, 114.93 (d, J ¼ 21.3 Hz), 117.92, 120.52,
123.94, 124.57, 131.20 (d, J ¼ 7.5 Hz), 132.37, 140.75, 141.89, 143.51,
145.97,159.38,162.65 (d, J ¼ 242.5 Hz); MS (EI, 70 eV) m/z: 323.2(Mþ);
HRMS (EI) m/z: calc. for C19H14FNO3: 323.0958; found: 323.0951.
Fig. 7. Treatment with 10a induced apoptotic pathways in A498 cells. A498 cells were
treated with 0.5 M 10a for the indicated times and lysed for protein extraction.
Protein samples (20 g protein/lane) were separated using 10% SDS-PAGE and sub-
jected to immunoblotting with antibodies specific to p53, AIF, Endo G, Apaf-1, cyto-
m
m
chrome c, caspase 9, caspase 3, and
b-actin (n ¼ 3 independent experiments).
b-actin
was used as a loading control.
4.2.1.6. Methyl 4-(4-fluorobenzyl)-4H-furo[3,2-b]indole-2-carboxylate
at room temperature. A substituted-benzyl halide or hetero-
arylmethyl halide (4 equiv) was then added dropwise. The mixture
was stirred at room temperature for 20 min. To this mixture was
added 50 mL of ice cold water, and the solution was extracted with
EtOAc. The organic phase was separately dried over anhydrous
MgSO4, filtered, and evaporated under vacuum. The residue was
purified by column chromatography on silica gel eluted with
EtOAc:n-hexane (1:3, v/v) and then recrystallized from n-hexane/
EtOAc to obtain the pure compound (5aeo, 9aec).
(5f). Yield: 56%; white cubic crystals; mp: 175e177 ꢃC; IR (KBr)
n
(cmꢀ1): 1718 (C]O); 1H NMR (500 MHz, DMSO-d6)
d (ppm): 3.87 (s,
3H), 5.53 (s, 2H), 7.12e7.17 (m, 2H), 7.20 (td, J ¼ 7.2, 1.0 Hz, 1H), 7.32e
7.38 (m, 3H), 7.66 (s, 1H), 7.72 (d, J ¼ 8.5 Hz, 1H), 7.82 (d, J ¼ 7.7 Hz,
1H); 13C NMR (125 MHz, DMSO-d6)
d (ppm): 47.48, 52.32, 106.68,
111.87, 112.88, 115.93 (d, J ¼ 21.3 Hz), 117.89,120.43, 124.52, 130.14 (d,
J ¼ 8.8 Hz), 132.26, 134.08, 141.84, 143.51, 145.91, 159.39, 162.05 (d,
J ¼ 242.5 Hz); MS (EI, 70 eV) m/z: 323.1 (Mþ); HRMS (EI) m/z: calc. for
C19H14FNO3: 323.0958; found: 323.0963.
4.2.1.1. Methyl 4-methyl-4H-furo[3,2-b]indole-2-carboxylate (5a).
4.2.1.7. Methyl 4-(2-chlorobenzyl)-4H-furo[3,2-b]indole-2-carboxylate
Yield: 51%; white needle crystals; mp: 135e136 ꢃC; IR (KBr)
n
(5g). Yield: 42%; yellow needle crystals; mp: 157e159 ꢃC; IR (KBr)
(cmꢀ1): 1716 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 3.85
n d (ppm): 3.85
(cmꢀ1): 1708 (C]O); 1H NMR (400 MHz, DMSO-d6)
(s, 3H), 3.88 (s, 3H), 7.19 (t, J ¼ 7.6 Hz,1H), 7.35 (t, J ¼ 7.6 Hz,1H), 7.58
(s, 3H), 5.62 (s, 2H), 6.99 (d, J ¼ 7.2 Hz, 1H), 7.21 (d, J ¼ 7.4 Hz, 1H), 7.28
(d, J ¼ 7.4 Hz, 1H), 7.31e7.36 (m, 3H), 7.53 (d, J ¼ 8.0 Hz, 1H), 7.60 (d,
J ¼ 8.4 Hz, 1H), 7.86 (d, J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
(d, J ¼ 8.4 Hz, 1H), 7.75 (s, 1H), 7.81 (d, J ¼ 7.6 Hz, 1H); 13C NMR
(50 MHz, DMSO-d6)
d (ppm): 31.45, 52.26, 106.39, 111.31, 112.46,
117.67, 120.03, 124.23, 133.07, 142.49, 143.01, 145.75, 159.44; MS (EI,
70 eV) m/z: 229.1 (Mþ); HRMS (EI) m/z: calc. for C13H11NO3:
229.0739; found: 229.0743.
d (ppm): 46.44, 52.34, 106.53, 111.75, 112.79, 117.90, 120.59, 124.64,
128.05, 129.88, 130.15, 132.23, 134.65, 142.02, 143.50, 145.79, 159.34;
MS (EI, 70 eV) m/z: 339.1 (Mþ); HRMS (EI) m/z: calc. for C19H14ClNO3:
339.0662; found: 339.0658.
4.2.1.2. Methyl 4-ethyl-4H-furo[3,2-b]indole-2-carboxylate (5b).
Yield: 28%; white cubic crystals; mp: 122e123 ꢃC; IR (KBr)
n
(cmꢀ1):
4.2.1.8. Methyl 4-(3-chlorobenzyl)-4H-furo[3,2-b]indole-2-carboxylate
1714 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 1.38 (t,
(5h). Yield: 25%; yellow crystals; mp: 135e137 ꢃC; IR (KBr)
n
J ¼ 7.2 Hz, 3H), 3.88 (s, 3H), 4.33 (q, J ¼ 7.2 Hz, 2H), 7.18 (t, J ¼ 7.6 Hz,
(cmꢀ1): 1710 (C]O); 1H NMR (400 MHz, DMSO-d6)
3H), 5.55 (s, 2H), 7.18e7.38 (m, 6H), 7.70 (d, J ¼ 6.0 Hz, 1H), 7.71 (s,
d (ppm): 3.87 (s,
1H), 7.34 (t, J ¼ 8.0 Hz, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.78 (s, 1H), 7.81

474
S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
Fig. 8. Effect of 10a on tumor cell growth in an in vivo model. (A) Mean tumor weightetime profiles. (B) Mean tumor volumeetime profiles, and (C) Mean body weightetime
profiles in A498 xenograft nude mice (n ¼ 5) following ip dosing of 10a at 30 mg/kg or 60 mg/kg QD for 4 consecutive weeks.
1H), 7.83 (d, J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
d
(ppm):
4.2.1.11. Methyl 4-(3-methoxybenzyl)-4H-furo[3,2-b]indole-2-
47.49, 52.36, 106.50, 111.70, 112.81, 117.86, 120.62, 124.69, 126.42,
127.50, 128.08, 131.05, 132.19, 133.67, 140.24, 141.80, 143.49, 145.90,
159.46; MS (EI, 70 eV) m/z: 339.1 (Mþ); HRMS (EI) m/z: calc. for
carboxylate (5k). Yield: 58%; brown crystals; mp: 71e73 ꢃC; IR
(KBr)
n
(cmꢀ1): 1716 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 3.74 (s, 3H), 3.85 (s, 3H), 5.48 (s, 2H), 6.79e7.34 (m, 6H),
C19H14ClNO3: 339.0662; found: 339.0670.
7.62 (s, 1H), 7.69 (d, J ¼ 8.4 Hz, 1H), 7.82 (d, J ¼ 8.0 Hz, 1H); 13C
NMR (50 MHz, DMSO-d6)
d (ppm): 48.12, 52.31, 55.43, 106.65,
4.2.1.9. Methyl 4-(4-chlorobenzyl)-4H-furo[3,2-b]indole-2-carboxylate
111.87, 112.77, 113.03, 113.98, 114.12, 114.24, 117.83, 119.99, 120.39,
124.49, 130.26, 132.37, 139.33, 141.92, 145.84, 159.79; MS (EI,
70 eV) m/z: 335.2 (Mþ); HRMS (EI) m/z: calc. for C20H17NO4:
335.1158; found: 335.1160.
(5i). Yield: 64%; pale yellow cubic crystals; mp: 152e154 ꢃC; IR (KBr)
n
(cmꢀ1): 1718 (C]O); 1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.87
(s, 3H), 5.54 (s, 2H), 7.19 (t, J ¼ 7.2 Hz,1H), 7.30 (d, J ¼ 8.4 Hz, 2H), 7.33
(t, J ¼ 8.0 Hz, 1H), 7.38 (d, J ¼ 8.4 Hz, 2H), 7.69 (s, 1H), 7.70 (d,
J ¼ 9.2 Hz, 1H), 7.83 (d, J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
4.2.1.12. Methyl 4-(4-methoxybenzyl)-4H-furo[3,2-b]indole-2-
d
(ppm): 47.45, 52.32, 106.62, 111.80, 112.86, 117.88, 120.48, 124.54,
carboxylate (5l). Yield: 43%; white needle crystals; mp: 88e90 ꢃC;
129.08, 129.76, 132.26, 132.70, 136.82, 141.81, 143.48, 145.89, 159.37;
MS (EI, 70 eV) m/z: 339.1 (Mþ); HRMS (EI) m/z: calc. for C19H14ClNO3:
339.0662; found: 339.0672.
IR (KBr)
(ppm): 3.69 (s, 3H), 3.85 (s, 3H), 5.43 (s, 2H), 6.87 (d, J ¼ 8.4 Hz,
n
(cmꢀ1): 1714 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
2H), 7.18 (t, J ¼ 7.6 Hz, 1H), 7.26 (d, J ¼ 8.4 Hz, 2H), 7.33 (t, J ¼ 7.6 Hz,
1H), 7.57 (s, 1H), 7.72 (d, J ¼ 8.4 Hz, 1H), 7.80 (d, J ¼ 8.0 Hz, 1H); 13
C
4.2.1.10. Methyl 4-(2-methoxybenzyl)-4H-furo[3,2-b]indole-2-
NMR (50 MHz, DMSO-d6) d (ppm): 47.71, 52.31, 55.46, 106.66,
carboxylate (5j). Yield: 33%; white crystals; mp: 124e126 ꢃC; IR
111.85, 112.72, 114.41, 117.79, 120.30, 124.44, 129.54, 130.83, 132.15,
141.81, 143.45, 145.77, 159.18, 159.41; MS (EI, 70 eV) m/z: 335.2
(Mþ); HRMS (EI) m/z: calc. for C20H17NO4: 335.1158; found:
335.1162.
(KBr) n d (ppm):
(cmꢀ1): 1714 (C]O); 1H NMR (400 MHz, DMSO-d6)
3.78 (s, 3H), 3.86 (s, 3H), 5.45 (s, 2H), 6.87 (t, J ¼ 7.2 Hz, 1H), 7.03 (d,
J ¼ 8.4 Hz, 1H), 7.07 (d, J ¼ 6.4 Hz, 1H), 7.17 (t, J ¼ 7.2 Hz, 1H), 7.27e
7.33 (m, 2H), 7.42 (s, 1H), 7.64 (d, J ¼ 8.4 Hz, 1H), 7.81 (d, J ¼ 8.0 Hz,
1H); 13C NMR (50 MHz, DMSO-d6)
d
(ppm): 43.87, 52.28, 55.81,
4.2.1.13. Methyl 4-(3,4-(methylenedioxy)benzyl)-4H-furo[3,2-b]indole-
106.72, 111.46, 111.87, 112.60, 117.72, 120.18, 120.80, 124.32, 125.04,
129.53, 129.81, 132.48, 141.94, 143.26, 145.67, 157.49, 159.40; MS (EI,
70 eV) m/z: 335.2 (Mþ); HRMS (EI) m/z: calc. for C20H17NO4:
335.1158; found: 335.1165.
2-carboxylate (5m). Yield: 42%; white flocculence crystals; mp: 96e
98 ꢃC; IR (KBr) (cmꢀ1): 1706 (C]O); 1H NMR (400 MHz, DMSO-d6)
n
d
(ppm): 3.87 (s, 3H), 5.42 (s, 2H), 5.97 (s, 2H), 6.85 (s, 2H), 6.91 (s,
1H), 7.19 (t, J ¼ 7.6 Hz,1H), 7.34 (t, J ¼ 8.0 Hz, 1H), 7.68 (s, 1H), 7.75 (d,

S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
475
J ¼ 8.4 Hz, 1H), 7.82 (d, J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
4.2.2. General procedure for synthesizing 4-substituted 4H-furo
[3,2-b]indole-2-carboxylic acid (3, 6aec)
d
(ppm): 48.05, 52.30, 101.49, 106.76, 108.68, 108.76, 111.95, 112.82,
117.84, 120.33, 121.67, 124.44, 131.52, 132.21, 141.80, 143.47, 145.85,
147.20,147.84,159.39; MS (EI, 70 eV) m/z: 349.1 (Mþ); HRMS (EI) m/z:
calc. for C20H15NO5: 349.0950; found: 349.0946.
Compound (1, 5aec) (1 equiv) was dissolved in 20 mL of a 10%
sodium hydroxide solution. The mixture was heated under reflux
for 2 h and then cooled and acidified with dilute HCl. The precipi-
tate was collected and recrystallized from n-hexane/EtOAc to yield
the pure compound (3, 6aec).
4.2.1.14. Methyl 4-(3-(methoxycarbonyl)benzyl)-4H-furo[3,2-b]indole-
2-carboxylate (5n). Yield: 36%; pale yellow flocculence crystals; mp:
132e133 ꢃC; IR (KBr)
(400 MHz, DMSO-d6)
n
(cmꢀ1): 1695 (C]O), 1726 (C]O); 1H NMR
(ppm): 3.81 (s, 3H), 3.86 (s, 3H), 5.64 (s, 2H),
4.2.2.1. 4H-Furo[3,2-b]indole-2-carboxylic acid (3). Yield: 66%; pale
d
yellow crystals; mp: 242e243 ꢃC; IR (KBr) (cmꢀ1): 1672 (C]O),
n
7.20 (t, J ¼ 7.2 Hz, 1H), 7.33 (t, J ¼ 7.6 Hz, 1H), 7.47 (t, J ¼ 7.6 Hz, 1H),
7.52 (d, J ¼ 7.6 Hz, 1H), 7.66 (s, 1H), 7.69 (d, J ¼ 8.4 Hz, 1H), 7.84 (d,
J ¼ 7.6 Hz, 1H), 7.86 (d, J ¼ 7.2 Hz, 1H), 7.90 (s, 1H); 13C NMR
3427 (OH); 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 7.15 (t, J ¼ 7.6 Hz,
1H), 7.27 (t, J ¼ 8.0 Hz, 1H), 7.49 (s, 1H), 7.50 (d, J ¼ 10.4 Hz, 1H), 7.78
(d, J ¼ 8.0 Hz, 1H), 11.12 (s, 1H); 13C NMR (50 MHz, DMSO-d6)
(50 MHz, DMSO-d6)
d
(ppm): 47.74, 52.32, 52.65, 106.56, 111.76,
d (ppm): 106.44, 112.67, 113.39,117.32, 119.95, 123.94, 130.51, 141.99,
112.87, 117.90, 120.53, 124.60, 128.34, 128.86, 129.67, 130.43, 132.33,
132.64, 138.66, 141.89, 143.49, 145.92, 159.37, 166.42; MS (EI, 70 eV)
m/z: 363.2 (Mþ); HRMS (EI) m/z: calc. for C21H17NO5: 363.1107;
found: 363.1099.
144.03, 147.12, 160.51; MS (EI, 70 eV) m/z: 201.1 (Mþ); HRMS (EI) m/
z: calc. for C11H7NO3: 201.0426; found: 201.0422.
4.2.2.2. 4-Methyl-4H-furo[3,2-b]indole-2-carboxylic acid (6a). Yield:
36%; white crystals; mp: 237e239 ꢃC; IR (KBr) (cmꢀ1): 1664 (C]O);
n
4.2.1.15. Methyl 4-(4-(methoxycarbonyl)benzyl)-4H-furo[3,2-b]indole-
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 3.86 (s, 3H), 7.18 (t, J ¼ 7.2 Hz,
2-carboxylate (5o). Yield: 47%; white crystals; mp: 142e144 ꢃC; IR
1H), 7.33 (t, J ¼ 7.6 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.64 (s, 1H), 7.79 (d,
(KBr)
n
(cmꢀ1): 1701 (C]O), 1716 (C]O); 1H NMR (400 MHz,
J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
d (ppm): 31.47, 105.82,
DMSO-d6)
d
(ppm): 3.82 (s, 3H), 3.86 (s, 3H), 5.65 (s, 2H), 7.20 (t,
111.26, 112.63, 117.50, 119.93, 123.91, 133.20, 142.23, 142.59, 147.13,
160.43; MS (EI, 70 eV) m/z: 215.1 (Mþ); HRMS (EI) m/z: calc. for
C12H9NO3: 215.0582; found: 215.0585.
J ¼ 7.6 Hz, 1H), 7.32 (t, J ¼ 8.0 Hz, 1H), 7.38 (d, J ¼ 8.0 Hz, 2H), 7.66
(d, J ¼ 10.0 Hz, 1H), 7.67 (s, 1H), 7.84 (d, J ¼ 8.0 Hz, 1H), 7.90 (d,
J ¼ 8.4 Hz, 2H); 13C NMR (50 MHz, DMSO-d6)
d (ppm): 47.86, 52.32,
52.57, 106.64, 111.79, 112.89, 117.92, 120.53, 124.58, 128.01, 129.33,
130.02, 132.40, 141.89, 143.29, 143.48, 145.91, 159.37, 166.35; MS (EI,
70 eV) m/z: 363.1 (Mþ); HRMS (EI) m/z: calc. for C21H17NO5:
363.1107; found: 363.1115.
4.2.2.3. 4-Ethyl-4H-furo[3,2-b]indole-2-carboxylic acid (6b). Yield:
71%; brown flocculence crystals; mp: 203e205 ꢃC; IR (KBr)
n
(cmꢀ1):
d (ppm): 1.37
1670 (C]O), 3448 (OH); 1H NMR (400 MHz, DMSO-d6)
(t, J ¼ 7.2 Hz, 3H), 4.31 (q, J ¼ 7.2 Hz, 2H), 7.16 (t, J ¼ 7.6 Hz, 1H), 7.31 (t,
J ¼ 7.2 Hz, 1H), 7.61 (d, J ¼ 8.4 Hz, 1H), 7.66 (s, 1H), 7.78 (d, J ¼ 8.0 Hz,
4.2.1.16. Methyl 4-((5-(methoxycarbonyl)furan-2-yl)methyl)-4H-furo
1H); 13C NMR (50 MHz, DMSO-d6)
d (ppm): 14.94, 106.18, 111.33,
[3,2-b]indole-2-carboxylate (9a). Yield: 37%; pale yellow cubic
112.68, 117.64, 119.92, 123.97, 131.86, 141.31, 143.00, 147.03, 160.43; MS
(EI, 70 eV) m/z: 229.1 (Mþ); HRMS (EI) m/z: calc. for C13H11NO3:
229.0739; found: 229.0733.
crystals; mp: 141e143 ꢃC; IR (KBr)
n
(cmꢀ1): 1707 (C]O), 1728
(C]O), 2954 (CH); 1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.75 (s,
3H), 3.88 (s, 3H), 5.66 (s, 2H), 6.68 (d, J ¼ 3.6 Hz, 1H), 7.22 (t,
J ¼ 7.6 Hz, 1H), 7.24 (d, J ¼ 3.6 Hz, 1H), 7.37 (t, J ¼ 7.6 Hz, 1H), 7.66
(s, 1H), 7.77 (d, J ¼ 8.4 Hz, 1H), 7.83 (d, J ¼ 8.0 Hz, 1H); 13C NMR
4.2.2.4. 4-Benzyl-4H-furo[3,2-b]indole-2-carboxylic acid (6c). Yield: 46%;
brown cubic crystals; mp: 141e143 ꢃC; IR (KBr) (cmꢀ1): 3439 (OH); 1H
n
(50 MHz, DMSO-d6)
d
(ppm): 52.24, 52.34, 106.62, 111.55, 111.76,
NMR (400 MHz, DMSO-d6)
7.25e7.34 (m, 6H), 7.48 (s, 1H), 7.69 (d, J ¼ 8.4 Hz, 1H), 7.80 (d, J ¼ 8.0 Hz,
1H); 13C NMR (50 MHz, DMSO-d6)
(ppm): 48.24, 106.11, 111.79, 112.96,
d
(ppm): 5.52 (s, 2H), 7.18 (t, J ¼ 7.6 Hz, 1H),
113.00, 117.89, 119.66, 120.70, 124.62, 132.13, 141.87, 143.59,
144.00, 145.94, 155.32, 158.52, 159.34; MS (EI, 70 eV) m/z: 353.1
(Mþ); HRMS (EI) m/z: calc. for C19H15NO6: 353.0899; found:
353.0890.
d
117.66, 120.24, 124.12, 127.99, 129.09, 132.47, 137.84, 141.66, 143.01, 147.17,
160.33; MS (EI, 70 eV) m/z: 291.10 (Mþ); HRMS (EI) m/z: calc. for
C18H13NO3: 291.0895; found: 291.0893.
4.2.1.17. Methyl 4-((5-(methoxycarbonyl)thiophen-2-yl)methyl)-4H-
furo[3,2-b]indole -2-carboxylate (9b). Yield: 35%; pale yellow crys-
4.2.3. General procedure for synthesizing 4-substituted 4H-furo
[3,2-b]indole-2-methanol (7aeo, 10aec)
tals; mp: 140e142 ꢃC; IR (KBr)
n
(cmꢀ1): 1726 (C]O); 1H NMR
(400 MHz, DMSO-d6)
d
(ppm): 3.74 (s, 3H), 3.88 (s, 3H), 5.82 (s, 2H),
Compound (5aeo, 9aec) (1 equiv) was dissolved in a homoge-
nous solution of Ca(BH4)2 (10 equiv) in THF (50 mL). The mixture
was heated under reflux for 10 h and then filtered. The solvent was
evaporated, and the residues were extracted with CH2Cl2. The
organic phase was separated, dried over anhydrous MgSO4, filtered,
and evaporated under vacuum. The residues were purified by col-
umn chromatography on silica gel eluted with EtOAc:n-hexane
(1:1, v/v) and then recrystallized from n-hexane/EtOAc to obtain
the pure compound (7aeo, 10aec).
7.22 (t, J ¼ 7.6 Hz, 1H), 7.26 (d, J ¼ 3.6 Hz, 1H), 7.37 (t, J ¼ 8.0 Hz, 1H),
7.65 (d, J ¼ 3.6 Hz, 1H), 7.78 (s, 1H), 7.79 (d, J ¼ 8.8 Hz, 1H), 7.83 (d,
J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
d (ppm): 43.16, 52.36,
52.64, 106.74, 111.90, 113.24, 117.99, 120.82, 124.67, 128.25, 132.00,
132.44, 134.08, 141.72, 143.79, 146.02, 147.97, 159.34, 161.97; MS (EI,
70 eV) m/z: 369.1 (Mþ); HRMS (EI) m/z: calc. for C19H15NO5S:
369.0671; found: 369.0677.
4.2.1.18. Methyl 4-((5-(methoxycarbonyl)selenophen-2-yl)methyl)-
4H-furo[3,2-b]indole-2-carboxylate (9c). Yield: 34%; pale yellow
4.2.3.1. 4H-Furo[3,2-b]indole-2-methanol (2). Yield: 22%; brown
crystals; mp: 129e131 ꢃC; IR (KBr)
n
(cmꢀ1): 1701 (C]O); 1H NMR
needle crystals; mp: 105e107 ꢃC; IR (KBr) (cmꢀ1): 3394 (OH); 1H
n
(400 MHz, DMSO-d6)
d
(ppm): 3.72 (s, 3H), 3.87 (s, 3H), 5.83 (s, 2H),
(ppm): 45.44,
NMR (400 MHz, DMSO-d6)
d
(ppm): 4.53 (d, J ¼ 3.2 Hz, 2H), 5.33 (t,
7.21e7.86 (m, 7H); 13C NMR (50 MHz, DMSO-d6)
d
J ¼ 3.2 Hz, 1H), 6.63 (s, 1H), 7.05e7.10 (m, 2H), 7.42 (d, J ¼ 7.6 Hz,
52.36, 52.75, 106.73, 111.88, 113.28, 117.98, 120.87, 124.70, 130.21,
131.92, 136.34, 137.73, 141.73, 143.89, 145.99, 155.34, 159.36, 163.29;
MS (EI, 70 eV) m/z: 417.1 (Mþ); HRMS (EI) m/z: calc. for
1H), 7.59 (d, J ¼ 7.6 Hz, 1H), 10.79 (s, 1H); 13C NMR (50 MHz, DMSO-
d6)
d (ppm): 57.25, 97.71, 112.92, 113.68, 115.51, 119.28, 121.16,
131.22, 139.54, 140.51, 159.58; MS (EI, 70 eV) m/z: 187.1 (Mþ); HRMS
(EI) m/z: calc. for C11H9NO2: 187.0633; found: 187.0627.
C
19H15NO5Se: 417.0115; found: 411.0110.

476
S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
4.2.3.2. 4-Methyl-4H-furo[3,2-b]indole-2-methanol (7a). Yield: 54%;
brown oil; IR (KBr)
7.18 (m, 2H), 7.25 (t, J ¼ 7.2 Hz, 1H), 7.34 (td, J ¼ 7.6, 1.2 Hz, 1H), 7.53
(dd, J ¼ 5.2, 2.4 Hz, 2H), 7.66 (d, J ¼ 7.2 Hz, 1H); 13C NMR (50 MHz,
n
(cmꢀ1): 2924 (CH), 3448 (OH); 1H NMR
(400 MHz, DMSO-d6)
d
(ppm): 3.80 (s, 3H), 4.55 (d, J ¼ 4 Hz, 2H), 5.38
DMSO-d6) d (ppm): 46.26, 57.19, 97.29, 111.19, 113.91, 115.93, 119.80,
(s, 1H), 6.75 (s, 1H), 7.09 (t, J ¼ 7.2 Hz, 1H), 7.17 (t, J ¼ 7.2 Hz, 1H), 7.49
121.55, 127.95, 129.54, 129.92, 130.01, 132.72, 133.15, 135.19, 139.46,
139.71, 159.78; MS (EI, 70 eV) m/z: 311.1 (Mþ); HRMS (EI) m/z: calc.
for C18H14ClNO2: 311.0713; found: 311.0710.
(d, J ¼ 8.0 Hz, 1H), 7.61 (d, J ¼ 8.0 Hz, 1H); 13C NMR (50 MHz, DMSO-
d6) d (ppm): 31.44, 57.28, 96.90, 110.79, 113.58, 115.70, 119.29, 121.14,
134.01, 139.17, 139.85, 159.75; MS (EI, 70 eV) m/z: 201.0 (Mþ); HRMS
(EI) m/z: calc. for C12H11NO: 201.0790; found: 201.0787.
4.2.3.9. 4-(3-Chlorobenzyl)-4H-furo[3,2-b]indole-2-methanol (7h).
Yield: 58%; brown oil; IR (KBr)
NMR (400 MHz, DMSO-d6)
n
(cmꢀ1): 2924 (CH), 3446 (OH); 1H
(ppm): 4.53 (d, J ¼ 5.6 Hz, 2H), 5.38 (t,
4.2.3.3. 4-Ethyl-4H-furo[3,2-b]indole-2-methanol (7b). Yield: 63%;
d
brown oil; IR (KBr)
n
(cmꢀ1): 2924 (CH), 3417 (OH); 1H NMR
J ¼ 5.6 Hz, 1H), 5.50 (s, 2H), 6.70 (s, 1H), 7.09e7.34 (m, 6H), 7.62 (d,
(400 MHz, DMSO-d6)
d
(ppm): 1.36 (t, J ¼ 7.2 Hz, 3H), 4.27 (q,
J ¼ 8.8 Hz, 1H), 7.64 (d, J ¼ 8.8 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
J ¼ 7.2 Hz, 2H), 4.55 (d, J ¼ 5.6 Hz, 2H), 5.37 (t, J ¼ 5.6 Hz,1H), 6.78 (s,
1H), 7.08 (t, J ¼ 7.6 Hz, 1H), 7.16 (t, J ¼ 7.2 Hz, 1H), 7.53 (d, J ¼ 8.0 Hz,
d (ppm): 47.49, 57.24, 97.24, 111.26, 113.95, 115.93, 119.77, 121.52,
126.31, 127.45, 127.88, 130.98, 133.20, 133.61, 139.28, 139.66, 140.91,
1H), 7.61 (d, J ¼ 7.6 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
d
(ppm):
159.92; MS (EI, 70 eV) m/z: 311.1 (Mþ); HRMS (EI) m/z: calc. for
15.03, 57.25, 97.31, 110.82, 113.62, 115.80, 119.22, 121.15, 132.61,
138.87, 139.55,159.65; MS (EI, 70 eV) m/z: 215.0 (Mþ); HRMS (EI) m/
z: calc. for C13H13NO2: 215.0946; found: 215.0949.
C18H14ClNO2: 311.0713; found: 311.0718.
4.2.3.10. 4-(4-Chlorobenzyl)-4H-furo[3,2-b]indole-2-methanol (7i).
Yield: 64%; brown oil; IR (KBr)
(cmꢀ1): 2924 (CH), 3417 (OH); 1H
NMR (400 MHz, DMSO-d6)
n
4.2.3.4. 4-Benzyl-4H-furo[3,2-b]indole-2-methanol (7c). Yield: 62%;
d
(ppm): 4.52 (d, J ¼ 5.6 Hz, 2H), 5.36 (t,
brown oil; IR (KBr)
n
(cmꢀ1): 2924 (CH), 3454 (OH); 1H NMR
J ¼ 5.6 Hz,1H), 5.48 (s, 2H), 6.66 (s,1H), 7.10 (t, J ¼ 7.2 Hz,1H), 7.15 (t,
J ¼ 8.0 Hz, 1H), 7.24 (d, J ¼ 8.0 Hz, 2H), 7.37 (d, J ¼ 8.4 Hz, 2H), 7.60
(d, J ¼ 8.0 Hz, 1H), 7.63 (d, J ¼ 7.6 Hz, 1H); 13C NMR (50 MHz, DMSO-
(400 MHz, DMSO-d6)
d
(ppm): 4.52 (d, J ¼ 5.2 Hz, 2H), 5.36 (t,
J ¼ 5.2 Hz,1H), 5.47 (s, 2H), 6.63 (s, 1H), 7.09 (t, J ¼ 7.2 Hz, 1H), 7.15 (t,
J ¼ 7.2 Hz, 1H), 7.23e7.32 (m, 5H), 7.61 (d, J ¼ 7.6 Hz, 1H), 7.63 (d,
d6) d (ppm): 47.42, 57.23, 97.27, 111.27, 113.94, 115.90, 119.71, 121.46,
J ¼ 6.8 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
d
(ppm): 48.20, 57.24,
129.02, 129.56, 132.50, 133.18, 137.35, 139.27, 139.66, 159.86; MS (EI,
70 eV) m/z: 311.1 (Mþ); HRMS (EI) m/z: calc. for C18H14ClNO2:
311.0713; found: 311.0707.
97.32, 111.29, 113.88, 115.85, 119.58, 121.37, 127.76, 127.89, 129.01,
133.28, 138.28,139.33,139.61,159.78; MS (EI, 70 eV) m/z: 277.2 (Mþ);
HRMS (EI) m/z: calc. for C18H15NO2: 277.1103; found: 277.1105.
4.2.3.11. 4-(2-Methoxybenzyl)-4H-furo[3,2-b]indole-2-methanol
4.2.3.5. 4-(2-Fluorobenzyl)-4H-furo[3,2-b]indole-2-methanol (7d).
(7j). Yield: 60%; brown oil; IR (KBr)
n
(cmꢀ1): 2924, 2954 (CH),
Yield: 52%; brown oil; IR (KBr)
NMR (500 MHz, DMSO-d6)
n
(cmꢀ1): 2924 (CH), 3448 (OH); 1H
3427 (OH); 1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.83 (s, 3H), 4.51
d
(ppm): 4.52 (d, J ¼ 5 Hz, 2H), 5.37 (t,
(d, J ¼ 5.6 Hz, 2H), 5.34 (t, J ¼ 5.6 Hz, 1H), 5.39 (s, 2H), 6.51 (s, 1H),
6.84 (t, J ¼ 7.6 Hz,1H), 6.91 (d, J ¼ 6.4 Hz,1H), 7.04 (d, J ¼ 8.4 Hz,1H),
7.09 (t, J ¼ 7.2 Hz, 1H), 7.14 (t, J ¼ 7.2 Hz, 1H), 7.27 (td, J ¼ 1.6 and
8.0 Hz, 1H), 7.55 (d, J ¼ 8.4 Hz, 1H), 7.62 (d, J ¼ 7.2 Hz, 1H); 13C NMR
J ¼ 5.5 Hz, 1H), 5.53 (s, 2H), 6.58 (s, 1H), 7.10e7.25 (m, 6H), 7.62 (d,
J ¼ 7.0 Hz, 1H), 7.64 (d, J ¼ 6.5 Hz, 1H); 13C NMR (125 MHz, DMSO-
d6)
d (ppm): 42.41, 57.23, 97.32, 111.21, 113.90, 115.89, 115.97 (d,
J ¼ 20 Hz), 119.74, 121.49, 124.84 (d, J ¼ 15 Hz), 125.09 (d, J ¼ 2.5 Hz),
130.37 (d, J ¼ 10 Hz), 130.44, 133.12, 139.30, 139.66, 159.79, 160.64
(d, J ¼ 243.8 Hz); MS (EI, 70 eV) m/z: 295.2 (Mþ); HRMS (EI) m/z:
calc. for C18H14FNO2: 295.1009; found: 295.1000.
(50 MHz, DMSO-d6) d (ppm): 43.58, 55.83, 57.21, 97.47, 111.26,
111.36, 113.71, 115.76, 119.43, 120.73, 121.26, 125.58, 129.02, 129.50,
133.42, 139.36, 157.33, 159.59; MS (EI, 70 eV) m/z: 307.2 (Mþ);
HRMS (EI) m/z: calc. for C19H17NO3: 307.1208; found: 307.1200.
4.2.3.6. 4-(3-Fluorobenzyl)-4H-furo[3,2-b]indole-2-methanol (7e).
4.2.3.12. 4-(3-Methoxybenzyl)-4H-furo[3,2-b]indole-2-methanol
Yield: 55%; brown oil; IR (KBr)
NMR (500 MHz, DMSO-d6)
n
(cmꢀ1): 2924 (CH), 3415 (OH); 1H
(7k). Yield: 79%; yellow oil; IR (KBr)
n
(cmꢀ1): 2924, 2954 (CH),
d
(ppm): 4.54 (d, J ¼ 3.5 Hz, 2H), 5.40 (t,
3448 (OH); 1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.69 (s, 3H), 4.52
J ¼ 3.0 Hz, 1H), 5.52 (s, 2H), 6.71 (s, 1H), 7.04e7.38 (m, 6H), 7.63 (d,
(d, J ¼ 5.2 Hz, 2H), 5.35 (t, J ¼ 5.6 Hz, 1H), 5.43 (s, 2H), 6.65 (s, 1H),
6.76 (d, J ¼ 7.6 Hz, 1H), 6.82e6.83 (m, 2H), 7.09 (t, J ¼ 7.6 Hz, 1H),
7.15 (t, J ¼ 7.2 Hz, 1H), 7.21 (t, J ¼ 8.4 Hz, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H),
J ¼ 9.5 Hz, 1H), 7.65 (d, J ¼ 10.5 Hz, 1H); 13C NMR (125 MHz, DMSO-
d6)
d
(ppm): 47.63, 57.28, 97.29,111.29,113.99,114.48 (d, J ¼ 21.25 Hz),
114.74 (d, J ¼ 21.25 Hz), 115.94, 119.78, 121.53, 123.73 (d, J ¼ 2.5 Hz),
131.11 (d, J ¼ 7.5 Hz), 133.26, 139.35, 139.71, 141.27 (d, J ¼ 7.5 Hz),
159.94, 162.65 (d, J ¼ 242.5 Hz); MS (EI, 70 eV) m/z: 295.2 (Mþ);
HRMS (EI) m/z: calc. for C18H14FNO2: 295.1009; found: 295.1011.
7.62 (d, J ¼ 8.0 Hz,1H); 13C NMR (50 MHz, DMSO-d6)
d (ppm): 48.10,
55.43, 57.24, 97.33, 111.31, 112.15, 112.81, 113.86, 115.85, 119.60,
119.82, 121.38, 130.15, 133.32, 139.36, 139.59, 139.86, 159.79; MS (EI,
70 eV) m/z: 307.0 (Mþ); HRMS (EI) m/z: calc. for C19H17NO3:
307.1208; found: 311.1205.
4.2.3.7. 4-(4-Fluorobenzyl)-4H-furo[3,2-b]indole-2-methanol
Yield: 44%; white flocculence crystals; mp: 97e99 ꢃC; IR (KBr)
(cmꢀ1): 2933 (CH), 3213 (OH); 1H NMR (400 MHz, DMSO-d6)
(7f).
n
4.2.3.13. 4-(4-Methoxybenzyl)-4H-furo[3,2-b]indole-2-methanol
(7l). Yield: 17%; orange cubic crystals; mp: 95e96 ꢃC; IR (KBr)
n
d
(ppm): 4.53 (d, J ¼ 5.6 Hz, 2H), 5.37 (t, J ¼ 5.6 Hz, 1H), 5.46 (s, 2H),
(cmꢀ1): 3491 (OH); 1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.70 (s,
6.65 (s, 1H), 7.08e7.31 (m, 6H), 7.62 (d, J ¼ 8.0 Hz, 2H); 13C NMR
3H), 4.52 (d, J ¼ 4.8 Hz, 2H), 5.34e5.38 (m, 3H), 6.60 (s, 1H), 6.86 (d,
(50 MHz, DMSO-d6) d (ppm): 47.42, 57.20, 97.31,111.24,113.89,115.80
J ¼ 8.4 Hz, 2H), 7.08 (t, J ¼ 7.6 Hz, 1H), 7.15 (t, J ¼ 7.6 Hz, 1H), 7.21 (d,
(d, J ¼ 21 Hz), 115.88, 116.01, 119.69, 121.48, 129.82 (d, J ¼ 8 Hz),
133.10,134.43,139.23,139.66,159.72; MS (EI, 70 eV) m/z: 295.2 (Mþ);
HRMS (EI) m/z: calc. for C18H14FNO2: 295.1009; found: 295.1002.
J ¼ 8.4 Hz, 2H), 7.61 (d, J ¼ 6.8 Hz, 1H), 7.63 (d, J ¼ 7.6 Hz, 1H); 13
C
NMR (50 MHz, DMSO-d6) d (ppm): 47.71, 55.49, 57.24, 97.38, 111.33,
113.85, 114.38, 115.82, 119.49, 121.30, 129.30, 130.14, 133.15, 139.24,
139.59,159.09,159.70; MS (EI, 70 eV) m/z: 307.1 (Mþ); HRMS (EI) m/
z: calc. for C19H17NO3: 307.1208; found: 307.1201.
4.2.3.8. 4-(2-Chlorobenzyl)-4H-furo[3,2-b]indole-2-methanol (7g).
Yield: 61%; brown oil; IR (KBr)
NMR (400 MHz, DMSO-d6)
(ppm): 4.50 (d, J ¼ 5.6 Hz, 2H), 5.35 (t,
J ¼ 5.6 Hz,1H), 5.56 (s, 2H), 6.44 (s,1H), 6.88 (d, J ¼ 7.2 Hz,1H), 7.10e
n
(cmꢀ1): 2924 (CH), 3441 (OH); 1H
d
4.2.3.14. 4-(3,4-(Methylenedioxy)benzyl)-4H-furo[3,2-b]indole-2-
methanol (7m). Yield: 62%; brown oil; IR (KBr) n
(cmꢀ1): 2924 (CH),

S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
477
3441 (OH); 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 4.53 (d, J ¼ 5.6 Hz,
145.70,154.85, 159.74; MS (EI, 70 eV) m/z: 361.1 (Mþ); HRMS (EI) m/
z: calc. for C17H15NO3Se: 361.0217; found: 361.0214.
2H), 5.34e5.37 (m, 3H), 5.96 (s, 2H), 6.66 (s, 1H), 6.78 (d, J ¼ 8.0 Hz,
1H), 6.83 (s,1H), 6.84 (d, J ¼ 8.4 Hz,1H), 7.09 (t, J ¼ 7.6 Hz,1H), 7.16 (t,
J ¼ 7.6 Hz,1H), 7.62 (d, J ¼ 7.6 Hz,1H), 7.64 (d, J ¼ 8.4 Hz,1H); 13C NMR
4.3. Biological evaluation
(50 MHz, DMSO-d6)
d (ppm): 47.98, 57.23, 97.39, 101.43, 108.43,
108.70, 111.35, 113.87, 115.83, 119.56, 121.34, 132.01, 133.11, 139.21,
139.60, 147.04, 147.79, 159.75; MS (EI, 70 eV) m/z: 321.0 (Mþ); HRMS
(EI) m/z: calc. for C19H15NO4: 321.1001; found: 321.1005.
4.3.1. Cell culture and treatment
Human cancer cell lines were purchased from ATCC (Manassas,
VA) or the BCRC. Human leukemia HL-60, non-small-cell-lung
cancer H460, and COLO 205 cancer cells were maintained in an
RPMI-1640 medium supplemented with 10% fetal bovine serum
(FBS) (GIBCO/BRL), penicillin (100 U/mL)/streptomycin (100 g/mL)
4.2.3.15. 4-(3-(Hydroxymethyl)benzyl)-4H-furo[3,2-b]indole-2-
methanol (7n). Yield: 42%; orange cubic crystals; mp: 92e94 ꢃC; IR
(KBr)
n
(cmꢀ1): 2918 (CH), 3277 (OH); 1H NMR (400 MHz, DMSO-d6)
(GIBCO/BRL) and 1% L
-glutamine (GIBCO/BRL) at 37 ꢃC in a hu-
d
(ppm): 4.43 (d, J ¼ 5.6 Hz, 2H), 4.52 (d, J ¼ 5.6 Hz, 2H), 5.15 (t,
midified atmosphere containing 5% CO2. Human hepatoma Hep 3B
and human fetal skin fibroblasts Detroit 551 cells were maintained
in a DMEM supplemented with 10% FBS, penicillin (100 U/mL)/
J ¼ 5.6 Hz, 1H), 5.36 (t, J ¼ 5.6 Hz, 1H), 5.46 (s, 2H), 6.62 (s, 1H), 7.08e
7.28 (m, 6H), 7.59 (d, J ¼ 8.0 Hz, 1H), 7.63 (d, J ¼ 7.6 Hz, 1H); 13C NMR
(50 MHz, DMSO-d6)
d
(ppm): 48.29, 57.23, 63.12, 97.34,111.27, 113.81,
streptomycin (100 g/mL) and 1% L
-glutamine at 37 ꢃC in a humid-
115.84, 119.55, 121.37, 125.75, 126.06, 126.13, 128.76, 133.29, 138.10,
139.31, 139.55, 143.37, 159.71; MS (EI, 70 eV) m/z: 307.1 (Mþ); HRMS
(EI) m/z: calc. for C19H17NO3: 307.1208; found: 307.1216.
ified atmosphere containing 5% CO2. Logarithmically growing
cancer cells were used for all experiments. The A498 renal cancer
cells were maintained in an MEM supplemented with 10% FBS,
penicillin (100 U/mL)/streptomycin (100 g/mL), and 1% L-glutamine
4.2.3.16. 4-(4-(Hydroxymethyl)benzyl)-4H-furo[3,2-b]indole-2-
at 37 ꢃC in a humidified atmosphere containing 5% CO2.
methanol (7o). Yield: 47%; pale red flocculence crystals; mp: 140e
141 ꢃC; IR (KBr)
n
(cmꢀ1): 2933 (CH), 3275 (OH); 1H NMR (400 MHz,
4.3.2. Cytotoxicity assay
DMSO-d6)
d
(ppm): 4.44 (d, J ¼ 5.6 Hz, 2H), 4.52 (d, J ¼ 5.6 Hz, 2H),
Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-
2,5- diphenyltetrazolium bromide (MTT) assay [16]. HL-60, Hep 3B,
H460 and Detroit 551 cells were treated with vehicle or test com-
pounds for 48 h. After treatment, the cells were washed once with
5.11 (t, J ¼ 5.6 Hz,1H), 5.36 (t, J ¼ 5.6 Hz,1H), 5.44 (s, 2H), 6.63 (s,1H),
7.09 (t, J ¼ 7.2 Hz, 1H), 7.15 (t, J ¼ 7.2 Hz, 1H), 7.20 (d, J ¼ 8.0 Hz, 2H),
7.24 (d, J ¼ 8.0 Hz, 2H), 7.61 (d, J ¼ 5.2 Hz, 1H), 7.62 (d, J ¼ 4.4 Hz,1H);
13C NMR (50 MHz, DMSO-d6)
d
(ppm): 48.06, 57.24, 63.05, 97.38,
PBS and incubated with 50
mL MTT for 2 h. The formazan precipitate
111.33, 113.86, 115.85, 119.55, 121.35, 127.13, 127.63, 133.24, 136.57,
139.30, 139.59, 142.27, 159.73; MS (EI, 70 eV) m/z: 307.1 (Mþ); HRMS
(EI) m/z: calc. for C19H17NO3: 307.1208; found: 307.1201.
was then dissolved in 150
measured on an ELISA reader at a wavelength of 570 nm.
m
L DMSO, and the absorbance was
4.3.3. Cell morphology
4.2.3.17. (5-((2-(Hydroxymethyl)-4H-furo[3,2-b]indol-4-yl)methyl)
The A498 cells were plated at a density of 5 ꢂ 105 cells per well
furan-2-yl) methanol (10a). Yield: 60%; pale yellow cubic crystals;
in a 10 cm dish and then incubated with 0.5 mM of 10a for 12e48 h.
mp: 121e123 ꢃC; IR (KBr)
n
(cmꢀ1): 2918, 3188 (CH), 3294 (OH); 1H
Cells were directly examined and photographed under a phase
contrast microscope.
NMR (600 MHz, DMSO-d6)
d
(ppm): 4.29 (d, J ¼ 4.8 Hz, 2H), 4.53 (d,
J ¼ 4.8 Hz, 2H), 5.15 (t, J ¼ 5.4 Hz, 1H), 5.38 (t, J ¼ 5.4 Hz, 1H), 5.42 (s,
2H), 6.22 (d, J ¼ 3.6 Hz,1H), 6.42 (d, J ¼ 3.0 Hz,1H), 6.60 (s,1H), 7.10 (t,
J ¼ 7.8 Hz, 1H), 7.17 (t, J ¼ 8.4 Hz, 1H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.67 (d,
4.3.4. Quantification of apoptosis
The A498 cells (5 ꢂ 105 cells/dish) were fluorescently labeled for
J ¼ 8.4 Hz, 1H); 13C NMR (150 MHz, DMSO-d6)
d
(ppm): 41.05, 55.59,
detecting apoptotic and necrotic cells by adding 100 mL of a binding
56.78, 96.90, 107.72, 109.41, 110.79, 113.43, 115.35, 119.20, 120.90,
132.54,138.74,139.14,149.64,155.53,159.23; MS (EI, 70 eV) m/z: 297.1
(Mþ); HRMS (EI) m/z: calc. for C17H15NO4: 297.1001; found: 297.1007.
buffer, 2
samples were mixed gently and incubated at room temperature in
the dark for 15 min. In total, 300 L of the binding buffer was
mL of annexin V-FITC, and 2 mL of PI to each sample. The
m
immediately added-to each sample before flow cytometric analysis.
A minimum of 10 000 cells within the gated region were analyzed.
The Annexin V-FITC Apoptosis Detection Kit was obtained from
Strong Biotech Corporation (Strong Biotech, Taiwan).
4.2.3.18. (5-((2-(Hydroxymethyl)-4H-furo[3,2-b]indol-4-yl)methyl)
thiophen-2-yl)methanol (10b). Yield: 37%; pale brown flocculence
crystals; mp: 143e145 ꢃC; IR (KBr) (cmꢀ1): 3190 (CH), 3288 (OH); 1H
n
NMR (400 MHz, DMSO-d6)
d
(ppm): 4.49 (d, J ¼ 5.6 Hz, 2H), 4.54 (d,
J ¼ 6.0 Hz, 2H), 5.33 (t, J ¼ 5.6 Hz, 1H), 5.39 (t, J ¼ 6.0 Hz, 1H), 5.61 (s,
2H), 6.72 (s,1H), 6.76 (d, J ¼ 3.6 Hz,1H), 7.00 (d, J ¼ 3.6 Hz,1H), 7.10 (t,
J ¼ 7.6 Hz, 1H), 7.18 (t, J ¼ 7.6 Hz, 1H), 7.61 (d, J ¼ 8.0 Hz, 1H), 7.68 (d,
4.3.5. Flow cytometric analysis for cell-cycle effects [17]
Cells were fixed in 70% ethanol overnight and re-suspended in
PBS containing 20
mg/mL PI (Sigma Chemical Co., St. Louis, MO,
J ¼ 8.4 Hz, 1H); 13C NMR (50 MHz, DMSO-d6)
d
(ppm): 43.32, 57.23,
USA), 0.2 mg/mL RNase A (Sigma Chemical Co., St. Louis, MO, USA),
and 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO, USA) in a
dark room. Following 30 min of incubation at 37 ꢃC, cell-cycle
distribution was analyzed using ModFit LT software (Verity Soft-
ware House, Topsham, USA) in a BD FACSCanto flow cytometer
(Becton Dickinson, San Jose, CA).
58.74, 97.41,111.35,114.11,115.86,119.76,121.41,123.91,126.46,132.89,
139.09,139.39,139.82,146.71, 159.70; MS (EI, 70 eV) m/z: 313.1 (Mþ);
HRMS (EI) m/z: calc. for C17H15NO3S: 313.0773; found: 313.0778.
4.2.3.19. (5-((2-(Hydroxymethyl)-4H-furo[3,2-b]indol-4-yl)methyl)
selenophen-2-yl)methanol (10c). Yield: 55%; pale yellow floccu-
lence crystals; mp: 153e154 ꢃC; IR (KBr)
n
(cmꢀ1): 3190 (CH), 3286
4.3.6. Mitochondrial membrane potential analysis
(OH); 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 4.50 (d, J ¼ 5.6 Hz,
Cells were plated in 6-well plates at 5.0 ꢂ 105 cells/dish and
2H), 4.54 (d, J ¼ 6.0 Hz, 2H), 5.38 (t, J ¼ 5.6 Hz,1H), 5.42 (t, J ¼ 5.6 Hz,
1H), 5.63 (s, 2H), 6.71 (s, 1H), 6.88 (d, J ¼ 3.6 Hz, 1H), 7.08e7.19 (m,
3H), 7.61 (d, J ¼ 7.6 Hz, 1H), 7.66 (d, J ¼ 8.0 Hz, 1H); 13C NMR
treated with 0.5 mM 10a for 6e48 h. Mitochondrial membranes
were stained by adding 0.5 mL of a JC-1 working solution (JC-1
according to the protocol on a BDÔ MitoScreen as described in the
section Methods for Staining Cells with JC-1 and Analyzing by Flow
Cytometry) to each sample. Samples were incubated for 10e15 min
(50 MHz, DMSO-d6)
d (ppm): 45.69, 57.23, 60.89, 97.43, 111.38,
114.20, 115.87, 119.76, 121.37, 124.65, 128.40, 132.88, 139.13, 139.90,

478
S.-H. Zhuang et al. / European Journal of Medicinal Chemistry 66 (2013) 466e479
at 37 ꢃC in the dark. The mitochondrial membrane potential was
measured using the BD FACSCanto flow cytometer (Becton Dick-
inson, San Jose, CA).
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4.3.7. Western blot assay
Treated cells were collected and washed with PBS. After centri-
fugation, cells were lysed in a lysis buffer. The lysates were incubated
on ice for 30 min and centrifuged at 12 000g for 20 min. Supernatants
were collected, and protein concentrations were then determined
using the Bradford Assay. After adding a 5ꢂ sample loading buffer
containing 625 mM TriseHCl (Sigma ChemicalCo. St. Louis, MO, USA),
pH¼ 6.8, 500 mM dithiothreitol(BIO-RAD),10%SDS (BIO-RAD), 0.06%
bromophenol blue (Merck), and 50% glycerol (AMRESCO), the protein
samples were electrophoresed on 10% SDS-polyacrylamide gels and
transferred to a nitrocellulose membrane (Amersham Pharmacia
Biotech, Piscataway, NJ, USA). Immunoreactivity was detected using
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4.3.8. Statistical analysis
Statistical analysis was performed using analysis of variance
(ANOVA) followed by Turkey’s test. All data were expressed as the
mean
ꢁ
SD from at least three independent experiments.
*P < 0.001 was indicative of a statistically significant difference.
4.3.9. In vivo antitumor activity assay
Male BALB/cAnN.Cg-Foxn1nu/CrlNarl nude mice (18e20 g; 4e6
weeks of age) were purchased from the National Animal Center and
maintained in pressurized ventilated cages according to institu-
tional regulations. Nude mice were subcutaneously inoculated with
A498 cells at 2 ꢂ 106 cells per mouse in 0.1 mL PBS by using a 24G
needle. After the appearance of a 100 mm3 tumor nodule, tumor-
bearing mice were randomly assigned to several groups (eight
animals in each group). The mice were administered a single ip
dose 5 times per week for 4 consecutive weeks at 30 mg/kg or
60 mg/kg. Body weight and tumor size were measured and recor-
ded every 7 days during the experiment period of 28 days. Tumor
volume was calculated using the following formula: 1/2 (L þ W2),
where L is the length and W is the width [18,19]. At the end of the
experiments, the animals were euthanized with carbon dioxide
before cervical dislocation. The tumors were excised, weighed, and
sectioned, and the tumor sections were embedded in an OCT
compound and frozen at ꢀ70 ꢃC.
Acknowledgments
This investigation was supported by research grants from the
National Science Council of the Republic of China awarded to L.J.
Huang (NSC 98-2628-B-039-018-MY3; NSC 101-2320-B-039-009-
MY3). Experiments and data analysis were partially performed
using the Medical Research Core Facilities Center, Office of Research
& Development, China Medical University, Taichung, Taiwan, ROC.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.012.
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