Antitumor agents 270. Novel substituted 6-phenyl-4H-furo[3,2-c]pyran-4-one derivatives as potent and highly selective anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmc.2009.11.049

6-Phenyl-4H-furo[3,2-c]pyran-4-one derivatives based on neo-tashinlactone (1) were synthesized and evaluated as novel anti-breast cancer agents. Compounds 10-13, 23, 25, and 27 showed potent inhibition against the SK-BR-3 breast cancer cell line. Importantly, 25 and 27 showed the highest cancer cell line selectivity, being approximately 100-250-fold more potent against SK-BR-3 (ED₅₀ 0.28 and 0.44 μM, respectively) compared with other cancer cell lines tested. In addition, 25 displayed low cytotoxicity against normal breast cell lines 184A1 and MCF10A. Compounds 25 and 27 merit further investigation in our continuing program to generate and develop selective anti-breast cancer agents.

Full text of DOI:10.1016/j.bmc.2009.11.049

Bioorganic & Medicinal Chemistry 18 (2010) 803–808
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antitumor agents 270. Novel substituted 6-phenyl-4H-furo[3,2-c]pyran-4-one
derivatives as potent and highly selective anti-breast cancer agents
a
a
a
Yizhou Dong a, Qian Shi a, , Kyoko Nakagawa-Goto , Pei-Chi Wu , Susan L. Morris-Natschke ,
*
c
Arnold Brossi a, Kenneth F. Bastow b, , Jing-Yu Lang , Mien-Chie Hung c,d, Kuo-Hsiung Lee a,
*
*
a Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, United States
b Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, United States
c Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, United States
d Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University and Hospital, Taichung, Taiwan 404, China
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Phenyl-4H-furo[3,2-c]pyran-4-one derivatives based on neo-tashinlactone (1) were synthesized and
evaluated as novel anti-breast cancer agents. Compounds 10–13, 23, 25, and 27 showed potent inhi-
bition against the SK-BR-3 breast cancer cell line. Importantly, 25 and 27 showed the highest cancer
cell line selectivity, being approximately 100–250-fold more potent against SK-BR-3 (ED50 0.28 and
Received 24 September 2009
Revised 19 November 2009
Accepted 21 November 2009
Available online 2 December 2009
0.44 lM, respectively) compared with other cancer cell lines tested. In addition, 25 displayed low
cytotoxicity against normal breast cell lines 184A1 and MCF10A. Compounds 25 and 27 merit fur-
ther investigation in our continuing program to generate and develop selective anti-breast cancer
agents.
Keywords:
6-Phenyl-4H-furo[3,2-c]pyran-4-ones
Neo-tanshinlactone
Breast cancer
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Natural products have been the most important source of
new medicinal leads.9–11 However, the structural complexity of
Breast cancer is the most common cancer among women.1–3
According to the American Cancer Society, the disease accounts
for more than one quarter of cancers diagnosed in US women. In
2007, it was estimated that 18,000 new cases of invasive breast
cancer would be diagnosed in women, as well as an estimated
60,000 additional cases of in situ breast cancer.4
Most clinically used anticancer drugs cause general toxicity to
proliferating cells, which can severely limit the therapeutic value
of these drugs.5 Thus, much effort has been made to increase tis-
sue, cell, and target selectivity for chemotherapy.6–8 However,
although new cytotoxic agents with unique mechanisms of action
have been developed continuously, many of them have not been
therapeutically useful due to low tumor selectivity.5 These facts
prompted us to design and develop novel potent and selective
anti-breast cancer agents.
natural products, such as intricate ring systems and numerous
chiral centers, may lead to limited supplies and hamper mecha-
nism of action studies and clinical development.12 For this rea-
son, structural simplification of natural products is a powerful
and highly productive tool for lead development and analog de-
sign.13 A well-known example is the simplification of morphine,
which led to the clinically used medicines levophanol and
meperidine.14 Neo-tanshinlactone (1) is a steroid-like tetracyclic
natural product originally isolated from the traditional Chinese
medicine Tanshen (Fig. 1). Compound 1 and its first generation
analog 2 with various substituents around the molecular scaf-
fold were totally synthesized and previously studied for biolog-
ical activity. Compound 1 was reported as a highly selective
inhibitor of the growth of breast cancer cell lines SK-BR-3
(HER2 over-expressing breast cancer) and ZR-75-1 (estrogen
receptor positive breast cancer).2,15 Although previous studies
provided much information about the structure–activity rela-
tionships, several questions remained unanswered: how does
the skeletal planarity affect activity and selectivity, how do each
of the four individual rings contribute to activity, and how will
the activity and selectivity change by simplification of the tetra-
cyclic molecule of 1. We used chemical and biological strategies
to investigate structurally simplified 1-analogs to answer these
questions.
Abbreviations: ER, estrogen receptor; HER2, human epidermal growth factor
receptor 2; SAR, structure–activity relationship; TAM, tamoxifen; DIEA, diisopro-
pylethylamine; DMAP, 4-(dimethyl amino) pyridine; LDA, lithium diisopropyl-
amide; TMEDA, tetramethylethylenediamine.
* Corresponding authors. Tel.: +1 919 843 6325 (Q.S.); +1 919 966 7633 (K.F.B.);
+1 919 962 0066; fax: +919 966 3893 (K.H.L.).
E-mail addresses: qshi1@email.unc.edu (Q. Shi), ken_bastow@unc.edu (K.F.
Bastow), khlee@unc.edu (K.-H. Lee).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.049

804
Y. Dong et al. / Bioorg. Med. Chem. 18 (2010) 803–808
O
2. Chemistry
O
C
O
O
B
All target compounds 9–30 were synthesized through a three to
five-step sequence (Scheme 1). Various substituted esters 5 were
reacted with a dianion intermediate generated from ethyl acetoac-
etate with LDA and TMEDA to give triketoesters 6 as tautomeric
mixtures. Pyrones 7 were prepared by heating 6 in a 170 °C oil bath
under reduced pressure. The resulting yellow solid was isolated by
vacuum filtration, and the compound used directly in the next
step.18 Target compounds 9–11, 14–17, 19, 21–22, 24–25, and
27–29 were obtained via a tandem alkylation/ intramolecular Aldol
reaction of 7.17,19 Removal of the methyl group in 11, 17, 19, 22,
and 25 by BBr3 gave 13, 18, 20, 23, and 26. Compound 12 was ob-
tained by treatment of 13 with iodoethane under basic conditions
(Scheme 1). Compound 25 was reacted with Lawesson’s reagent to
afford 30.20
D
O
O
A
Et
Me
Neo-tanshinlactone (1)
2
HOOC
OH
O
O
O
O
Et
R
3. Results and discussion
4
3
Together with 1, the newly synthesized 6-phenyl-4H-furo[3,2-
c]pyran-4-one analogs 9–30 were evaluated for in vitro anti-breast
cancer activity against the SK-BR-3 human tumor cell line. Results
from 9–27 (Table 1) showed that different substituents around the
phenyl ring were critical to the potency and selectivity. Modifica-
tions in the furopyranone ring system were also explored with
28–30 (Table 1). Selected active compounds with ED50 values less
Figure 1. Structures of neo-tanshinlactone (1), a first generation neo-tanshinlac-
tone analog 2, a second generation optimized analog 3, and a newly designed
scaffold 4.
In our prior paper, we reported a study on how the individual A,
C, and D rings influence in vitro anti-breast cancer activity.16,17 The
results revealed that 2-(furan-2-yl)-naphthalen-1-ol derivatives
(e.g., 3), in which ring-C of 1 is missing, are a new class of potent
and selective anti-breast cancer agents. These results encouraged
us to further simplify the scaffold of 1. Herein, we report a new
chemical entity, substituted 6-phenyl-4H-furo[3,2-c]pyran-4-one
derivatives (4), the synthesis of these 4-analogs, and their cytotoxic
activity against a human tumor cell line panel.
than 10 lM against SK-BR-3 were further examined against ZR-7-
51, MDA-MB-231 (estrogen receptor negative breast cancer), A549
(human lung cancer), DU145 (prostate cancer), KB (nasopharyn-
geal carcinoma), and KB-vin (vincristine-resistant KB subline) can-
cer cell lines (Table 2).
Structurally, both 1 and 10 have a methyl substituent at corre-
sponding positions on their phenyl rings. Thus, the two compounds
O
O
O
O
O
O
R4
R3
R4
R3
R4
OMe
OEt
OH
a)
b)
R1
R1
R3
R1
R2
R2
R2
3
7
5
6
O
O
R5
4
5
O
O
2
R6
R4
R3
R4
R3
O
d)
11, 17, 19, 22, 25
6
c)
O
1
7
R1
R1
R2
R2
e)
12
, R2 = OEt
13
: R2 = OH; R1,R3-R4= H
9-11, 14-17, 19, 21-22, 24-25, 27-29
18: R3= OH; R1-R2, R4= H
20: R1= R2 = OH; R3-R4= H
S
25 f)
23
: R2= R3 = OH; R1,R4= H
26: R2= R4 = OH; R1,R3=H
O
MeO
O
OMe
30
O
Cl
R6
Conditions: a) Ethyl acetoacetate, LDA, TMDA, then HOAc; b) 170 oC, 5 mmHg; c) R5
NH4OAc; d) BBr3, CH2Cl2; e) EtI, K2CO3, acetone; f) Lawesson's reagent
, HOAc,
R1, R2, R3, R4 are found in Table 1; for 9-27 R5=Me, R6=H; for 28 R5=Et, R6=H; for 29 R5=R6=Me
Scheme 1.

Y. Dong et al. / Bioorg. Med. Chem. 18 (2010) 803–808
805
Table 1
displayed slightly greater activity (ED50 0.67
lM) than 1. In con-
Cytotoxicity of 9–27 against SK-BR-3 tumor cell linea
trast, fluorine at the R2-position (14), as well as R3-position (15),
led to somewhat decreased potency compared with the unsubsti-
tuted analog 9. Addition of methyl, methoxy, or hydroxyl at the
phenyl R3-position (16–18) reduced potency significantly. Com-
pounds 19–26 and 27 are di- and tri-substituted derivatives,
respectively, with one substituent always present at the phenyl
R2-position. Neither R1, R2-disubstituted compounds (19, 20)
showed significant activity, leading us to speculate that a substitu-
ent in the R1-position may have a steric effect on the orientation of
the lactone ring and reduce the ligand–receptor interaction. Ana-
logs with the same substituent at both the R2- and R3-positions
showed increased potency relative to the corresponding R3-mono-
substituted analog (16 vs 21, 17 vs 22, 18 vs 23). Thus, alkyl, alk-
oxy, and hydroxy groups are favored at R2-position, while they
are not favored at the R3-position. Comparison of 25 with 24 and
26 indicated that a methoxy group is favored at R4-position, while
methyl and hydroxy groups are not. Furthermore, the R2, R3, R4-tri-
methoxy (27) and R2, R4-dimethoxy (25) analogs showed dramat-
ically enhanced potency compared with the R2-methoxy
compound (11), while the R2, R3-dimethoxy (22) and R1, R2-dime-
thoxy (19) analogs showed decreased potency. In fact, the R2,
O
O
O
O
C
D
O
MeO
R4
R3
O
A
R1
OMe
R2
9-27
28
O
S
O
O
MeO
MeO
O
O
OMe
29
OMe
30
Compd
R1
R2
R3
R4
SK-BR-3
0.95 0.01
1
9
—
H
H
H
H
H
H
H
H
H
H
OMe
OH
H
H
H
H
H
—
H
—
H
H
H
H
H
H
F
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R4-dimethoxy analog 25 (ED50 0.28 lM) was the most active
analog among the 19 substituted phenyl A-ring analogs (9–27). It
was also approximately threefold more potent than 1.
15.49 0.60
1.50 0.04
2.58 0.15
0.67 0.03
1.61 0.09
20.49 1.50
19.26 0.65
>83.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Me
OMe
OEt
OH
F
We also investigated the cytotoxic activity of 28–30, which
have a modified ring-C or -D, as shown in Table 1. Insertion of an
ethyl (28) or two methyl (29) groups rather than a single methyl
group on the furan, as well as bioisosteric replacement of sulfur
(thiolactone 30) for oxygen in the lactone carbonyl led to greatly
reduced or no anti-breast cancer activity (Table 1). More SAR stud-
ies of ring-C and -D are in progress and will be reported in a future
publication.
To examine the human tumor-tissue-type selectivity, active
compounds 10–13, 23, 25, and 27 were tested against a limited
but diverse panel of human cancer cell lines, using 1 as a positive
control (Table 2). Compounds 10–13 and 23 displayed similar inhi-
bition of the ZR-75-1 and SK-BR-3 cell lines. Interestingly, 25 and
27 showed very weak activity against ZR-75-1. Except for 23, all
lead compounds had weak activity or no activity against MDA-
MB-231 breast cancer or the remaining four cancer cell lines
tested, which suggested high tumor-tissue-type selectivity. Fur-
thermore, 23 showed only moderate inhibition. Importantly, 25
and 27 showed unique selectivity against the SK-BR-3 breast can-
cer cell line (HER2+), with approximately 100–250-fold differences
compared with the other cancer cell lines tested. The unique selec-
tivity of these novel lead compounds could be exploited to develop
novel anti-breast cancer trials candidates and explore the mecha-
nism(s) of action.
H
H
H
H
Me
OMe
OH
H
>78.1
35.95 1.50
65.03 2.50
34.11 1.15
14.57 0.63
>69.9
0.47 0.01
22.44 0.95
0.28 0.01
38.37 0.95
0.44 0.01
55.57 1.65
>66.7
OMe
OH
Me
OMe
OH
Me
OMe
OH
OMe
—
H
Me
OMe
OH
H
Me
OMe
OH
OMe
—
H
H
H
H
—
—
—
OMe
—
—
—
—
—
—
—
>66.2
a
Mean ED50+SEM (lM), from 2 or more independent tests.
are identical, except that 10 has no ring-B. Interestingly, 10 showed
potent activity with an ED50 value of 1.50 M, which is slightly less
l
active than 1. The unsubstituted analog 9 was less potent than
either 1 or 10. Addition of methyl (10), methoxy (11), ethoxy
(12), and hydroxyl (13) at the R2-position of the phenyl ring in-
creased activity against the SK-BR-3 cell line, compared with 9.
The rank order of potency of the five compounds was
12 > 13 > 11 > 10 > 9. Compound 12, with a R2-ethoxyphenyl ring,
Compound 25 was tested independently against cell lines
derived from normal breast tissue (MCF10A and 184A1) versus
SK-BR-3 as a positive breast cancer cell line control, and results
Table 2
Cytotoxicity of selected compounds against tumor cell line panela
Compd
ZR-75-1
MDA-MB-231
A549
DU145
KB
KBvin
1
10
11
12
13
23
25
27
0.9 0.02
6.3 0.4
5.5 0.2
2.2 0.06
0.9 0.03
1.2 0.07
30.8 1.7
29.1 0.8
37.9 1.4
>83.3
>39.1
>37.0
>41.3
22.9 0.65
>69.9
>31.6
54.2 3.4
53.8 0.8
58.2 1.7
57.8 3.7
68.6 2.9
19.4 0.71
>69.9
58.3 2.0
24.6 1.5
>78.1
74.1 5.6
82.6 3.4
25.6 1.2
65.4 3.1
>63.3
>37.9
43.8 1.3
>78.1
64.4 3.0
65.3 0.9
20.2 0.5
>69.9
>75.8
40.0 1.1
71.1 2.1
>74.1
60.3 2.9
23.3 0.39
>69.9
63.3 2.7
>63.3
>63.3
a
Mean ED50+SEM (lM), from 2 or more independent tests.

806
Y. Dong et al. / Bioorg. Med. Chem. 18 (2010) 803–808
tained from Aldrich, Inc. and Fisher, Inc. Preparation of intermedi-
100
80
60
40
20
0
ates 6 and 7 were reported by Douglas, etc.17
5.2. Cell growth inhibition assay
All stock cultures are grown in T-25 flasks. Freshly trypsinized
cell suspensions were seeded in 96-well microtiter plates with
compounds added from DMSO-diluted stock. The plates were
incubated for an additional 72 h after attachment and drug addi-
tion, and the assay was terminated by 10% TCA. Then, 0.4% SRB
dye in 1% HOAc was added to stain the cells for 10 min. Unbound
dye was removed by repeated washing with 1% HOAc and the
plates were air dried. Bound stain was subsequently solved with
10 mM trizma base, and the absorbance read at 515 nm. Growth
inhibition of 50% (ED50) is calculated as the drug concentration,
which caused a 50% reduction in the net protein increase during
the drug incubation. The mean ED50 is the concentration of agent
that reduces cell growth by 50% under the experimental condi-
tions and is the average with SEM from at least two independent
determinations. Variation between replicates was no more than
5% of the mean. The following human tumor cell lines were used
in the assay: A549 (non small cell lung cancer, ZR-75-1 (estrogen
receptor positive breast cancer), MDA MB-231 (estrogen receptor
negative breast cancer), SK-BR-3 (HER2 over-expressing breast
cancer), KB (nasopharyngeal carcinoma), KB-VIN (vincristine-
resistant KB subline). All cell lines were obtained from the
Lineberger Cancer Center (UNC-CH) or from ATCC (Rockville,
MD). Cells propagated in RPMI-1640 supplemented with 10%
SK-Br3
MCF10A
184A1
0
0.005 0.01 0.05
0.1
0.5
1
5
10
Drug concentration (ug/ml)
Figure 2. Selective in vitro anticancer activity of 25 against SK-BR-3 breast cancer
versus normal breast tissue-derived cell lines (MCF10A and 184A1). Legend: Cell
line description, source and activity determination using the MTT-dye assay are
described in Section 5. Graphical data are the mean and standard deviation of
values obtained from replicates in a single experiment.
are shown in Figure 2. The interpolated ED50 values were 1.0, 16.8
and >35.0 lM against SK-BR-3, 184A1, and MCF10A cells, respec-
tively, showing that 25 is selective for a sub-set of breast cancer-
derived cell lines and is significantly less active against normal
breast-derived tissue.
4. Conclusions
In conclusion, this study discovered a novel class of promising
anti-breast cancer agents, substituted 6-phenyl-4H-furo[3,2-c]pyr-
an-4-one derivatives. The ED50 values of the two most potent
FBS, penicillin (100 IU/mL), streptomycin (1
lg/mL), and ampho-
tericin B (0.25 g/mL), and were cultured at 37 °C in a humidified
l
atmosphere of 95% air/5% CO2.
analogs (25 and 27) against SK-BR-3 were 0.28 and 0.44 lM,
respectively. More importantly, 25 and 27 showed extremely high
cancer cell line selectivity, being approximately 100–250-fold
more potent against SK-BR-3 compared with six additional tested
cancer cell lines. Furthermore, 25 displayed much greater potency
against the SK-BR-3 breast cancer cell line compared with normal
breast cell lines 184A1 and MCF10A. Preliminary SAR studies led to
the following observations. (1) R2-Methyl, methoxy, ethoxy, and
hydroxy groups, but not a R2-fluoro group, could increase potency.
(2) Among disubstituted phenyl compounds, R1-, R3-, or R4-methyl
groups, R1- or R3-methoxy groups, and R4-hydroxy groups de-
creased potency; while a R3-hydroxy or R4-methoxy group in-
creased potency. (3) Current modifications in ring-C and -D were
not preferred. The SAR profile established from the current study
is different from that with the neo-tanshinlactone series, which
is a four-ring system. Thus, skeletal planarity is not indispensable
for the entire molecule, though it may be important to some
extent. Focused studies will continue to develop promising novel
analogs as clinical trials candidates for anti-breast cancer
treatment.
5.2.1. General preparation of9–11, 14–17, 19, 21–22, 24–25, 27–
29
To a solution of 7 (1.04 mmol) in toluene (9 mL) was added a
mixture of HOAc (0.30 ml, 5.20 mmol) and NH4OAc (400 mg,
5.20 mmol) in EtOH (3 mL) and chloroacetone (0.42 mL,
5.20 mmol). The mixture was stirred for 30 min at rt, and then
heated to 60 °C for 30 min. Subsequently, it was refluxed for
24 h. After cooling, the mixture was diluted with H2O and ex-
tracted with EtOAc. The organic layer was dried over Na2SO4, fil-
tered, and evaporated in vacuo. The residue was purified by
column chromatography to give a solid.
5.2.2. 3-Methyl-6-phenyl-4H-furo[3,2-c]pyran-4-one (9)
50% Yield; mp 105–107 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.33 (d, J = 1.2 Hz, 3H, CH3), 7.00 (s, 1H, OCH), 7.28–7.29 (m, 1H,
C7-H), 7.43–7.46 (m, 3H, aromatic), 7.83–7.87 (m, 2H, aromatic);
HRMS for (M++H): calcd 227.0708, found: 227.0696.
5.2.3. 3-Methyl-6-m-tolyl-4H-furo[3,2-c]pyran-4-one (10)
52% Yield; mp 135–137 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.33 (d, J = 1.2 Hz, 3H, CH3), 2.42 (s, 3H, CH3), 6.99 (s, 1H, C7-H),
7.23–7.36 (m, 3H, aromatic), 7.62 (d, J = 7.2 Hz, 1H, aromatic),
7.69 (d, J = 1.5 Hz, 1H, OCH); HRMS for (M++H): calcd 241.0865,
found: 241.0851.
5. Experimental section
5.1. Materials and methods
Melting points were measured with a Fisher Johns melting
apparatus without correction. 1H NMR spectra were measured on
a 300 MHz Varian Gemini 2000 spectrometer using TMS as internal
standard. The solvent used was CDCl3 unless indicated. Mass spec-
tra were measured on a Shimadzu LC-MS2010 instrument. Thin-
layer chromatography (TLC) and preparative TLC were performed
on precoated silica gel GF plates purchased from Merck, Inc. Bio-
tage Flash+ or Isco Companion systems were used for flash chro-
matography. Silica gel (200–400 mesh) from Aldrich, Inc. was
used for column chromatography. All other chemicals were ob-
5.2.4. 6-(3-Methoxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-4-
one (11)
44% Yield; mp 119–121 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.34 (d, J = 1.5 Hz, 3H, CH3), 3.87 (s, 3H, OCH3), 6.96–7.00 (m,
2H), 7.29–7.44 (m, 4H); 13C NMR (300 MHz, CDCl3, ppm): d 8.52,
55.47, 93.64, 109.91, 110.68, 116.36, 117.82, 119.55, 129.91,
133.07, 140.76, 157.71, 159.39, 160.01, 161.93; HRMS for
(M++H): calcd 257.0814, found: 257.0800.

Y. Dong et al. / Bioorg. Med. Chem. 18 (2010) 803–808
807
5.2.5. 6-(3-Fluorophenyl)-3-methyl-4H-furo[3,2-c]pyran-4-one
(14)
1H, C7-H), 6.98 (s, 1H, aromatic), 6.99 (d, J = 2.7 Hz, 2H, aromatic),
7.30 (d, J = 1.5 Hz, 1H, OCH); HRMS for (M++H): calcd 287.0919,
found: 287.0898.
34% Yield; mp 158–160 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.34 (d, J = 1.5 Hz, 3H, CH3), 7.01 (s, 1H, C7–H), 7.10–7.17 (m, 1H,
aromatic), 7.31 (d, J = 1.2 Hz, 1H, OCH), 7.39–7.46 (m, 1H, aro-
matic), 7.54–7.58 (m, 1H, aromatic), 7.61–7.65 (m, 1H, aromatic);
HRMS for (M++H): calcd 245.0614, found: 245.0603.
5.2.14. 3-Methyl-6-(3,4,5-trimethoxyphenyl)-4H-furo[3,2-
c]pyran-4-one (27)
40% Yield; mp 201–203 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.34 (d, J = 1.5 Hz, 3H, CH3), 3.91 (s, 3H, OCH3), 3.95 (s, 6H,
OCH3), 6.94 (s, 1H, C7–H), 7.06 (s, 2H, aromatic), 7.29 (d,
J = 1.2 Hz, 1H, OCH); HRMS for (M++H): calcd 317.1025, found:
317.1037.
5.2.6. 6-(4-Fluorophenyl)-3-methyl-4H-furo[3,2-c]pyran-4-one
(15)
52% Yield; mp 175–177 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.34 (d, J = 1.5 Hz, 3H, CH3), 6.94 (s, 1H, C7–H), 7.12–7.18 (m, 2H,
aromatic), 7.29 (d, J = 1.2 Hz, 1H, OCH), 7.82–7.87 (m, 2H, aro-
matic); HRMS for (M++H): calcd 245.0614, found: 245.0603.
5.2.15. 6-(3,5-Dimethoxyphenyl)-3-ethyl-4H-furo[3,2-c]pyran-
4-one (28)
51% Yield; mp 131–133 °C; 1H NMR (300 MHz, CDCl3, ppm): d
1.31 (t, J = 7.2 Hz, 3H, CH2CH3), 2.77 (q, J = 7.5 Hz, 2H, CH2CH3),
3.86 (s, 6H, OCH3), 6.54 (t, J = 2.1 Hz, 1H, C7–H), 6.98 (d,
J = 2.1 Hz, 3H, aromatic), 7.30 (t, J = 1.2 Hz, 1H, OCH); HRMS for
(M++H): calcd 301.1076, found: 301.1057.
5.2.7. 3-Methyl-6-p-tolyl-4H-furo[3,2-c]pyran-4-one (16)
62% Yield; mp 153–155 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.33 (d, J = 1.2 Hz, 3H, CH3), 2.40 (s, 3H, CH3), 6.95 (s, 1H, C7-H),
7.24–7.27 (m, 3H, aromatic & OCH), 7.74 (d, J = 8.1 Hz, 2H, aro-
matic); HRMS for ([M+H]+): calcd 241.0865, found: 241.0848.
5.2.16. 6-(3,5-Dimethoxyphenyl)-2,3-dimethyl-4H-furo[3,2-
c]pyran-4-one (29)
5.2.8. 6-(4-Methoxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-4-
one (17)
12% Yield; mp 163–165 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.25 (d, J = 0.6 Hz, 3H, CH3), 2.33 (d, J = 0.6 Hz, 3H, CH3), 3.86 (s,
6H, OCH3), 6.53 (t, J = 2.1 Hz, 1H, aromatic), 6.93 (s, 1H, OCH),
6.97 (d, J = 2.1 Hz, 2H, aromatic); HRMS for ([M+H]+): calcd
301.1071, found: 301.1067.
60% Yield; mp 146–148 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.33 (d, J = 1.5 Hz, 3H, CH3), 3.87 (s, 3H, OCH3), 6.88 (s, 1H, C7–
H), 6.97 (d, J = 9.0 Hz, 2H, aromatic), 7.26 (d, J = 1.5 Hz, 1H, OCH),
7.80 (d, J = 9.0 Hz, 2H, aromatic); HRMS for ([M+H]+): calcd
257.0808, found: 257.0816.
5.3. General preparation of 13, 20, 23, and 26
5.2.9. 6-(2,3-Dimethoxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-
4-one (19)
To a solution of 13, 20, 23, or 26 (0.2 mmol) in DCM (3 ml) was
added BBr3 (0.6 ml, 0.6 mmol) dropwise at 0 °C. The reaction mix-
ture was stirred overnight. Water was added to quench the reac-
tion. The solution was extracted with CHCl3 and concentrated.
The residue was purified by column chromatography to give a
white solid.
40% Yield; mp 111–113 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.34 (d, J = 1.2 Hz, 3H, CH3), 3.87 (s, 3H, OCH3), 3.92 (s, 3H,
OCH3), 7.00 (dd, J = 1.2, 8.1 Hz, 1H, aromatic), 7.16 (t, J = 8.1 Hz,
1H, aromatic), 7.34 (q, J = 1.2 Hz, 1H, OCH), 7.48 (s, 1H, C7–H),
7.54 (dd, J = 1.2, 8.1 Hz, 1H, aromatic); HRMS for (M++H): calcd
287.0919, found: 287.0906.
5.3.1. 6-(3-Hydroxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-4-
one (13)
5.2.10. 6-(3,4-Dimethylphenyl)-3-methyl-4H-furo[3,2-c]pyran-
4-one (21)
78% Yield; mp 225–227 °C; 1H NMR (300 MHz, CD3OD, ppm): d
2.29 (d, J = 1.5 Hz, 3H, CH3), 6.86–6.90 (m, 1H, C7–H), 7.26–7.31 (m,
3H, aromatic & OCH), 7.35–7.39 (m, 1H, aromatic), 7.52 (dd, J = 1.2,
2.7 Hz, 1H, aromatic); HRMS for (M++H): calcd 243.0657, found:
243.0659.
67% Yield; mp 182–184 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.30 (s, 3H, CH3) 2.32 (s, 3H, CH3), 2.33 (d, J = 1.5 Hz, 3H, CH3),
6.95 (s, 1H, C7–H), 7.20 (d, J = 8.1 Hz, 1H, aromatic), 7.26 (d,
J = 1.2 Hz, 1H, OCH), 7.57 (d, J = 8.1 Hz, 1H, aromatic), 7.64 (s, 1H,
aromatic); HRMS for ([M+H]+): calcd 255.1016, found: 255.1010.
5.3.2. 6-(4-Hydroxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-4-
one (18)
5.2.11. 6-(3,4-Dimethoxyphenyl)-3-methyl-4H-furo[3,2-
c]pyran-4-one (22)
80% Yield; mp 258–260 °C; 1H NMR (300 MHz, CD3OD, ppm): d
2.27 (d, J = 1.5 Hz, 3H, CH3), 6.86 (d, J = 9.0 Hz, 2H, aromatic) 7.18
(s, 1H, C7–H), 7.46 (d, J = 1.2 Hz, 1H, OCH), 7.75 (d, J = 9.3 Hz, 2H,
aromatic); HRMS for ([M+H]+): calcd 243.0657, found: 243.0641.
83% Yield; mp 154–156 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.33 (d, J = 1.5 Hz, 3H, CH3), 3.94 (s, 3H, OCH3), 3.98 (s, 3H,
OCH3), 6.90 (s, 1H, C7–H), 6.92 (d, J = 8.7 Hz, 1H, aromatic), 7.27
(t, J = 1.5 Hz, 1H, OCH), 7.34 (d, J = 2.1 Hz, 1H, aromatic), 7.43 (dd,
J = 2.1, 8.4 Hz, 1H, aromatic); HRMS for (M++H): calcd 287.0919,
found: 287.0900.
5.3.3. 6-(2,3-Dihydroxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-
4-one (20)
66% Yield; mp 239–241 °C; 1H NMR (300 MHz, CD3OD, ppm): d
2.28 (d, J = 0.9 Hz, 3H, CH3), 6.76 (t, J = 1.5 Hz, 1H, aromatic), 6.86
(dd, J = 1.5, 1.8 Hz, 1H, aromatic), 7.38 (dd, J = 1.2, 1.5 Hz, 1H, aro-
matic), 7.48 (q, J = 1.2 Hz, 1H, OCH), 7.75 (s, 1H, C7–H); HRMS for
(M++H): calcd 259.0606, found: 259.0602.
5.2.12. 6-(3,5-Dimethylphenyl)-3-methyl-4H-furo[3,2-c]pyran-
4-one (24)
30% Yield; mp 171–173 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.33 (d, J = 0.9 Hz, 3H, CH3), 2.37 (s, 6H, CH3), 6.98 (s, 1H, C7–H),
7.07 (s, 1H, aromatic), 7.28 (d, J = 0.9 Hz, 1H, OCH), 7.48 (s, 2H, aro-
matic); HRMS for (M++H): calcd 255.1021, found: 255.1010.
5.3.4. 6-(3,4-Dihydroxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-
4-one (23)
5.2.13. 6-(3,5-Dimethoxyphenyl)-3-methyl-4H-furo
[3,2-c]pyran-4-one (25)
60% Yield; mp 259–261 °C; 1H NMR (300 MHz, CD3OD, ppm): d
2.69 (d, J = 1.5 Hz, 3H, CH3), 6.83 (d, J = 7.8 Hz, 3H, C7–H), 7.12 (s,
1H, aromatic), 7.26–7.31 (m, 2H, aromatic), 7.46 (dd, J = 1.2 Hz,
1H, OCH); HRMS for (M+ÀH): calcd 257.0450, found: 257.0464.
38%, Yield; mp 153–155 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.34 (d, J = 1.5 Hz, 3H, CH3), 3.86 (s, 6H, OCH3), 6.54 (t, J = 2.1 Hz,

808
Y. Dong et al. / Bioorg. Med. Chem. 18 (2010) 803–808
5.3.5. 6-(3,5-Dihydroxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-
4-one (26)
night incubation. After 72 h of incubation at 37 °C in 5% CO2, 20 lL
of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide] reagent was added to each well and incubation contin-
ued for 2 h. The amount of formazan product was measured at
an OD of 570 nM using a plate-reader.
70% Yield; mp >300 °C; 1H NMR (300 MHz, CD3OD, ppm): d 2.86
(d, J = 1.2 Hz, 3H, CH3), 6.35 (t, J = 2.1 Hz, 1H, aromatic), 6.80 (d,
J = 2.1 Hz, 2H, aromatic), 7.21 (s, 1H, C7–H), 7.51 (d, J = 1.2 Hz,
1H, OCH); HRMS for ([M+H]+): calcd 259.0601, found: 259.0594.
Acknowledgement
5.3.6. 6-(3-Ethoxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-4-one
(12)
This work was supported by a NIH Grant CA-17625 from the
National Cancer Institute, awarded to K.H. Lee.
To a mixture of 13 (212 mg, 1.00 mmol), K2CO3 (300 mg,
2.17 mmol) in acetone (8 mL) was added iodoethane (0.4 mL,
5.00 mmol). The mixture was stirred for 12 h. The mixture was
concentrated and diluted with H2O and extracted with EtOAc.
The organic layer was dried over Na2SO4, filtered, and evaporated.
The residue was purified by column chromatography to give a
white solid.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.11.049.
35% Yield; mp 128–130 °C; 1H NMR (300 MHz, CDCl3, ppm): d
1.45 (t, J = 6.9 Hz, 3H, CH2CH3), 2.33 (d, J = 1.5 Hz, 3H, CH3), 4.10
(d, J = 7.2 Hz, 2H, CH2CH3), 6.95–6.96 (m, 1H, aromatic), 6.98 (s,
1H, C7–H), 7.29 (d, J = 1.5 Hz, 1H, OCH), 7.32–7.43 (m, 3H, aro-
matic); HRMS for ([M+H]+): calcd 271.0965, found: 271.0962.
References and notes
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2561.
5.3.7. 6-(3,5-Dimethoxyphenyl)-3-methyl-4H-furo[3,2-c]pyran-
4-thione (30)
A mixture of 25 (0.1 mmol) and Lawesson’s reagent (81 mg,
0.2 mmol) in dry toluene (5 mL) was heated to reflux for 12 h. Tol-
uene was removed and the red residue was dissolved in EtOAc and
partitioned with H2O. The organic phase was separated and dried
over Na2SO4. Removal of solvent in vacuo afforded an oily, residue
which was purified by column chromatography resulting in a yel-
low solid.
60% Yield; mp 147–149 °C; 1H NMR (300 MHz, CDCl3, ppm): d
2.46 (d, J = 1.5 Hz, 3H, CH3), 3.87 (s, 6H, OCH3), 6.57 (t, J = 2.1 Hz,
1H, C7–H), 7.02 (s, 1H, aromatic), 7.03 (s, 1H, aromatic), 7.17 (s,
1H, aromatic), 7.32 (d, J = 1.5 Hz, 1H, OCH); HRMS for (M++H):
calcd 303.0691, found: 303.0702.
13. Raghavan, B.; Balasubramanian, R.; Steele, J. C.; Sackett, D. L.; Fecik, R. A. J. Med.
Chem. 2008, 51, 1530.
14. Wolff, M. E. Burger’s Medicinal Chemistry and Drug Discovery, 5th ed.; Wiley
Interscience, 1994; Vol. 1, p 1064.
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K. H. J. Med. Chem. 2006, 49, 5631.
5.4. Methodology of MTT assay
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S. L.; Lee, K. H. Bioorg. Med. Chem. Lett. 2009, 19, 6289.
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G. M.; Morgan, C. D.; Shih, R. A.; Mueller, K. L.; Seurer, L. M.; Johnson, E. W.;
Hsung, R. P. Tetrahedron 1999, 55, 13683.
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20. Boeckman, R. K., Jr.; Ge, P.; Reed, J. E. Org. Lett. 2001, 3, 3647.
The MTT assay was used to independently test the potent activ-
ity of 25 against SK-BR-3 and to investigate antitumor selectivity
versus activity against two normal breast cancer cell lines:
184A1 and MCF10A (CRL-10317) purchased from ATCC (Rockville,
MD). Cells were seeded into 96-well plates at a density of 5000
cells per well in the recommended growth medium. The drug
was dissolved in DMSO. The drug was added into wells after over-