Design and synthesis of 2,3-dihydro- and 5-chloro-2,3-dihydro-naphtho-[1,2-b]furan-2-carboxylic acid N-(substitutedphenyl)amide analogs and their biological activities as inhibitors of NF-κB activity and anticancer agents

Article abstract of DOI:10.1007/s12272-016-0737-5

A series of 2,3-dihydro- and 5-chloro-2,3-dihydro-naphtho-[1,2-b]furan-2-carboxylic acid N-(substitutedphenyl)amide analogs (1a–k and 2a–i) were designed and synthesized for developing novel naphthofuran scaffolds as anticancer agents and inhibitors of NF-κB activity. Compound 1d, which had a 4′-chloro group on the N-phenyl ring, exhibited inhibitory activity of NF-κB. Compound 2g, which had a 5′-chloro group on the naphthofuran ring and a 3′,5′-bistrifluoromethane group on the N-phenyl ring, had the best NF-κB inhibitory activity. In addition, the novel analogs exhibited potent cytotoxicity at low concentrations against HCT-116, NCI-H23, and PC-3 cell lines. The two electron-withdrawing groups, especially at the 3′,5′-position on the N-phenyl ring, increased anticancer activity and NF-κB inhibitory activity. However, only 5-chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic N-(3′,5′-bis(trifluoromethyl)phenyl)amide (2g) exhibited both outstanding cytotoxicity and NF-κB inhibitory activities. This novel lead scaffold may be helpful for investigation of new anticancer agents by inactivation of NF-κB.

Full text of DOI:10.1007/s12272-016-0737-5

Arch. Pharm. Res.
DOI 10.1007/s12272-016-0737-5
RESEARCH ARTICLE
Design and synthesis of 2,3-dihydro- and 5-chloro-2,3-
dihydro-naphtho-[1,2-b]furan-2-carboxylic acid
N-(substitutedphenyl)amide analogs and their biological activities
as inhibitors of NF-jB activity and anticancer agents
Minho Choi1 Hyeju Jo1 Dayoung Kim1 Jieun Yun2 Jong-Soon Kang2
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Youngsoo Kim1 Jae-Kyung Jung1 Jin Tae Hong1 Jungsook Cho3
Jae-Hwan Kwak4 Heesoon Lee1
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•
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Received: 29 February 2016 / Accepted: 14 March 2016
Ó The Pharmaceutical Society of Korea 2016
Abstract A series of 2,3-dihydro- and 5-chloro-2,3-di-
hydro-naphtho-[1,2-b]furan-2-carboxylic acid N-(substi-
tutedphenyl)amide analogs (1a–k and 2a–i) were designed
and synthesized for developing novel naphthofuran scaf-
folds as anticancer agents and inhibitors of NF-jB activity.
Compound 1d, which had a 40-chloro group on the N-phenyl
ring, exhibited inhibitory activity of NF-jB. Compound 2g,
which had a 50-chloro group on the naphthofuran ring and a
30,50-bistrifluoromethane group on the N-phenyl ring, had
the best NF-jB inhibitory activity. In addition, the novel
analogs exhibited potent cytotoxicity at low concentrations
against HCT-116, NCI-H23, and PC-3 cell lines. The two
electron-withdrawing groups, especially at the 30,50-position
on the N-phenyl ring, increased anticancer activity and NF-
jB inhibitory activity. However, only 5-chloro-2,3-dihy-
dronaphtho[1,2-b]furan-2-carboxylic N-(30,50-bis(trifluo-
romethyl)phenyl)amide (2g) exhibited both outstanding
cytotoxicity and NF-jB inhibitory activities. This novel lead
scaffold may be helpful for investigation of new anticancer
agents by inactivation of NF-jB.
Keywords Anticancer activity Á Inhibition of NF-jB
transcriptional activity Á 2,3-Dihydronaphtho-[1,2-b]furan
scaffolds
Introduction
Cancer is a large group of diseases that represent out-of-
control proliferation of a particular cell type caused by
oncogenes such as mutated genes, which allow unregulated
cell growth (Campisi 2001). Oncogenes generate a large
number of proteins involved in cancer cell signaling, which
is triggered by the exposure to carcinogens such as chemi-
cals, viruses, ultraviolet light, and radicals (Levy 1966;
McCann et al. 1975; Sun 1990; Brash et al. 1991). In addi-
tion, oncogene up-regulation can lead to uncontrolled tumor
growth through aberrant activation of cell signaling path-
ways by hormones, cytokines, and chemokines (Mallette
et al. 2007; Mantovani et al. 2008; Borsig et al. 2014).
Oncogenes are also activated by various transcription factors
such as nuclear factor-jB (NF-jB) and signal transducer and
activator of transcription (STAT) family proteins (Silva
2004; Gilmore 2006; Grivennikov and Karin 2009). NF-jB,
an inducible transcription factor, is present in an inactive
form in the cytoplasm in most cell types. Upon activation by
external stimuli or abnormal signaling, NF-jB translocates
to the nucleus and binds DNA or oncogenes. NF-jB binding
regulates the expression of genes related to inflammation and
unregulated cell growth (Gilmore 2006). Therefore, tran-
scription factors such as NF-jB are regarded as key regula-
tors of tumorigenesis. STAT3 is also one of transcription
factors increasing the expression of proliferative and sur-
vival genes in cancers (Grivennikov and Karin 2009).
& Jae-Hwan Kwak
jhkwak@ks.ac.kr
& Heesoon Lee
medchem@chungbuk.ac.kr
1
Department of Pharmacy, Chungbuk National University,
Chungbuk 28644, Republic of Korea
2
Korea Research Institute of Bioscience and Biotechnology,
Ochang 28116, Republic of Korea
3
College of Pharmacy, Dongguk University, Goyang 10326,
Republic of Korea
4
The STAT family, which consists of 7 proteins (STAT1–
STAT4, STAT5A, STAT5B, and STAT6) in humans, is
College of Pharmacy, Kyungsung University, Busan 48434,
Republic of Korea
123

M. Choi et al.
considered to be important for cell survival and proliferation
(Yu and Jove 2004). STAT3 regulates cell proliferation, cell
cycle progression, apoptosis, angiogenesis, and immune
evasion and influences cancer stem cells (Xiong et al. 2014).
In interleukin-6 (IL-6)–stimulated hepatocytes, STAT3 acts
as a DNA-binding factor mediated by the IL-6 family of
cytokines and growth factors including epidermal growth
factor (EGF) and hepatocyte growth factor (HGF) (Zhang
et al. 2002; Aggarwal et al. 2009; Rebe et al. 2013). In breast,
colon, gastric, lung, head and neck, skin, and prostate tumor
cells, STAT3 is activated through phosphorylation of Janus
kinase (JAK), which subsequently phosphorylates STAT3
(Siddiquee and Turkson 2008). Aberrant STAT3 activation in
cancer cells increases resistance to apoptosis and chemother-
apies aimed at initiating apoptosis (Mali 2015). Therefore, to
suppress the proliferation of human cancer cells, various
studies suggested agents that regulate oncogenes through
inactivation of transcription factors such as NF-jB and
STAT3 (Darnell 2002).
coumarin analogs and CX4152 also have anticancer activity
(Chougala et al. 2015; Ha et al. 2015). Therefore, in this
study we have designed and synthesized novel 2,3-dihy-
dronaphtho-[1,2-b]furan-2-carboxamide analogs to identify
those with most potent anticancer and NF-jB inhibitory
activities (Fig. 1c).
Materials and methods
Chemistry
All commercial chemicals were used as obtained and all
solvents were purified prior to use by applying standard
procedures. Air and moisture sensitive reactions were per-
formed under an nitrogen atmosphere. All the products
obtained were purified by column chromatography using
silica gel (100–200 mesh). Thin layer chromatography was
performed on E Merck silica gel GF-254 pre-coated plates;
identification was performed under UV illumination. Flash
column chromatography was performed on E Merck silica
gel (230–400 mesh). Hexane was used as a co-eluent. 1H and
13C NMR were recorded in Bruker 400, 500 and 100,
125 MHz spectrometer respectively. The chemical shifts are
reported in ppm downfield to TMS (d = 0) for 1H NMR and
relative to the central CDCl3 resonance (d = 77.0) for 13C
NMR. IR spectra recorded on JASCO FT/IR-4100. High
resolution mass spectra (HRMS) was obtained by using
Agilent 1290 Infinity HPLC system and Agilent 6530
Q-TOF LC/MS system. Melting points were recorded on an
Electro thermal IA 9100 apparatus and are uncorrected.
As part of our continuous efforts to design and develop
novel scaffolds with anticancer activity and NF-jB inhibi-
tory activity, we recently reported a series of novel benzo-
furan- and 2,3-dihydrobenzofuran-2-carboxamide analogs
and explored their NF-jB inhibitory activities and cytotox-
icity against various human cancer cell lines (Choi et al.
2015). Moreover, we reported that 5-chloro-2,3-dihydron-
aphtho [1,2-b]furan-2-carboxylic N-(30,50-bis(trifluoromethyl)
phenyl)amide (2g) significantly inhibits liver tumor growth
through activation of hepatocyte nuclear factor 4a (HNF4a)
and inhibition of STAT3 (Kwon et al. 2015).
In our previous studies, chroman-, indoline- and dihy-
drobenzofuran-2-carboxamide analogs were designed and
synthesized (Fig. 1a), and their NF-jB inhibitory activity
and anticancer effects were evaluated (Kwak et al. 2007,
2008, 2009a, b, 2010; Choi et al. 2015). Among these
analogs, KL-1156, the first hit compound, was reported as
an inhibitor of NF-jB translocation to the nucleus in LPS-
stimulated RAW 264.7 macrophage cells (Kim et al. 2004).
Recently, it was confirmed that benzofuran- and 2,3-dihy-
drobenzofuran-2-carboxylic acid N-(substitutedphenyl)amide
analogs inhibited NF-jB in LPS-stimulated RAW 264.7 cells
and had anticancer effects against six human cancer cell
lines: ACHN (renal), HCT15 (colon), MM231 (breast),
NUGC-3 (gastric), NCI-H23 (lung), and PC-3 (prostate). To
obtain compounds with potent NF-jB inhibitory and anti-
cancer activities, we designed novel scaffolds on the basis of
them (Choi et al. 2015).
1-(Allyloxy)naphthalene
A mixture of 1-naphthol (3) (300 mg, 2.08 mmol), K2CO3
(575 mg, 4.16 mmol) and acetone (6.00 mL) was stirred for
30 min, then allyl bromide (0.216 mL, 2.50 mmol) was
added at room temperature. The mixture was stirred for 2 h at
50–60 °C. After 2 h, the solvent was evaporated. Purification
via flash column chromatography (EtOAc/hexanes = 1:9)
provided 1-(allyloxy)naphthalene (98.4 %). 1H NMR
(CDCl3, 400 MHz) d 8.32 (m, 1H), 7.80 (m, 1H), 7.50–7.46
(m, 2H), 7.43 (d, 1H, J = 7.8 Hz), 7.36 (t, 1H, J = 7.8 Hz),
6.82 (d, 1H, J = 7.8 Hz), 6.19 (tdd, 1H, J = 17.3, 10.9,
5.2 Hz), 5.55 (dd, 1H, J = 17.3, 1.6 Hz), 5.35 (dd, 1H,
J = 10.5, 1.6 Hz), 4.73 (dd, 2H, J = 5.2, 1.6 Hz).
In many studies, various compounds containing the
dihydronaphthofuran moiety as a core structure exhibited
anti-proliferation effects against human cancer cell lines. For
instance, 2-nitronaphthofuran and its analogs exhibit anti-
cancer effects against hepatoma cells by activating HNF4a
(Fig. 1b) (Le Guevel et al. 2009). 3-(3-Naphthofuranyl)-
2-Allylnaphthalen-1-ol (4)
A mixture of 1-(allyloxy)naphthalene (205 mg, 1.11 mmol)
and xylene (1.00 mL) was stirred for 16 h. After stirring for
16 h, the solvent was evaporated then purification via flash
column chromatography (EtOAc/hexanes = 1:9) provided
123

Design and synthesis of 2,3-dihydro- and 5-chloro-2,3-dihydro-naphtho-[1,2-b]furan-2-carboxylic…
Fig. 1 a Structures of chroman, indoline, and 2,3-dihydrobenzofuran. b Biologically active scaffolds of naphthobenzofuran. c Design of 2,3-
dihydronaphtho-[1,2-b]furan-2-carboxamide analogs
2-allylnaphthalen-1-ol (87.6 %). 1H NMR (CDCl3,
400 MHz) d 8.17 (m, 1H), 7.78 (m, 1H), 7.48–7.44 (m, 2H),
7.41 (d, 1H, J = 8.4 Hz), 7.22 (d, 1H, J = 8.4 Hz), 6.09
(tdd, 1H, J = 17.1, 10.2, 6.2 Hz), 5.26 (dd, 1H, J = 17.1,
1.6 Hz), 5.24 (dd, 1H, J = 10.2, 1.6 Hz), 3.58 (dd, 2H,
J = 6.2, 1.6 Hz).
then washed with H2O (3 9 20 mL) and brine (20 mL),
dried over anhydrous MgSO4. Purification via flash column
chromatography (EtOAc/hexanes = 1:8) provided 2-((1-
(benzyloxy)naphthalen-2-yl)methyl)oxirane (72.9 %). 1H
NMR (CDCl3, 500 MHz) d 8.14 (d, 1H, J = 8.5 Hz), 7.86
(d, 1H, J = 8.5 Hz), 7.64 (d, 1H, J = 8.5 Hz), 7.55 (d, 2H,
J = 7.4 Hz), 7.52–7.43 (m, 5H), 7.39 (t, 1H, J = 7.4 Hz),
5.07 (s, 2H), 3.21 (m, 1H), 3.10 (dd, 1H, J = 14.4, 5.4 Hz),
3.04 (dd, 1H, J = 14.4, 5.4 Hz), 2.79 (dd, 1H, J = 5.1,
4.3 Hz), 2.59 (dd, 1H, J = 5.1, 2.5 Hz).
2-Allyl-1-(benzyloxy)naphthalene
A
mixture of 2-allylnaphthalen-1-ol (4) (436 mg,
2.37 mmol), K2CO3 (655 mg, 4.74 mmol) and acetone
(50.0 mL) was stirred for 30 min. Then benzyl bromide
(0.582 mL, 2.67 mmol) was added at room temperature. The
mixture was stirred for 8 h at 50–60 °C. After stirring for 8 h,
the solvent was evaporated and the reaction mixture was
diluted by DCM. The mixture was washed with H2O
(3 9 20 mL), dried over anhydrous MgSO4. Purification via
flash column chromatography (EtOAc/hexanes = 1:9) pro-
(2,3-Dihydronaphtho[1,2-b]furan-2-yl)methanol (6)
A mixture of 2-((1-(benzyloxy)naphthalen-2-yl)methyl)oxi-
rane (5) (631 mg, 2.17 mmol), 10 % Pd/C (39.5 mg),
K2CO3 (8.98 mg, 0.065 mmol), Et3N (catalytic amount) and
MeOH (20.0 mL) was stirred for 8 h at room temperature.
After stirring for 8 h, the reaction mixture was filtered by
Celite then solvent was evaporated. Purification via flash
column chromatography (EtOAc/hexanes = 1:3) provided
(2,3-dihydronaphtho[1,2-b]furan-2-yl)methanol (93.7 %).
1H NMR (CDCl3, 500 MHz) d 7.96 (d, 1H, J = 8.3 Hz),
7.81 (d, 1H, J = 8.3 Hz), 7.46–7.41 (m, 2H), 7.39 (d, 1H,
J = 8.3 Hz), 7.32 (d, 1H, J = 8.3 Hz), 5.14 (m, 1H), 3.95
(dd, 1H, J = 12.3, 3.2 Hz), 3.84 (dd, 1H, J = 12.3, 6.3 Hz),
3.44 (dd, 1H, J = 15.4, 9.7 Hz), 3.20 (dd, 1H, J = 15.4,
7.6 Hz).
1
vided 2-allyl-1-(benzyloxy)naphthalene (98.7 %). H NMR
(CDCl3, 500 MHz) d 8.19 (d, 1H, J = 8.4 Hz), 7.88 (d, 1H,
J = 8.4 Hz), 7.66 (d, 1H, J = 8.4 Hz), 7.61 (d, 2H,
J = 8.4 Hz), 7.55–7.48 (m, 4H), 7.43 (t, 1H, J = 8.4 Hz),
7.39 (d, 1H, J = 8.4 Hz), 6.07 (tdd, 1H, J = 17.7, 9.4,
6.4 Hz), 5.15 (dd, 1H, J = 17.7, 1.6 Hz), 5.15 (dd, 1H,
J = 9.4, 1.6 Hz), 5.08 (s, 2H), 3.66 (dd, 2H, J = 6.4, 1.6 Hz).
2-((1-(Benzyloxy)naphthalen-2-yl)methyl)oxirane (5)
A mixture of 2-allyl-1-(benzyloxy)naphthalene (455 mg,
1.66 mmol), m-CPBA (891 mg, 3.97 mmol) and dichlor-
omethane (20.0 mL) was stirred for 6 h at 50–60 °C. After
stirring for 6 h, the reaction mixture was diluted by DCM
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid (7)
To a stirred solution of (2,3-dihydronaphtho[1,2-b]furan-2-
yl)methanol (6) (194 mg, 0.970 mmol) in CH3CN/H2O (1/
123

M. Choi et al.
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(4-
chlorophenyl)amide (1d) Yield 17.1 %; IR 3057, 1684,
743, 669 cm-1; MS(ESI) m/z 346.1 (M ? Na?); 1H NMR
(CDCl3, 500 MHz) d 8.04 (d, 1H, J = 8.5 Hz), 7.87 (d,
1H, J = 8.5 Hz), 7.55 (t, 1H, J = 8.5 Hz), 7.52–7.49 (m,
4H), 7.36 (d, 1H, J = 8.5 Hz), 7.28 (d, 2H, J = 8.8 Hz),
5.47 (dd, 1H, J = 11.0, 6.2 Hz), 3.86 (dd, 1H, J = 16.0,
11.0 Hz), 3.70 (dd, 1H, J = 16.0, 6.2 Hz); 13C NMR
(CDCl3, 100 MHz) d 170.0, 153.1, 135.3, 134.0, 129.9,
129.2, 129.1, 128.2, 126.1, 122.6, 122.2, 121.3, 120.6,
120.4, 118.7, 81.1, 35.0.
1, 2 mL) was added TEMPO (30.3 mg, 0.194 mmol),
BAIB (687 mg, 2.13 mmol). After stirring for 5–6 h, the
mixture was basicfied with sat. NaHCO3 to pH 10–11 and
washed with diethyl ether (10 ml 9 3). The combined
extracts were acidified with 1 N HCl to pH 2–3 then the
mixture was extracted with EtOAc (3 9 20 mL). The
combined extracts were dried over anhydrous Na2SO4.
After evaporation, the product was dried in vacuo to afford
the desired 2,3-dihydronaphtho[1,2-b]furan-2-carboxylic
acid (65.8 %) as a brown oil. 1H NMR (CDCl3, 500 MHz)
d 8.01 (d, 1H, J = 8.2 Hz), 7.83 (d, 1H, J = 8.2 Hz), 7.50-
7.44 (m, 3H), 7.32 (d, 1H, J = 8.2 Hz), 5.46 (dd, 1H,
J = 11.0, 6.6 Hz), 3.81 (dd, 1H, J = 15.7, 11.1 Hz), 3.62
(dd, 1H, J = 15.7, 6.6 Hz).
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(3,4-
dichlorophenyl)amide (1e) Yield 15.3 %; m. p. 146.9–
150.8 °C; IR 2923, 1684, 743, 669 cm-1; MS(ESI) m/z
358.2 (M ? H?); 1H NMR(CDCl3, 400 MHz) d 8.04(d, 1H,
J = 8.1 Hz), 7.87 (d, 1H, J = 8.0 Hz), 7.80 (s, 1H),
7.55–7.49 (m, 3H), 7.37–7.34 (m, 3H), 5.46 (dd, 1H,
J = 11.0, 6.5 Hz), 3.86 (dd, 1H, J = 16.0, 11.0 Hz), 3.69
(dd, 1H, J = 16.0, 6.5 Hz); 13C NMR (CDCl3, 100 MHz) d
170.1, 153.0, 136.2, 134.0, 132.9, 130.5, 128.3, 128.1, 126.2,
122.6, 122.5, 122.3, 121.7 120.6, 120.4, 119.2, 118.6, 81.1,
35.0.
General procedure for the synthesis of 2,3-
dihydronaphtho[1,2-b]furan-2-carboxylic acid N-
(substitutedphenyl)amide analogs (1a–1b, 1d–g and 1i)
To a mixture of acid (1.0 eq) and 1,10-carbodiimidazole
(1.2 eq) in anhydrous THF was stirred for 1 h then sub-
stituted aniline (1.0 eq) was added at room temperature.
After stirring for 12–16 h, the reaction mixture was washed
with H2O (3 9 10 mL) then acidified with 1 N-HCl to pH
2. The mixture was extracted with EtOAc (3 9 10 mL)
and the combined extracts were dried over anhydrous
Na2SO4. After evaporation, the residue was purified by
column chromatography (EtOAc/hexanes = 1:3–1:10).
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(3,5-
dichlorophenyl)amide
(1f) Yield
28.2 %;
m.
p. 169.9–170.8 °C; IR 2924, 1684, 750, 669 cm-1
;
MS(ESI) m/z 358.1 (M ? H?); 1H NMR (CDCl3,
400 MHz) d 8.04 (d, 1H, J = 8.2 Hz), 7.87 (d, 1H,
J = 8.2 Hz), 7.55 (td, 1H, J = 8.2, 1.7 Hz), 7.52–7.49 (m,
4H), 7.35 (d, 1H, J = 8.2 Hz), 7.10 (t, 1H, J = 1.7 Hz),
5.45 (dd, 1H, J = 11.0, 6.4 Hz), 3.85 (dd, 1H, J = 16.0,
11.0 Hz), 3.69 (dd, 1H, J = 16.0, 6.4 Hz); 13C NMR
(CDCl3, 100 MHz) d 170.2, 153.0, 138.5, 135.3, 134.0,
128.3, 126.2, 126.1, 124.8, 122.5, 122.3, 120.6, 120.4,
118.6, 118.2, 81.0, 35.0.
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-pheny-
lamide (1a) Yield 30.6 %; m. p. 144.1–149.4 °C; IR 3057,
1
1683, 751 cm-1; LRMS (ESI) m/z 312.2 (M ? Na?); H
NMR (CDCl3, 400 MHz) d 8.06 (d, 1H, J = 8.2 Hz), 7.87
(d, 1H, J = 8.2 Hz), 7.56–7.54 (m, 3H), 7.51–7.49 (m, 2H),
7.37–7.30 (m, 3H), 7.12 (t, 1H, J = 7.5 Hz), 5.48 (dd, 1H,
J = 10.9, 6.5 Hz), 3.86 (dd, 1H, J = 16.0, 10.9 Hz), 3.72
(dd, 1H, J = 16.0, 6.5 Hz); 13C NMR (CDCl3, 100 MHz) d
169.9, 153.2, 136.8, 134.0, 129.1, 128.2, 126.1, 126.2, 124.9,
122.7, 122.1, 120.7, 120.4, 120.0, 118.8, 81.2, 35.0.
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(3,5-
bis(trifluoromethyl)phenyl)amide (1g) Yield 9.8 %; m.
p. 163.1–165.7 °C; IR 2924, 1683, 1177, 1131, 752 cm-1
;
MS(ESI) m/z 448.2 (M ? Na?); 1H NMR (CDCl3,
500 MHz) d 8.08–8.07 (m, 3H), 7.88 (d, 1H, J = 8.2 Hz),
7.63 (s, 1H), 7.58 (t, 1H, J = 8.2 Hz), 7.53–7.50 (m, 2H),
7.36 (d, 1H, J = 8.3 Hz), 5.50 (dd, 1H, J = 11.0, 6.7 Hz),
3.89 (dd, 1H, J = 16.0, 11.0 Hz), 3.72 (dd, 1H, J = 16.0,
6.7 Hz); 13C NMR (CDCl3, 100 MHz) d 170.6, 153.0,
138.2, 134.1, 132.7, 132.3, 128.3, 126.3, 126.2, 122.5,
122.4, 120.6, 120.4, 119.8 118.5 118.2, 81.0, 35.0.
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(4-
methoxyphenyl)amide
(1b) Yield
19.5 %;
m.
p. 142.3–146.2 °C; IR 3061, 1681, 1245, 742 cm-1
;
MS(ESI) m/z 342.2 (M ? Na?); 1H NMR (CDCl3,
400 MHz) d 8.05 (d, 1H, J = 8.2 Hz), 7.86 (d, 1H,
J = 8.2 Hz), 7.56–7.47 (m, 3H), 7.45 (d, 2H,
J = 9.0 Hz), 7.36 (d, 1H, J = 8.2 Hz), 6.85 (d, 2H,
J = 9.0 Hz), 5.47 (dd, 1H, J = 10.9, 6.5 Hz), 3.85 (dd,
1H, J = 16.0, 10.9 Hz), 3.78 (s, 2H), 3.78 (s, 3H) 3.71
(dd, 1H, J = 16.0, 6.5 Hz); 13C NMR (CDCl3, 100 MHz)
d 169.6, 156.8, 153.2, 134.0, 129.9, 129.8, 128.2, 126.0,
122.7, 122.0, 121.8, 120.7, 120.4, 118.9, 114.2, 81.2,
55.5, 35.0.
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(2-tri-
fluoromethylphenyl)amide (1h) Yield 17.9 %; m.
p. 154.8–158.7 °C; IR 3057, 1700, 1171, 1104, 744 cm-1
;
MS(ESI) m/z 358.2 (M ? H?); 1H NMR (CDCl3,
123

Design and synthesis of 2,3-dihydro- and 5-chloro-2,3-dihydro-naphtho-[1,2-b]furan-2-carboxylic…
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(2-hy-
400 MHz) d 8.41 (d, 1H, J = 8.3 Hz), 8.05 (d, 1H,
J = 8.3 Hz), 7.85 (d, 1H, J = 8.3 Hz), 7.59–7.53 (m, 3H),
7.49 (d, 1H, J = 8.3 Hz), 7.49 (t, 1H, J = 8.3 Hz), 7.35 (d,
1H, J = 8.3 Hz), 7.21 (t, 1H, J = 8.3 Hz), 5.50 (dd, 1H,
J = 11.2, 5.6 Hz), 3.88 (dd, 1H, J = 16.0, 11.2 Hz), 3.72
(dd, 1H, J = 16.0, 5.6 Hz); 13C NMR (CDCl3, 100 MHz) d
170.5, 153.0, 134.6, 134.5, 134.0, 133.0, 128.0, 126.2, 126.1,
124.5, 123.0, 122.5, 122.4, 122.2, 120.8, 120.5, 118.4, 81.2,
34.9.
droxyphenyl)amide(1j) Yield 4.7 %; m. p. 170.5–179.9 °C;
IR 3361, 3056, 1648, 1120, 748 cm-1; MS(ESI) m/z 328.3
1
(M ? Na?); H NMR (CDCl3, 400 MHz) d 8.05 (d, 1H,
J = 7.9 Hz), 7.87 (d, 1H, J = 7.9 Hz), 7.57–7.48 (m, 4H),
7.36 (d, 1H, J = 8.2 Hz), 7.12 (t, 1H, J = 8.2 Hz), 7.06 (d,
1H, J = 8.2 Hz), 7.00 (d, 1H, J = 8.2 Hz), 6.85 (t, 1H,
J = 8.2 Hz), 5.54 (dd, 1H, J = 11.0, 6.4 Hz), 3.89 (dd, 1H,
J = 16.1, 11.0 Hz), 3.72 (dd, 1H, J = 16.1, 6.4 Hz); 13C
NMR (CDCl3, 100 MHz) d 171.5, 170.8, 153.0, 148.5,
134.1, 128.2, 127.5, 126.2, 126.1, 124.4, 122.5, 122.4, 122.3,
120.6, 120.4, 119.7, 118.5, 80.8, 35.1.
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(4-tri-
fluoromethylphenyl)amide
(1i) Yield
14.9 %;
m.
p. 135.0–137.8 °C; IR 3057, 1685, 1164, 1113, 741 cm-1
;
MS(ESI) m/z 715.8 (2 M ? H?); 1H NMR (CDCl3,
400 MHz) d 8.06 (d, 1H, J = 8.1 Hz), 7.88 (d, 1H,
J = 8.1 Hz), 7.69 (d, 2H, J = 8.5 Hz), 7.57 (d, 2H,
J = 8.5 Hz), 7.56 (t, 1H, J = 8.1 Hz), 7.53–7.49 (m, 2H),
7.36 (d, 1H, J = 8.1 Hz), 5.49 (dd, 1H, J = 11.0, 6.4 Hz),
3.87 (dd, 1H, J = 16.0, 11.0 Hz), 3.71 (dd, 1H, J = 16.0,
6.4 Hz); 13C NMR (CDCl3, 100 MHz) d 170.3, 153.0, 139.8,
134.0, 128.3, 126.5, 126.4, 126.3, 126.2, 126.1, 122.6, 122.3,
120.6, 120.4, 119.7, 118.7, 81.1, 35.0.
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(4-
hydroxyphenyl)amide (1k) Yield 4.6 %; IR 3362, 2924,
1683, 1219, 735 cm-1; MS(ESI) m/z 328.1 (M ? Na?);
1H NMR (CDCl3, 400 MHz) d 8.04 (d, 1H, J = 8.2 Hz),
7.87 (d, 1H, J = 8.2 Hz), 7.55–7.47 (m, 3H), 7.39 (d, 2H,
J = 8.9 Hz), 7.36 (d, 1H, J = 8.2 Hz), 6.78 (d, 2H,
J = 8.9 Hz), 5.47 (dd, 1H, J = 11.0, 6.5 Hz), 3.85 (dd,
1H, J = 16.1, 11.0 Hz), 3.72 (dd, 1H, J = 16.1, 6.5 Hz);
13C NMR (CDCl3, 100 MHz) d 171.3, 169.9, 153.2, 134.0,
129.5, 128.2, 126.1, 125.4, 122.7, 122.3, 122.0, 120.7,
120.4, 118.8, 115.8, 81.2, 35.0.
General procedure for the synthesis of 2,3-
dihydronaphtho[1,2-b]furan-2-carboxylic acid N-
(substitutedphenyl)amide analogs (1c, 1j and 1k)
General procedure for the synthesis of 5-chloro-2,3-
dihydronaphtho-[1,2-b]furan-2-carboxylic acid N-
(substitutedphenyl)amide derivatives (2 series)
To a mixture of acid (1.0 eq), HOBt (1.0 eq) and EDCI
(1.0 eq) in N,N-dimethylformamide was stirred for 10 min.
The substituted aniline (1.0 eq) was added then after
10 min DIPEA (2.0 eq) at room temperature. After stirring
for 12–16 h, the reaction mixture was washed with H2O
(3 9 10 mL) then acidified with 1 N HCl to pH 2. The
mixture was extracted with EtOAc (3 9 10 mL) and the
combined extracts were dried over anhydrous Na2SO4.
After evaporation, the residue was purified by column
chromatography (EtOAc/hexanes = 1:3–1:9).
To a stirred solution of (2,3-dihydronaphtho[1,2-b]furan-2-
yl)methanol (221 mg, 1.10 mmol) in THF/sat. NaHCO3 (1/1,
2 mL) was added TEMPO (34.2 mg, 0.219 mmol), KBr
(39.2 mg, 0.329 mmol), and NaOCl aq (4.8 mL) at 0 °C.
After stirring for 1–6 h, the mixture was was washed with
diethyl then the combined extracts were acidified with
1 N HCl to pH 2–3. The mixture was extracted with EtOAc
(3 9 10 mL) and the combined extracts were dried over
anhydrous Na2SO4. After evaporation, to a mixture of
5-chloro-2,3-dihydronaphtho-[1,2-b]furan-2-carboxylic acid
(1.0 eq) and 1,10-carbodiimidazole (1.2 eq) in anhydrous
THF was stirred for 1 h then substituted aniline (1.0 eq) was
added at room temperature the reaction mixture was washed
with H2O (3 9 10 mL) then acidified with 1 N-HCl to pH 2.
The mixture was extracted with EtOAc (3 9 10 mL) and the
combined extracts were dried over anhydrous MgSO4. After
evaporation, the residue was purified by column chromatog-
raphy (EtOAc/hexanes = 1:3–1:10).
2,3-Dihydronaphtho[1,2-b]furan-2-carboxylic acid N-(3-chlor-
ophenyl)amide (1c) Yield 5.8 %; m. p. 108.3–113.9 °C; IR
2923, 1684, 745, 650 cm-1
; MS(ESI) m/z 346.2
1
(M ? Na?); H NMR (CDCl3, 400 MHz) d 8.05 (d, 1H,
J = 8.2 Hz), 7.86 (d, 1H, J = 8.2 Hz), 7.66 (t, 1H,
J = 2.0 Hz), 7.58 (td, 1H, J = 8.2, 2.0 Hz), 7.50 (d, 1H,
J = 8.2 Hz), 7.52 (td, 1H, J = 8.2, 2.0 Hz), 7.40 (dd, 1H,
J = 8.2, 2.0 Hz), 7.35 (d, 1H, J = 8.2 Hz), 7.23 (t, 1H,
J = 8.2 Hz), 7.10 (dd, 1H, J = 8.2, 2.0 Hz), 5.46 (dd, 1H,
J = 11.0, 6.6 Hz), 3.82 (dd, 1H, J = 16.0, 10.8 Hz), 3.71
(dd, 1H, J = 16.0, 6.6 Hz); 13C NMR (CDCl3, 100 MHz) d
170.0, 153.1, 135.3, 134.7, 134.0, 129.9, 129.1, 128.2, 126.2,
126.1, 124.9, 122.6, 122.2, 121.3, 120.6, 120.4, 118.7, 118.0,
81.1, 35.0.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-phenylamide (2a) Yield 28.1 %; m. p. 173.3–176.7 °C;
IR 3189, 3070, 1666, 755 cm-1; MS(ESI) m/z 326.2
123

M. Choi et al.
1
(M ? Na?); H NMR (CDCl3, 400 MHz) d 8.22 (m, 1H),
(CDCl3, 100 MHz) d 169.5, 153.0, 136.1, 133.0, 130.6,
127.9, 127.6, 127.3, 127.0, 126.3, 125.3, 122.8, 121.7,
121.1, 121.0, 119.2, 144.9, 81.3, 34.8.
8.07 (m, 1H), 7.67 (s, 1H), 7.64–7.60 (m, 2H), 7.54 (d, 2H,
J = 8.7 Hz), 7.32 (t, 2H, J = 8.4 Hz), 7.13 (t, 1H,
J = 8.4 Hz), 5.49 (dd, 1H, J = 10.8, 6.6 Hz), 3.86 (dd,
1H, J = 16.2, 10.8 Hz), 3.73 (dd, 1H, J = 16.2, 6.6 Hz);
13C NMR (CDCl3, 100 MHz) d 169.9, 153.2, 136.8, 134.0,
129.1, 128.2, 126.9, 126.1, 124.9, 122.7, 122.1, 120.7,
120.0, 119.7, 118.8, 81.2, 35.0.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(3,5-dichlorophenyl)amide (2f) Yield 21.7 %; m.
p. 158.3–163.7 °C; IR 3306, 1684, 1187, 737, 668 cm-1
;
MS(ESI) m/z 416.2 (M ? Na?); 1H NMR (CDCl3,
400 MHz) d 8.23 (m, 1H), 8.04 (m, 1H), 7.67 (s, 1H),
7.64–7.61 (m, 2H), 7.52 (d, 2H, J = 1.8 Hz), 7.12 (t, 1H,
J = 1.8 Hz), 5.47 (dd, 1H, J = 11.0, 6.5 Hz), 3.86 (dd,
1H, J = 16.2, 11.0 Hz), 3.70 (dd, 1H, J = 16.2, 6.5 Hz);
13C NMR (CDCl3, 100 MHz) d 169.6, 152.9, 138.4, 135.4,
131.9, 127.9, 127.6, 127.0, 126.3, 125.0, 121.3, 121.1,
119.4, 118.3, 115.0, 81.2, 34.7.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(4-methoxyphenyl)amide (2b) Yield 42.7 %; m.
p. 156.3–157.1 °C; IR 3313, 1656, 1177, 736 cm-1
;
MS(ESI) m/z 379.9 (M ? Na?); 1H NMR (CDCl3,
400 MHz) d 8.20 (m, 1H), 7.84 (m, 1H), 7.62 (s, 1H),
7.59–7.53 (m, 2H), 6.89 (d, 2H, J = 8.6 Hz), 6.61 (d, 2H,
J = 8.6 Hz), 5.31 (dd, 1H, J = 11.1, 5.9 Hz), 3.73 (dd, 1H,
J = 16.0, 11.1 Hz), 3.71 (s, 1H), 3.56 (dd, 1H, J = 16.0,
5.9 Hz); 13C NMR (CDCl3, 100 MHz) d 171.1, 158.3, 153.3,
131.7, 130.2, 129.6, 127.6, 127.4, 126.5, 125.0, 124.3, 121.3,
121.2, 119.7, 114.0, 81.3, 55.2, 34.6.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(3,5-ditrifluoromethylphenyl)amide (2g) Yield 31.6 %;
m. p. 156.1–158.4 °C; IR 3309, 1672, 1175, 1129, 760,
721 cm-1; MS(ESI) m/z 482.92 (M ? Na?); 1H NMR
(CDCl3, 400 MHz) d 8.23 (m, 1H), 8.09–8.07 (m, 3H),
7.67 (s, 1H), 7.64–7.62 (m, 2H), 7.03 (s, 1H), 5.51 (dd, 1H,
J = 10.9, 6.6 Hz), 3.88 (dd, 1H, J = 16.2, 10.9 Hz), 3.72
(dd, 1H, J = 16.2, 6.6 Hz); 13C NMR (CDCl3, 100 MHz)
d 167.0, 153.3, 143.4, 138.1, 135.8, 132.7, 132.5, 132.0,
127.9, 127.7, 127.1, 126.3, 121.1, 119.8, 119.4, 115.1,
81.1, 34.7.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(3-chlorophenyl)amide (2c) Yield 19.4 %; m. p. 177.5–
179.8 °C; IR 3250, 1670, 1203, 737, 669 cm-1; MS(ESI)
m/z 390.2 (M ? Na?); 1H NMR (CDCl3, 400 MHz) d 8.27
(m, 1H), 8.10 (m, 1H), 7.71–7.70 (m, 2H), 7.67–7.64 (m,
2H), 7.43 (d, 1H, J = 8.2 Hz), 7.29 (t, 1H, J = 9.0 Hz),
7.15 (d, 1H, J = 8.2 Hz), 5.52 (dd, 1H, J = 10.9, 6.5 Hz),
3.90 (dd, 1H, J = 16.0, 10.9 Hz), 3.75 (dd, 1H, J = 16.0,
6.5 Hz); 13C NMR (CDCl3, 100 MHz) d 169.5, 153.0,
137.8, 134.8, 131.9, 130.1, 127.8, 127.6, 127.0, 126.4,
125.1, 121.3, 121.1, 120.1, 119.6, 118.0, 114.8, 81.3, 34.7.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(2-trifluoromethylphenyl)amide (2h) Yield 6.3 %; m.
p. 153.2–163.9 °C; IR 3410, 1698, 1163, 1106, 759,
680 cm-1; MS(ESI) m/z 414.2 (M ? H?); 1H NMR
(CDCl3, 400 MHz) d 8.40 (d, 1H, J = 8.3 Hz), 8.22 (m,
1H), 8.06 (m, 1H), 7.67 (s, 1H), 7.63–7.54 (m, 4H), 7.22 (t,
1H, J = 7.7 Hz), 5.51 (dd, 1H, J = 10.9, 5.8 Hz), 3.87
(dd, 1H, J = 16.0, 10.9 Hz), 3.75 (dd, 1H, J = 16.0,
5.8 Hz); 13C NMR (CDCl3, 100 MHz) d 170.5, 153.0,
134.6, 134.0, 133.0, 128.0, 126.2, 126.1, 124.5, 123.0,
122.4, 122.3, 122.2, 120.4, 120.1, 119.9, 119.5, 118.4,
81.0, 34.9.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(4-chlorophenyl)amide (2d) Yield 24.8 %; m. p. 147.8–
154.3 °C; IR 3249, 1669, 1185, 757, 669 cm-1; MS(ESI)
m/z 380.0 (M ? Na?); 1H NMR (MeOD, 400 MHz) d
8.19–8.16 (m, 2H), 7.75 (s, 1H), 7.65 (d, 2H, J = 9.0 Hz),
7.63-7.59 (m, 2H), 7.34 (d, 2H, J = 9.0 Hz), 5.55 (dd, 1H,
J = 10.6, 7.3 Hz), 3.81 (dd, 1H, J = 16.1, 10.6 Hz), 3.68
(dd, 1H, J = 16.1, 7.3 Hz); 13C NMR (CDCl3, 100 MHz)
d 169.4, 153.1, 135.2, 131.9, 130.0, 129.1, 127.8, 127.6,
126.9, 126.4, 121.3, 121.1, 119.6, 119.0, 114.8, 81.3, 34.7.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(4-trifluoromethylphenyl)amide (2i) Yield 27.6 %; m.
p. 156.8–162.5 °C; IR 3275, 1698, 1122, 1118, 763,
683 cm-1; MS(ESI) m/z 413.3 (M ? Na?); 1H NMR
(CDCl3, 400 MHz) d 8.25 (m, 1H), 8.07 (m, 1H), 7.68 (d,
1H, J = 8.2 Hz), 7.48 (s, 1H), 5.20 (dd, 1H, J = 10.9,
6.5 Hz), 3.87 (dd, 1H, J = 16.0, 10.9 Hz), 3.72 (dd, 1H,
J = 16.0, 6.5 Hz); 13C NMR (CDCl3, 100 MHz) d 170.3,
153.0, 139.8, 134.0, 128.3, 127.6, 126.4, 126.3, 126.2,
126.1, 122.6, 122.3, 120.6, 120.4, 119.7, 118.7, 81.4, 35.0.
5-Chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxylic acid
N-(3,4-dichlorophenyl)amide (2e) Yield 34.6 %; m.
p. 184.9–188.3 °C; IR 3313, 1684, 1186, 738, 669 cm-1
;
MS(ESI) m/z 416.4 (M ? Na?); 1H NMR (CDCl3,
400 MHz) d 8.25 (m, 1H), 8.05 (m, 1H), 7.79 (s, 1H),
7.64–7.61 (m, 2H), 7.37–7.36 (d, 2H, J = 1.4 Hz), 5.48
(dd, 1H, J = 10.9, 6.5 Hz), 3.85 (dd, 1H, J = 16.0,
11.0 Hz), 3.70 (dd, 1H, J = 16.0, 6.5 Hz); 13C NMR
123

Design and synthesis of 2,3-dihydro- and 5-chloro-2,3-dihydro-naphtho-[1,2-b]furan-2-carboxylic…
Biology
diethanolamine, and 1 mM MgCl2) in the dark at room
temperature for 1 h. As a reporter, SEAP activity was
measured as relative fluorescence units (RFU) with emission
449 nm and excitation 360 nm.
Cell lines
The three human cancer cell lines, HCT-116, NCI-H23 and
PC-3 were used in this study. All the cells were obtained
from the National Cancer Institute, U.S.A. These cells were
maintained in RPMI1640 media supplemented with 10 %
fetal calf serum at 37 °C under a humidified atmosphere of
5 % CO2.
Results and discussion
Chemistry
Various substituents on the N-phenyl rings of the target
compounds were introduced (H, OMe, Cl, CF3, and OH);
these substituents resulted in potent activity in the previous
studies. In addition, we attempted to establish structure–
activity relationships (SAR) according to the electronic and
hydrophobic characteristics of the substituents. The posi-
tional effects of the substituents were also explored by
examining biological activities of the compounds with
substituents at various positions (20-; 30-; 40-; 30,40-; and
30,50-) of the N-phenyl ring.
SRB assay
The number of cells was measured indirectly using the
sulforhodamine B (SRB) method according to the NCI
(USA) protocol. Briefly, the cells were plated into a 96 well
plate at a density of 2 9 103 cells per well. On the next day
(day 0), the compounds of interest dissolved in
DMSO/media were added in quadruplicate. The final
concentration of each compound ranged from 1 to 10 lM
and the final concentration of DMSO was \0.1 %.
Seventy-two hours later, the cells were fixed with 10 %
trichloroacetic acid (TCA) overnight at 4 °C. The TCA-
treated cells were washed extensively with distilled water
and dried in air. A SRB solution (0.4 % in 1 % acetic acid)
was then added to the well at room temperature for 1 hour.
The bound dye was dissolved in 10 mM Tris after washing
the wells with 1 % acetic acid. The absorbances were
measured at 690 nm using a micro plate reader. The
absorbance of the day 0 sample was subtracted from the
absorbance of the day 3 sample.
For the target naphtho-[1,2-b]furan-2-carboxylic acid N-
(substitutedphenyl)amides (series 1 and 2), the general
synthetic procedure is outlined in Scheme 1. The com-
mercially available starting material, 1-naphthol (3) was
treated with 2-allylphenol in the presence of K2CO3 and
then underwent Claisen rearrangement to obtain compound
4. To prepare the dihydronaphthofuran ring, epoxidation-
cyclization was performed with m-CPBA directly from
compound 4, but it resulted in low yield and side products.
Therefore, we redesigned the synthetic strategy to include
protection, epoxidation, and cyclization. The synthesis of
compound 6 containing a dihydronaphthofuran ring was
accomplished by benzylation using treatment of compound
4 with benzyl bromide and epoxidation with m-CPBA
followed by cyclization with hydrogenolysis using 10 %
Pd–C and H2 gas. For 2,3-dihydronaphtho[1,2-b]furan-2-
carboxylic acid (7), we attempted TEMPO oxidation with
NaOCl. However, carboxylic acid 8 having a chloro group in
the C-5 position was obtained instead of desired carboxylic
acid 7 without a chloro group. Therefore, 2,3-dihydronaph-
tho[1,2-b]furan-2-carboxylic acid 7 was prepared using
TEMPO and BAIB. After oxidation, 2,3-dihydronaphtho[1,2-
MTT assay
HCT-116 cancer cells were plated in 96-well plates
(5 9 104 cells/well) per 100 mL medium, and subconfluent
cells were subsequently treated with compounds (0–20 lg/
mL). To determine the appropriate dose that is not cyto-
toxic to the cells, the cytotoxic effect was evaluated in the
cells cultured for 72 h by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT; Sigma–Aldrich,
St. Louis, MO). Metabolic activity was quantified by
measuring light absorbance at 540 nm.
b]furan-2-carboxylic
acid
N-(substitutedphenyl)amide
derivatives (series 1) were obtained by using the coupling
agent CDI (1,1-carbonyldiimidazole) in anhydrous THF with
aniline and its analogs with various substituents such as H,
OH, OMe, Cl, and CF3 groups. In addition, 5-chloro-2,3-di-
hydronaphtho[1,2-b]furan-2-carboxylic acid N-(substituted-
phenyl)amide derivatives (series 2) were synthesized from
carboxylic acid 8 through treatment with 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDC) and hydroxyl-
benzotriazole (HOBt) (Scheme 1).
Measurement of NF-jB transcriptional activity
RAW 264.7 macrophages were stably transfected with NF-
jB-SEAP-NPT plasmid and then treated with 1 lg/mL LPS
plus sample for 16 h. Aliquots of the cell-free media were
heated at 65 °C for 5 min, and then reacted with SEAP
assay buffer (500 lM 4-methylumbelliferyl phosphate, 2 M
123

M. Choi et al.
Scheme 1 General methods to prepare carboxamide analogs. Reagents and conditions: a), allylbromide, K2CO3, acetone, reflux, 2 h, 98 %; b),
xylene, reflux, 16 h, 88 %; c), BnBr, K2CO3, acetone, reflux, 8 h, 99 %; d), m-CPBA, CH2Cl2, reflux, 6 h, 73 %; e), 10 % Pd–C, H2, K2CO3, Et3N,
MeOH, 8 h, 94 %; f), TEMPO, BAIB, CH3CN–H2O (50 % v/v), 6 h, 66 %; g), TEMPO, KBr, NaOCl, THF–sat. aq. NaHCO3 (50 % v/v), 6 h; h),
peptide coupling conditions: CDI, THF (1 a–b, 1d–g, 1i, and 2 series); HOBt, EDC, DIPEA, DMF (1c, 1j, and 1k); EDC, THF (1h); 5–40 %
Biology
Unlike series 1 compounds, analogs having a chloro
group at C-5 of the 2,3-dihydronaphtho[1,2-b]furan ring
closely resembled the series of indoline-2-carboxamide,
benzofuran-2-carboxamide, and 7-methychroman-2-car-
boxamde in NF-jB inhibitory activity (Kwak et al. 2009b,
2010; Choi et al. 2015). In compounds with a 5-membered
ring system such as indoline or benzofuran, introduction of
the trifluoromethyl group at C-30 and C-50 of the N-phenyl
ring enhanced their NF-jB inhibitory activity. The data for
the series of 5-chloro-2,3-dihydronaphtho[1,2-b]furan-2-
carboxamide were consistent with the results of previous
studies. Compound 2g, which had a 5-membered ring and a
trifluoromethyl group at C-30 and C-50 of the N-phenyl ring,
exhibited most potent activity among series 2 compounds.
In chroman analogs, only compounds containing a 30,50-
bistrifluoromethyl group on the N-phenyl ring and a sub-
stitution of a methyl group at the C-7 position of the
chroman moiety had NF-jB inhibitory activity (Fig. 2).
Among the compounds containing N-(40-chlor-
ophenyl)amide, the 2,3-dihydronaphtho[1,2-b]furan analog
had as potent activity as the (substituted)chroman analog
because of their structural similarity. However, the intro-
duction of substituents (such as a chloro group) at the C-5
position of the naphthofuran moiety abolished NF-jB inhi-
bitory activity. In the analogs having the 30,50-bistrifluo-
romethyl group, we confirmed that structural extension of
the plane ring and introduction of a substituent at the C-5
positions of dihydrobenzofuran moieties preserved their NF-
jB inhibitory activity. Interestingly, their activity was
The newly synthesized library of 2,3-dihydronaphtho-[1,2-
b]furan-2-carboxamide analogs (1a–k) and 5-chloro-2,3-di-
hydronaphtho[1,2-b]furan-2-carboxamide analogs (2a–i)
was evaluated for NF-jB inhibitory activity in LPS-stimu-
lated RAW 264.7 cells (Table 1). Among the compounds of
series 1, only 2,3-dihydronaphtho-[1,2-b]furan-2-carboxylic
N-(40-chlorophenyl) amide (1d) (IC50: 26 lM) was more
potent than the reference compounds KL-1156 (IC50:
37.2 lM) and PDTC (IC50: 34.5 lM). In series 2 (chloro
group in the C-5 position of the naphthofuran ring), 5-chloro-
2,3-dihydronaphtho [1,2-b]furan-2-carboxylic N-pheny-
lamide (2a) and 5-chloro-2,3-dihydronaphtho [1,2-b]furan-2-
carboxylic N-(30,50-bis(trifluoromethyl)phenyl)amide (2g)
had IC50 values of 27 lM and 12 lM, respectively. Com-
pound 2g, which contained 30,50-bistrifluoromethyl groups on
the N-phenyl ring, exhibited the highest NF-jB inhibitory
activity among the 2,3-dihydronaphtho-[1,2-b]furan-2-car-
boxamide analogs; its inhibitory activity was approximately
3 times those of KL-1156 and PDTC.
In the previous studies, chroman-2-carboxamide and
(substituted)chroman-2-carboxamide analogs with a chloro
group at C-40 of the N-phenyl ring generally were most
potent inhibitors of NF-jB in the respective series
(IC50 \ 30 lM) (Kwak et al. 2007, 2008, 2009b). Like-
wise, the best compound of series 1, 2,3-dihydronaphtho-
[1,2-b]furan-2-carboxyamide (1d), had a chloro group at
C-40 of the N-phenyl ring (Fig. 2).
123

Design and synthesis of 2,3-dihydro- and 5-chloro-2,3-dihydro-naphtho-[1,2-b]furan-2-carboxylic…
Table 1 Inhibition of LPS-induced NF-jB activation by 2,3-dihydronaphtho-[1,2-b]furan-2-carboxamide analogs
Substituent
R1
No.
% inhibition at
30 lM (ICa50
No.
% inhibition at
30 lM (ICa50
)
)
R2
R3
R4
H
H
H
H
1a
1b
1c
1d
1e
1f
46
2a
2b
2c
2d
2e
2f
56 (29 lM)
H
H
OMe
H
H
49
50
H
Cl
H
H
36
37
H
Cl
Cl
H
H
57 (26 lM)
42
H
Cl
Cl
CF3
H
H
19
43
30
41
39
38
42
37
H
Cl
CF3
H
44
H
H
1g
1h
1i
2g
2h
2i
88 (12 lM)
CF3
H
31
36
H
H
CF3
H
H
OH
H
H
1j
H
H
OH
H
1k
KL-1156 (Ref)
PDTC (Ref)
IC50: 37.2 lM
IC50: 34.5 lM
Compounds having more potent activity than others are in italics
a
IC50 values are means of three experiments of the concentrations exhibiting 50 % inhibition of LPS-induced NF-jB transcriptional activity
improved when a substituent was added at the C-7 position
of the chroman moiety and the C-5 position of the dihy-
dronaphthofuran moiety.
(Fig. 3). Compound 1e showed a GI50 value of 7.97 lg/mL
and compound 2g had a GI50 value of 2.68 lg/mL against
HCT-116 cells (Table 2). Most importantly, the inhibitory
activity of compound 2g was more potent than those of
5-fluorouracil (5-FU) and oxaliplatin, which were used as
reference anticancer compounds.
Recent study reported that naphthofuran compounds
regulate HNF4a, which is associated with proliferation,
progression, and metastasis of hepatocellular carcinoma (Le
Guevel et al. 2009). In particular, compound 2g inhibits liver
tumor growth through inhibition of STAT3 (Kwon et al.
2015). Therefore, we also evaluated synthesized compounds
(1 and 2 series) for in vitro cytotoxicity against human
cancer cell lines HCT-116, NCI-H23, and PC-3 by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) and sulforhodamine B assays.
However, cell growth inhibitory activity of the tested
compounds was not directly related to their NF-jB inhibi-
tory activity. Interestingly, only 5-chloro-2,3-dihydronaph-
tho [1,2-b]furan-2-carboxylic N-(30,50-bis(trifluoromethyl)
phenyl)amide (2g), which was reported as an outstanding
inhibitor of STAT3 (Kwon et al. 2015), had not only potent
NF-jB inhibitory activity but also cytotoxicity against HCT-
116 cells.
In the MTT assay, six 2,3-dihydronaphtho-[1,2-b]furan-2-
carboxamide analogs (1a, 1d–g, and 1i) and eight 5-chloro-
2,3-dihydronaphtho[1,2-b]furan-2-carboxamide analogs (2a
and 2c–i) inhibited cell growth of the colon cancer cell line
HCT-116 by more than 50 % at 25 lg/ml (Table 2). 2,3-
dihydronaphtho [1,2-b]furan-2-carboxylic N-(30,40-dichlor-
ophenyl)amide (1e) and 5-chloro-2,3-dihydronaphtho[1,2-b]
We also evaluated the growth inhibitory effects of
series 1 and 2 against human cancer cell lines NCI-H23
(lung) and PC-3 (prostate) by using the sulforhodamine B
assay (Table 3). Three 2,3-dihydronaphtho-[1,2-b]furan-2-
carboxamide analogs (1c, 1e, and 1h) and two 5-chloro-
2,3-dihydronaphtho[1,2-b]furan-2-carboxamide analogs (2f
and 2g) exhibited considerable activities against NCI-H23
cells. Compounds 1c (30-Cl on the N-phenyl ring), 1e (30,40-
diCl), and 1h (20-CF3) had moderate cytotoxicity with GI50
values of 4.50 lM, 3.15 lM, and 5.65 lM, respectively.
Compounds 2f (30,50-diCl) and 2 g (30,50-diCF3) showed
furan-2-carboxylic
N-(30,50-bis(trifluoromethyl)phenyl)
amide (2g) exhibited the best activity among the compounds
of the respective series (9.70 and 16.11 % cell viability,
respectively). In addition, compounds 1e and 2g exhibited
concentration-dependent inhibition of cancer cell growth
123

M. Choi et al.
Fig. 2 NF-jB inhibitory
activity (IC50 values) of
compounds possessing
a 40-chloro or 30,50-
bistrifluoromethyl group on the
N-phenyl ring. (Kwak et al.
2007, 2008, 2009a, b, 2010;
Choi et al. 2015)
Table 2 Viability of HCT-116
colorectal carcinoma cells
treated with 2,3-
No.
Viability at 25 lg/mL (%)
No.
Viability at 25 lg/mL (%)
(GI50 value)a
(GI50 value)a
dihydronaphtho-[1,2-b]furan-2-
carboxylic acid N-
(substitutedphenyl)amide
analogs
1a
1b
1c
1d
1e
1f
36.25
2a
47.11
78.12
2b
57.51
58.27
2c
23.05
33.26
2d
32.93
9.70 (7.97 lg/mL)
46.63
2e
44.37
2f
40.66
1g
1h
1i
44.92
2g
16.11 (2.68 lg/mL)
43.84
54.57
2h
22.38
2i
44.68
1j
74.64
5-FU
36.59 (at 10 lg/mL)
55.94 (at 10 lg/mL)
1k
82.18
Oxaliplatin
Compounds having more potent activity than others are in italics
a
GI50 values are means of three experiments and correspond to the agent’s concentration causing a 50 %
decrease in net cell growth
cytotoxic activity with GI50 values of 3.80 lM and
3.08 lM, respectively. Compound 2 g had the best activity,
but its activity against NCI-H23 cells was lower than those
of pyrrolidine, Bay-11-7082, and Adriamycin, which were
used as reference compounds. When tested with PC-3 cells,
only three compounds inhibited cancer cell growth: com-
pound 1e (GI50 = 6.51 lM), 2f (5.57 lM), and 2g
(3.30 lM). Overall, compound 2g had the best cytotoxic
activity among the prepared compounds, although it was
not as good as those of the reference compounds (Table 3).
To explore the SAR in order to achieve potent cytotoxic
activities against the three cancer cell lines, we substituted
various groups (R1, R2, R3, and R4) on the planar N-phenyl
ring of the carboxamide functionality. Compounds 1b, 1j,
123

Design and synthesis of 2,3-dihydro- and 5-chloro-2,3-dihydro-naphtho-[1,2-b]furan-2-carboxylic…
Fig. 3 Effect of compounds 2g
and 1e on viability of colon
cancer cells (HCT-116). 5-FU
and oxaliplatine were used as
reference compounds. After
treatment for 72 h, the cells
were harvested by trypsinization
and stained with MTT. The data
are expressed as the
mean SD of three
experiments
Table 3 Anticancer activities
No.
NCI-H23 (GI50, lM)a
PC-3 (GI50, lM)a
No.
NCI-H23 (GI50, lM)a
PC-3 (GI50, lM)a
of 2,3-dihydronaphtho-[1,2-
b]furan analogs against human
cancer cell lines
1a
1b
1c
1d
1e
1f
[10
[10
[10
[10
[10
6.51
[10
[10
[10
[10
[10
[10
2a
2b
2c
2d
2e
2f
[10
[10
[10
[10
[10
3.80
3.08
[10
[10
[10
[10
[10
[10
[10
5.57
3.30
[10
[10
[10
4.50
[10
3.15
[10
1g
1h
1i
[10
5.65
2g
2h
2i
[10
1j
[10
1k
[10
Pyrrolidine
Bay-11-7082
ADR
0.079
1.078
0.086
0.205
1.028
0.037
Compounds having more potent activity than others are in italics
a
GI50 values are means of three experiments and correspond to the agent’s concentration causing a 50 %
decrease in net cell growth
1k, and 2b with introduced electron-donating groups such
as methoxy or hydroxyl groups exhibited lower cytotoxic
activity than compounds with other substituents. Com-
pounds with anticancer activity generally had two intro-
duced electron-withdrawing groups (such as chloro or
trifluoro groups) on the N-phenyl ring. For instance, com-
pounds 1e with 30,40-dichloro group, 2f with 30,50-dichloro
group, and 2g with 30,50-bisfluoromethyl group on the N-
phenyl ring were more potent than compounds with other
substituents. In the derivatives of compounds 2f and
2 g that were cytotoxic against NCI-H23 and PC-3 cell
lines, the substituents were located at the C-30 and C-50
positions of the N-phenyl ring. Only compounds 1e and 2g
exhibited potent cell growth inhibitory activity against all
three lines tested (HCT-116, NCI-H23, and PC-3), and
compound 2g had the best cytotoxic activity. Moreover,
compound 2g inhibited the transcription factors NF-jB and
STAT3 (Kwon et al. 2015).
In conclusion, a series of 2,3-dihydronaphtho-[1,2-b]fu-
ran-2-carboxamide analogs (1a–k) and 5-chloro-2,3-dihy-
dronaphtho[1,2-b]furan-2-carboxamide analogs (2a–i) was
designed and synthesized from commercially available
starting materials via the well-known Claisen rearrange-
ment, epoxidation-cyclization, oxidation, and amidation
reactions using CDI or EDC. Compounds 1d and 2g
exhibited the best NF-jB inhibitory activity in LPS-stimu-
lated RAW 264.7 cells among the compounds of the
respective series. Compound 1d, which had a 40-chloro
group on the N-phenyl ring, exhibited NF-jB inhibitory
activity because of its structural similarity to a previously
reported (substituted)chroman analog. In contrast, as SAR of
indoline, benzofuran and 7-methylchroman moiety, com-
pound 2g with the introduced 5-chloro group on the naph-
thofuran ring and 30,50-bisfluoromethyl groups on the N-
phenyl ring had the best NF-jB inhibitory activity. The
in vitro anticancer activities of these analogs were evaluated
123

M. Choi et al.
Ha H, Debnath B, Odde S, Bensman T, Ho H, Beringer PM, Neamati
N (2015) Discovery of novel CXCR2 inhibitors using ligand-
based pharmacophore models. J Chem Inf Model 55:1720–1738
Kim BH, Reddy AM, Lee KH, Chung EY, Cho SM, Lee H, Min KR,
Kim Y (2004) Inhibitory mechanism of chroman compound on
LPS-induced nitric oxide production and nuclear factor-kappaB
activation. Biochem Biophys Res Commun 325:223–228
Kwak JH, Kim BH, Jung JK, Kim Y, Cho J, Lee H (2007) Structure-
activity relationships of 6-hydroxy-7-methoxychroman-2-car-
boxylic acid N-(substituted)phenylamides as inhibitors of nuclear
factor-kappaB activation. Arch Pharm Res 30:1210–1215
Kwak JH, Won SW, Kim TJ, Roh E, Kang HY, Lee HW, Jung JK,
Hwang BY, Kim Y, Cho J, Lee H (2008) Synthesis of chroman-
2-carboxylic acid N-(substituted)phenylamides and their inhibi-
tory effect on nuclear factor-kappaB (NF-kappaB) activation.
Arch Pharm Res 31:133–141
Kwak J-H, Won S-W, Kim T-J, Yi W, Choi E-H, Kim S-C, Park H,
Roh E, Jung J-K, Hwang B, Hong J, Kim Y, Cho J, Lee H
(2009a) Synthesis and nuclear factor-kB inhibitory activities of
6- or 7-methylchroman-2-carboxylic acid N-(substituted) pheny-
lamides. Arch Pharmacal Res 32:167–175
Kwak JH, Won SW, Kim TJ, Yi W, Choi EH, Kim SC, Park H, Roh
E, Jung JK, Hwang BY, Hong JT, Kim Y, Cho J, Lee H (2009b)
Synthesis and nuclear factor-kappaB inhibitory activities of 6- or
7-methylchroman-2-carboxylic acid N-(substituted) pheny-
lamides. Arch Pharm Res 32:167–175
against three human cancer cell lines: HCT-116 (colon),
NCI-H23 (lung), and PC-3 (prostate). 5-Chloro-2,3-dihy-
dronaphtho[1,2-b]furan-2-carboxylic N-(30,50-bis(trifluo-
romethyl)phenyl)amide (2g) exhibited the most potent
anticancer activity against the three tested cell lines. 2,3-
dihydronaphtho [1,2-b]furan-2-carboxylic N-(30,40-dichlor-
ophenyl)amide (1e) also exhibited promising growth inhi-
bitory activities against all these cell lines. In the SAR
studies, two electron-withdrawing groups at the planar N-
phenyl rings of compounds 1e, 2f, and 2g resulted in more
potent anticancer activities than those of all other analogs.
However, cytotoxicity of these analogs was not directly
related to their NF-jB inhibitory activities. Only compound
2g exhibited both outstanding anticancer and NF-jB inhi-
bitory activities. Our SAR studies have encouraged us to
investigate additional novel lead scaffolds exhibiting potent
anticancer activity through inactivation of NF-jB; this work
is in progress.
Acknowledgments This research was supported by the Basic Sci-
ence Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science and
Technology (NRF-2013R1A1A2009381), Medical Research Center
Program (2008-0,062,275) and the research grant of Chungbuk
National University in 2014.
Kwak JH, Kim Y, Park H, Jang JY, Lee KK, Yi W, Kwak JA, Park
SG, Kim H, Lee K, Kang JS, Han SB, Hwang BY, Hong JT,
Jung JK, Cho J, Lee H (2010) Structure-activity relationship of
indoline-2-carboxylic acid N-(substituted)phenylamide deriva-
tives. Bioorg Med Chem Lett 20:4620–4623
Kwon SM, Jung YY, Hwang CJ, Park MH, Yoon NY, Kim TM, Yu
JM, Kim DH, Seo DW, Youn HS, Seo HO, Chung IS, Han SB,
Hwang BY, Yoo HS, Jung JK, Lee H, Hong JT (2015) Anti-
cancer effect of N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2,3-
dihydronaphtho[1,2-b]furan-2-carboxamide, a novel synthetic
compound. Mol Carcinog. doi:10.1002/mc.22311 (article in
press)
Le Guevel R, Oger F, Lecorgne A, Dudasova Z, Chevance S, Bondon A,
Barath P, Simonneaux G, Salbert G (2009) Identification of small
molecule regulators of the nuclear receptor HNF4alpha based on
naphthofuran scaffolds. Bioorg Med Chem 17:7021–7030
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