Anti-influenza drug discovery: Structure-activity relationship and mechanistic insight into novel angelicin derivatives

Article abstract of DOI:10.1021/jm901570x

By using a cell-based high throughput screening campaign, a novel angelicin derivative 6a was identified to inhibit influenza A. (H1N1) virus induced cytopathic effect in Madin-Darby canine kidney cell culture in low micromolar range. Detailed structure-activity relationship studies of 6a revealed that the angelicin scaffold is essential for activity in pharmacophore B, while meta-substituted phenyl/2-thiophene rings are optimal in pharmacophore A and C. The optimized lead 4-methyl-9-phenyl-8-(thiophene-2-carbonyl)-furo[2,3-h] chromen-2-one (8g, IC₅₀ = 70 nM) showed 64-fold enhanced activity compared to the high throughput screening (HTS) hit 6a. Also, 8g was found effective in case of influenza A (H3N2) and influenza B virus strains similar to approved anti-influenza drug zanamivir (4). Preliminary mechanistic studies suggest that these compounds act as anti-influenza agents by inhibiting ribonucleoprotein (RNP) complex associated activity and have the potential to be developed further, which could form the basis for developing additional defense against influenza pandemics.

Full text of DOI:10.1021/jm901570x

J. Med. Chem. 2010, 53, 1519–1533 1519
DOI: 10.1021/jm901570x
Anti-Influenza Drug Discovery: Structure-Activity Relationship and Mechanistic Insight into
Novel Angelicin Derivatives
Jiann-Yih Yeh,†,O Mohane Selvaraj Coumar,†,O Jim-Tong Horng,§,O Hui-Yi Shiao,†,‡,O Fu-Ming Kuo,† Hui-Ling Lee,†
In-Chun Chen,† Chun-Wei Chang,† Wen-Fang Tang,§ Sung-Nain Tseng, ,^ Chi-Jene Chen,^ Shin-Ru Shih, John T.-A. Hsu,†,#
Chun-Chen Liao,‡,3 Yu-Sheng Chao,† and Hsing-Pang Hsieh*,†
†Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County
350, Taiwan, ROC, ‡Department of Chemistry, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 300, Taiwan, ROC,
§Department of Biochemistry, Department of Medical Biotechnology and Laboratory Science, and ^Research Center for Emerging Viral
Infections, Chang Gung University, 259 Wen-Hua first Road, Kweishan, Taoyuan 333, Taiwan, ROC, #Department of Biological Science and
Technology, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan, ROC, and 3Department of Chemistry, Chung Yuan
Christian University, 200 Chung-Pei Road, Chungli 320, Taiwan, ROC. O These authors contributed equally to this work.
Received June 29, 2009
By using a cell-based high throughput screening campaign, a novel angelicin derivative 6a was identified
to inhibit influenza A (H1N1) virus induced cytopathic effect in Madin-Darby canine kidney cell
culture in low micromolar range. Detailed structure-activity relationship studies of 6a revealed that the
angelicin scaffold is essential for activity in pharmacophore B, while meta-substituted phenyl/2-
thiophene rings are optimal in pharmacophore A and C. The optimized lead 4-methyl-9-phenyl-
8-(thiophene-2-carbonyl)-furo[2,3-h]chromen-2-one (8g, IC50 = 70 nM) showed 64-fold enhanced
activity compared to the high throughput screening (HTS) hit 6a. Also, 8g was found effective in case
of influenza A (H3N2) and influenza B virus strains similar to approved anti-influenza drug zanamivir
(4). Preliminary mechanistic studies suggest that these compounds act as anti-influenza agents by
inhibiting ribonucleoprotein (RNP) complex associated activity and have the potential to be developed
further, which could form the basis for developing additional defense against influenza pandemics.
Introduction
(H2N2) in 1957 had caused the death of 1 million people
worldwide. Besides, Hong Kong influenza (H3N2) in 1968
had reached pandemic proportions.3-5 The recent repeated
lethal outbreaks (2007) of the virulent H5N1 avian influenza
strains in humans has caused global health threat, as they may
adapt to become easily transmissible from human to human,
which could cause serious pandemics.6-8 Currently (2009),
swine influenza A (H1N1) outbreak in Mexicoand otherparts
of the world has led to issuances of pandemic alertness by the
WHO.9 As the impact of influenza pandemic is enormous,
a renewed drug discovery effort worldwide is essential to
counteract the disease more efficiently.
Presently vaccination is the mainstay, along with anti-
influenza drugs, to control the spread and treatment of the
disease. Because influenza virus continuously evolves to avoid
host detection systems by changing its antigenicity through
mutation of its surface glycoprotein (NA, HA) genes by a
process called “antigenic drift”, it could reinfect the same
person in the following seasons.10,11 Another major factor is
the reassortment (antigenic shift) of genetic materials between
different strains of the virus circulating in humans and avians
such as that which occurred during the pandemic of Asian
influenza (H2N2) in 1957.3,10 Because of the higher rate of
mutation to the influenza virus, continuous monitoring of
viralgenetics toidentifythe circulatingstrainof the virusevery
season prior to vaccine development so as to achieve maxi-
mum protection for that season is essential.4,10 To address this
issue, WHO experts every season identify the proper com-
bination of antigenic strains for which vaccine is developed.
Influenza, usually known as flu, is an infectious disease of
birds and mammals caused by ribonucleic acid (RNAa)
viruses of the family Orthomyxoviridae. It is a contagious,
acute, febrile, respiratory disease, which occurs seasonally in
epidemic and sometimes in pandemic proportions. On the
basis of the serological subtyping, three distinct types A, B,
and C are present, of which influenza A and B are of much
concern as human pathogens. Further, on the basis of the
antigenicity of the two surface glycoproteins hemagglutinin
(HA) and neuraminidase (NA), a total of 16 HA (H1-H16)
and 9 NA (N1-N9) subtypes are known, resulting in the
possibility of distinct strains of influenza virus such as H1N1,
H1N2..., H2N1, H2N2..., and so on.1,2 Influenza epidemic
and pandemics in the past century had caused serious impact
on global morbidity, mortality, and economy. For example,
the “Spanish flu” (H1N1) outbreak of 1918-1919 resulted in
the death of at least 20 million people, and the Asian influenza
*To whom correspondence should be addressed. Phone: þ886-37-246-
166 ext. 35708. Fax: þ886-37-586-456. E-mail: hphsieh@nhri.org.tw.
a Abbreviations: CPE, cytopathic effect; cRNA, complementary
RNA; DMEM, Dulbecco’s modified Eagle medium; DNA, deoxyribo-
nucleic acid; dsRNA, double-strand RNA; FBS, fetal bovine serum;
Fluc, firefly luciferase; HA, hemagglutinin; HBSS, Hanks’ balanced salt
solutions; HTS, high throughput screening; MDCK cells, Madin-
Darby canine kidney cells; MOI, multiplicity of infection; mRNA,
messenger RNA; NA, neuraminidase; NP, nucleoprotein; PFU, plaque
forming unit; RBC, red blood cells; Rluc, renilla luciferase; RNA,
ribonucleic acid; RNP, ribonucleoprotein; SAR, structure-activity
relationship; vRNA, viral RNA; WHO, World Health Organization.
r
2010 American Chemical Society
Published on Web 01/21/2010
pubs.acs.org/jmc

1520 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Yeh et al.
Figure 1. Inhibitors of influenza virus: M2 ion channel inhibitors (1 and 2), NA inhibitors (3-5), and HTS angelicin hit 6a.
Scheme 1. General Synthetic Method for the Preparation of Angelicin Derivatives 6-10a
a (a) R4COCl, Et3N, THF, rt, 16 h; (b) AlCl3, 170 ꢀC, 2 h; (c) R5COCH2Br, K2CO3, CH3CN, reflux, 16 h, 15-44% over three steps.
However, as there is a time gap of 9-12 months from the
recommendation to the actual use of vaccine, this could still
result in mismatch between the virus and the vaccine, which
could result in ineffectiveness.10,11 Because of these and other
complicating factors influencing the development of an all
effective flu vaccine, additional weapons in the form of
effective anti-influenza agents are a must in our fight against
seasonal or pandemic influenza that may arise in the future.4,8
Adamantane derivatives 1 (amantadine) and 2 (rimantadine)
are the first two antiviral drugs used for the treatment
of influenza, which target the M2 ion channels (Figure 1).
Recently, orally administered 3 (oseltamivir) and inhaled 4
(zanamivir) are approved for use as anti-influenza treatments,
while 5 (peramivir) is under clinical investigation; all the three
compounds target the neuraminidase (NA).1-3 However,
alternative therapeutics are needed because the development
of resistance strains to these drugs targeting NA enzyme,
particularly 3, has been reported as an increasing clinical
problem.12-14 Thus there is an urgent need to identify novel
compounds with inhibitory activity against influenza virus,
which is the aim of this study.
Using a cell-based assay, high throughput screening (HTS)
was carried out to identify novel compounds as anti-influenza
agents. The HTS assay measured the ability of the test
compounds to inhibit the virus-induced cytopathic effect
(CPE) on MDCK (Madin-Darby canine kidney) cells and
the results expressed as IC50 (concentration required to reduce
CPE by 50% relative to the virus control). Also, a concurrent
assay to determine the general cytotoxicity of the compounds
on MDCK cells were performed without virus infection and
the results expressed as CC50 (concentration required to
produce 50% cell death). Cytotoxicity measurement was
performed to identify compounds which selectively inhibit
virus replication without killing uninfected cells. The ratio
between CC50 and IC50 isan indicator of toxicity potential of a
compound and could guide in selecting appropriate com-
pounds for further development. Through the HTS screening
of about 20000 compounds, we have discovered a novel
angular coumarin derivative 6a (Figure 1) to inhibit influenza
virus, with an IC50 of 4.5 μM and a CC50>25 μM. Literature
survey revealed that linear furanocoumarin, in which the
furan ring is attached to the 6,7 positions of the coumarin
ring, such as psoralen, is highly phototoxic to many organisms
due to their ability to cross-link deoxyribonucleic acid (DNA)
after photoactivation. An angular furanocoumarin such as
angelicin, in which the furan ring is attached to the 7,8
positions of the coumarin ring, which because of their geo-
metry cannot cross-link with DNA, is less phototoxic.15 To
our knowledge, so far angelicin derivatives closely related to
6a are not reported as antiviral agents, let alone anti-influenza
agents. Moreover, only a few reports in the literature report
the synthesis of 6a and related compounds with angelicin
scaffold, making the synthetic exploration interesting. On the
basis of structural novelty and the necessity to develop anti-
influenza agents to combat any future influenza pandemic, we
started a MedChem program to investigate the potential of
this novel hit 6a, the results of which are described herein.
Chemistry
For the construction of the most of the desired angelicin
derivatives 6-10, appropriately substituted 7-hydroxy-
coumarin 11 (commercial source or synthesized by Pechmann
condensation of meta-hydroxy phenol with β-ketoesters
under acidic condition as reported by Lee et al.16,17) was
O-acylated with acid chloride (R4COCl) to give 12. Fries
rearrangement of 12 under acidic condition using anhydrous
AlCl3 at elevated temperaturegavethe keyintermediate8-acyl
substituted coumarins 13. The desired angelicin derivatives
6-10 were constructed from 13 and appropriate 2-bromo
ketones (R5COCH2Br) via a single step O-alkylation-
cyclization-elimination process under basic condition with
an overall yield of 15-44% over three steps (Scheme 1).18,19
For the preparation of 6b, coumarin 11a was first iodi-
niated20 and then reacted with 1-phenyl-2-propyn-1-ol using
copper mediated ring annulation21 to construct the furan ring
of 15. Oxidation of the secondary alcohol group of 15 using
Jones reagent provided the desired angelicin derivative 6b in
37% yield over three steps. For the construction of com-
pounds 8c and 8d without the ketone group, coumarin 11a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1521
Scheme 2. Synthesis of Angelicin Derivatives 6b and 8c,da
a (a) I2, HIO4/EtOH, 2 h, rt, 76%; (b) 1-phenyl-2-propyn-1-ol, CuI, Et3N, DMF, 60 ꢀC, 24 h, 51%; (c) Jones reagent, acetone, 0 ꢀC f rt, 0.5 h, 95%;
(d) K2CO3, CH3CN, reflux, 16 h; (e) H3PO4, xylene, reflux, 16 h, 18-21% over two steps (8c from 16a; 8d from 16b).
Scheme 3. Synthesis of Derivatives 7a,ba
a (a) (i) n-BuLi, PhCOCl, THF, 0 ꢀC f rt, 2 h; (ii) MeOH, HCl, reflux, 0.5 h, 75%. (b) PhCOCH2Br, K2CO3, CH3CN, reflux, 16 h, 72% for 19 from
18a, 93% for 7b from 18b. (c) BBr3, CH2Cl2, 0 ꢀC f rt, 12 h, 80%. (d) Crotonic anhydride, pyridine, imidazole, rt, 2 h, 81%.
Scheme 4. Synthesis of Derivative 7ca
benzoyl chloride gave 23. Without purification, 23 was
cyclized with 2-bromoacetophenone in basic conditions to
afford 7b in 8% yield over three steps (Scheme 4). For the
preparation of bioisosteric thienocoumarin derivative 7l,
Newman-Kwart thermal rearrangement was utilized to ob-
tain the thiophenol intermediate 26 from phenol 13a. Con-
densation of 26 with 2-bromoacetophenone afforded the
desired compound 7l, in an overall yield of 22% in four steps
starting from 13a (Scheme 5).
Reductionof the ketone group of 6f by sodiumborohydride
in THF gave the secondary alcohol 8a. Alternatively, when
the reduction process was carried out in the presence of TFA,
a (a) NaH, MeI, THF, 0ꢀCfrt, 12h;(b) AlCl3, CH2Cl2, PhCOCl, 16h;
(c) PhCOCH2Br, K2CO3, CH3CN, reflux, 16 h, 8% over three steps.
complete reduction of the keto function to the methylene
group was achieved to get the compound 8b (Scheme 6). The
phenol 8u was obtained from the methoxy derivative 8p by
demethylation using BBr3. The analogues 9a-f were synthe-
sized by alkylation of the phenol 8u under basic conditions
using the required bromo compound (R1Br). In the case of
compounds 9g,h, TBDMS protected bromoalkanol (R2Br)
was used and deprotection using TBAF in the final step
unmasked the OH group (Scheme 7).
was alkylated with either 2-bromoacetophenone or 2-bromo-
2-phenylacetophenone to give the intermediates 16a and 16b,
respectively. Cyclization under acidic conditions afforded the
desired products 8c,d with a yield of 18-21% over two steps
(Scheme 2).
For the preparation of lactone ring opened analogue 7a,
hydrogen abstraction by n-BuLi from MOM protected 3-
hydroxy anisole22 (17), followed by acylation with benzoyl
chloride, and then MOM deprotection in acidic conditions
gave 18a. Condensation of 2-bromoacetophenone with 18a
under basic conditions afforded 19. Demethylation using
BBr3 to give 20, followed by acylation with crotonic an-
hydride, gave the desired 7a. For the preparation compound
7b without the lactone ring, commercially available 18b was
condensed with 2-bromoacetophenone to afford 7b in 93%
yield (Scheme 3).
Results and Discussion
We initiated structure-activity relationship (SAR) studies
to explore the pharmacophore (molecular framework respon-
sible for a drug’s biological activity) requirements for anti-
influenza activity based on the HTS hit 6a. For convenience of
SAR discussion, the lead molecule 6a is divided into three
pharmacophore parts: A (3-methyl group on furan ring), B
(angelicin core), and C (2-benzoyl group on furan ring)
(Figure 1). In the pharmacophore A (Table 1), removal of
the methyl group led to slightly better activity for 6b than the
parent compound 6a. However, increasing the alkyl chain
Preparation of 7c without the lactone ring was carried out
as shown in Scheme 4. 2-Naphthol (21) was the first methyl-
ated using MeI to give 22, then Fridel-Crafts acylation with

1522 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Yeh et al.
Scheme 5. Synthesis of Bioisosteric Thienocoumarin Derivative 7la
a (a) Dimethylthiocarbamoyl chloride, NaH, DMAP, THF, rt, 24 h, 84%; (b) mesitylene, 280 ꢀC, sealed tube, 2 h, 65%; (c) NaOCH3, MeOH, reflux,
1 h; (d) PhCOCH2Br, K2CO3, CH3CN, reflux, 16 h, 40% over two steps.
Table 1. Optimization of Pharmacophore A:a In Vitro Anti-Influenza
A Virus Activity of Compounds 6a-v
Scheme 6. Synthesis of Angelicin Derivatives 8a,ba
compdb
R1
IC50, μMc
6a
6b
6c
6d
6e
6f
CH3
H
4.51 ( 0.24
2.43 ( 0.31
0.29 ( 0.03
0.65 ( 0.02
>25
n-C3H7
n-C6H13
n-C10H21
Ph
0.14 ( 0.01
0.88 ( 0.04
1.91 ( 0.23
0.15 ( 0.01
0.65 ( 0.18
0.82 ( 0.10
10.08 ( 0.47
0.76 ( 0.12
3.92 ( 1.70
0.46 ( 0.11
0.08 ( 0.01
0.73 ( 0.03
0.10 ( 0.02
0.10 ( 0.03
0.25 ( 0.05
1.41 ( 0.04
0.53 ( 0.02
6g
6h
6i
1-naphthyl
2-naphthyl
2-thienyl
2-furanyl
4-CH3-Ph
4-CH3O-Ph
4-NO2-Ph
4-Cl-Ph
a (a) NaBH4, THF, 0 ꢀC, 16 h, 60%; (b) NaBH4, THF, TFA, 0 ꢀC,
16 h, 72%.
6j
6k
6ld
6m
6n
6o
6p
6q
6r
6s
6t
Scheme 7. Synthesis of Angelicin Derivatives 8u and 9a-ha
4-Br-Ph
3-Br-Ph
2-Br-Ph
3,5-diBr-Ph
3-CH3-Ph
3-NO2-Ph
3-Cl-Ph
6u
6v
3-CN-Ph
a (a) BBr3, CH2Cl2, 2 h, rt, 92%; (b) for 9a-f: R1Br (R1 = CH3-
(CH2)n), or (CH3)2NCH2CH2), K2CO3, KI, CH3CN, reflux, 16 h,
25-38%; for 9g,h: (i) R2Br (R2 = TBDMSO(CH2)n), K2CO3, KI,
CH3CN, reflux, 16 h; (ii) TBAF, THF, 0 ꢀC, 1.5 h, 15-19%.
a Using influenza A/WSN/33 (H1N1) strain. b All the compounds
exhibited CC50 > 25 μM. c IC50: The concentration required for a test
compound to reduce the virus-induced cytopathic effect (CPE) by 50%
relative to the virus control. Values are expressed as the mean of at
least two independent determinations, each experiments performed in
triplicates. d Synthesis 6l was not possible by the standard method A
reported, due to demethylation of the methoxy group during Fries
rearrangement. Hence an alternative synthetic route for this and related
compounds were developed, which will be reported separately.
length resulted in significant improvement in activity, with
the n-propyl (6c) and n-hexyl (6d) analogues showing nano-
molar level activity. However, increasing the length further
decreased the activity, with the n-decyl (6e) analogue, showed
complete loss of activity. Next we replaced the methyl
group of the lead compound 6a with various aromatic
and heteroaromatic rings such as phenyl (6f), naphthyl
(6g,6h), thiophene (6i), and furan (6j). All the five analogues
showed improved activity compared to the HTS hit 6a,
showing that aromatic hydrophobic group at this position
is preferred. Of particular interest are the phenyl analogue
6f (IC50=140 nM) and the bioisosteric equivalent 2-
thiophene analogue 6i (IC50 =150 nM), both of which
showed over 30-fold improved activity compared to the HTS
hit 6a.
electron withdrawing NO2/Cl/Br groups (6m-o) at the para-
position of the phenyl group of 6f, led to the conclusion that
the para-Br substitution is better. Furthermore, it was found
that when the Br substitution was moved to meta-position in
6p, the activity improved almost 2-fold of that of the unsub-
stituted compound 6f, with the IC50 reaching a value of
80 nM. However Br substitution in ortho-position (6q) of
the phenyl ring led to decreased activity. Moreover, di-Br
substituted compound (6r) did not show further improvement
in activity. Because the meta-Br (6p) compound showed
higher potency than para-Br (6o) and ortho-Br (6q) analogues,
meta-CH3 (6s), meta-NO2 (6t), meta-Cl (6u), and meta-CN
(6v) derivatives were prepared. Even though these meta-
substituted analogues (6s-u) are more potent than their
With the identification of potent compound 6f, we contin-
ued the search for optimal phenyl ring substitution. Initial
attempts to introduce electron donating CH3/OCH3 (6k,l) or

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1523
Table 2. Optimization of Pharmacophore B:a In Vitro Anti-Influenza A
Virus Activity of Compounds 7a-l
Table 3. Optimization of Pharmacophore C:a In Vitro Anti-Influenza A
Virus Activity of Compounds 8a-u
compdb
R
IC50c, μM
6f
Ph
OH
H
0.14 ( 0.01
0.32 ( 0.01
0.63 ( 0.04
>25
8a
8b
8c
8d
8e
8f
H
compdb
R
IC50c, μM
Ph
CH3
>25
>25
6f
7a
7b
7c
7d
7e
7f
7g
7h
7i
4-CH3
OC(O)CHdCHCH3
0.14 ( 0.01
20.82 ( 1.79
>25
2-furanyl
2-thienyl
3-thienyl
2-pyridyl
4-CH3-Ph
4-OCH3-Ph
4-NO2-Ph
4-Cl-Ph
0.41 ( 0.09
0.07 ( 0.02
0.15 ( 0.01
0.61 ( 0.02
0.87 ( 0.01
1.67 ( 0.03
>6.25
H
8g
8h
8i
>25
H
3-CH3
17.6 ( 0.91
2.79 ( 0.39
7.76 ( 0.22
0.24 ( 0.04
0.47 ( 0.08
>25
8j
5-CH3
4-C2H5
8k
8l
4-C3H7
4-Ph
8m
8n
8o
8p
8q
8r
8s
8t
22.34 ( 1.22
0.26 ( 0.10
6.62 ( 1.17
0.11 ( 0.03
0.64 ( 0.01
1.71 ( 0.56
0.26 ( 0.01
0.29 ( 0.01
0.72 ( 0.11
3-CH3-Ph
2-CH3-Ph
3-OCH3-Ph
2-OCH3-Ph
3-NO2-Ph
3-Cl-Ph
7j
3-CH3, 4-CH3
3-CH2CH2CH2CH2-4
0.29 ( 0.04
>25
7k
7l
0.72 ( 0.04
a Using influenza A/WSN/33 (H1N1) strain. b All the compounds
exhibited CC50 > 25 μM, except 7b (CC50 = 5.27 ( 0.12 μM). c The
concentration required for a test compound to reduce the virus-induced
cytopathic effect (CPE) by 50% relative to the virus control was
expressed as IC50. Values are expressed as the mean of at least two
independent determinations, each experiments performed in triplicates.
3-F-Ph
3-OH-Ph
8u
a Using influenza A/WSN/33 (H1N1) strain. b All the compounds
exhibited CC50 > 25 μM, except 8l (CC50 = 3.68 ( 0.09). c The con-
centration required for a test compound to reduce the virus-induced
cytopathic effect (CPE) by 50% relative to the virus control was
expressed as IC50. Values are expressed as the mean of at least two
independent determinations, each experiments performed in triplicates.
corresponding para analogues (6k,m,n), these compounds are
not superior to 6p. These results suggest that substitution in
meta position could help in orienting the phenyl ring at an
angle to that of the flat furanocoumarin ring, which could
result in better interaction with the active site.
thiophene ring, which led to 5-fold loss of activity for 7l
compared to that of 6f. Thus SAR exploration at pharmaco-
phore B clearly shows that the angelicin scaffold is essential and
the 4-position methyl group is critical and optimal for activity.
Next, we turned our attention to pharmacophore C (2-
benzoyl group on furan ring) modifications (Table 3). To
examine the role of ketone group of lead 6f, the ketone group
was reduced to a secondary alcohol (8a) or to methylene (8b)
group, which led to 2-4-fold loss of activity. However, either
removal of 2-benzoyl group completely (8c), attaching the
phenyl group directly to the furan ring (8d), or replacement of
phenyl group with methyl group (8e), led to complete loss of
activity. Compounds 8a-b, which had the phenyl group
attached to the furan ring through one-carbon linker showed
only a maximum of 2-4-fold loss of activity compared to 6f;
while compounds 8c-e, either without the phenyl group or
withthe phenylgroupatdifferentspatialorientationthanlead
6f showed complete loss of activity. This clearly shows the
importance of 2-benzoyl group attached to the angelicin
scaffold for activity.
For the pharmacophore B (angelicin core) modification
(Table 2), we have used compound 6f as our template to which
all structural modification were compared. When we at-
tempted to either open (7a), remove (7b), or replace the
lactone ring with a phenyl ring (7c), complete loss of activity
was observed. This shows the critical requirement of the
coumarin ring in maintaining the activity. Next, by retaining
the coumarin ring intact, we tried to find the effect of
substitution at various positions. Removal of the 4-methyl
group (7d) or shifting it to the 3-position (7e) or the 5-position
(7f) of the coumarin ring led to decreased activitycompared to
6f. Particularly, removal of the 4-position methyl group of 6f
had led to drastic loss of activity in 7d. It indicates that the
methyl substituent at C-4 position has an essential role in
maintaining activity. Next, replacing the methyl group with
more bulky ethyl (7g), propyl (7h), and phenyl (7i) groups
decreased the activity, with the phenyl substituent 7i being
completely inactive, showing that methyl group is optimal for
activityatthe 4-position ofthe coumarin ring. Byretainingthe
essential 4-methyl group, we then introduced an additional 3-
methyl group (7j) in the coumarin ring, which resulted in
retention of activity with only 2-fold loss of activity. However,
cyclization of 3- and 4-position of coumarin ring (7k) through
a cyclohexyl ring led to complete loss of activity. Also,
we investigated the effect of replacing the furan ring with
Further, we attempted to replace the phenyl group of 6f on
pharmacophore C with different heteroaromatic groups such
as furan (8f), thiophene (8g,8h), and pyridine (8i). 2-Thio-
phene analogue 8g (IC50 = 70 nM) is of particular interest,
which showed a 2-fold improvement in activity than the
phenyl analogue 6f. However, substitution on the thiophene

1524 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Yeh et al.
ring of 8g did not further improve the activity (data not
shown). Hence, substitution effect on the phenyl ring of
pharmacophore C was explored in order to identify optimal
substituent in this region. Introduction of electron donating
CH3/OCH3 (8j,k) groups in the para-position led to decrease
in activity, while introduction of electron withdrawing NO2/
Cl (8l,m) groups in the para-position of the phenyl group led
to drastic loss of activity compared to the unsubstituted 6f.
When the electron donating CH3/OCH3 groups were moved
from the para-position to either the ortho- or meta-position
(8n-q), it led to the identification of a potent meta-OCH3
compound 8p with similar activity to that of unsubstituted 6f.
Because moving the substitution from para- to meta-position
improved the activity for CH3/OCH3 groups, NO2 (8r), Cl
(8s), F (8t), and OH (8u) groups were also introduced at the
meta-positionof the phenyl ring. Particularly, themeta-Cl (8s)
and meta-F (8t) substitution led only to a 2-fold loss of activity
compared to 6f, demonstrating that the meta-position could
tolerate both electron donating and withdrawing groups
much better than the ortho- or para-position of the phenyl
ring in pharmacophore C.
8p from one carbon to eight carbons (9a-9e) to identify
additional binding elements in this region (Table 4). Up to a
five-carbon O-alkyl chain the activity level was retained;
particularly, the n-butyl analogue 9c (IC50 = 80 nM) showed
a 2-fold improved activity over the lead 6f. Further increasing
the chain length to n-octyl (9e) led to drastic loss of activity.
However, introduction of hydrophilic groups at the end of the
alkyl chain, such as the N(CH3)2 (9f) or OH (9g,h) groups, led
to loss of activity. This SAR trend indicates the presence of a
tunnel-like hydrophobic cavity with steric limitation at the
active site to which pharmacophore C interacts.
Individual optimization of pharmacophores A-C has iden-
tified key structural features essential for optimal anti-influ-
enza activity in that region. On the basis of this information,
we next synthesized two compounds by bringing together these
optimized structural features into a single molecule (Table 5).
Thus, the 3-bromo phenyl (6p) was chosen as the pharmaco-
phore A optimized structure, and 3-methoxy phenyl (8p) and
2-thiophene rings (8g) were chosen as the pharmacophore C
optimized structures. Results showed that 10a and 10b dis-
played good anti-influenza activity, with the thiophene deri-
vative 10b being the most potent compound identified in this
study. Thus, an overall improvement of ∼75-fold anti-influ-
enza activity was achieved for 10b (IC50 = 60 nM) through
systematic SAR study on the HTS hit 6a (IC50 = 4.51 μM).
Having identified potent anti-influenza angelicin deriva-
tives, we investigated in detail the potential of a representative
With the identification of analogue 8p, further SAR explora-
tion was carried out by increasing the O-alkyl chain length of
Table 4. Optimization of Pharmacophore C:a In Vitro Anti-Influenza A
Virus Activity of 9a-h
Table 5. Optimization of Pharmacophore A and C Together:a In Vitro
Anti-Influenza A Virus Activity of Compounds 10a,b
compdb
R
IC50, μMc
6f
H
OCH3
OCH2CH3
0.14 ( 0.01
0.11 ( 0.03
0.12 ( 0.02
0.15 ( 0.01
0.08 ( 0.01
0.14 ( 0.01
7.97 ( 2.96
5.28 ( 0.03
6.09 ( 0.67
2.65 ( 0.39
8p
9a
9b
9c
9d
9e
9f
compdb
R1
R2
IC50, μMc
O(CH2)2CH3
O(CH2)3CH3
O(CH2)4CH3
O(CH2)7CH3
O(CH2)2N(CH3)2
O(CH2)2OH
O(CH2)3OH
6p
8p
Br
H
Ph
0.08 ( 0.01
0.11 ( 0.03
0.07 ( 0.02
0.09 ( 0.01
0.06 ( 0.02
0.06 ( 0.02
3-OCH3-Ph
2-thiophene
3-OCH3-Ph
2-thiophene
8g
H
10a
10b
Br
Br
9g
9h
4 (Zanamivir)
a Using influenza A/WSN/33 (H1N1) strain. b All the compounds
exhibited CC50 >25 μM, except 9f (CC50 =17.65 ( 2.08). c The con-
centration required for a test compound to reduce the virus-induced
cytopathic effect (CPE) by 50% relative to the virus control was
expressed as IC50. Values are expressed as the mean of at least two
independent determinations.
a Using influenza A/WSN/33 (H1N1) strain. b All the compounds
exhibited CC50>25 μM. c The concentration required for a test com-
pound to reduce the virus-induced cytopathic effect (CPE) by 50%
relative to the virus control was expressed as IC50. Values are expressed
as the mean of at least two independent determinations, each experi-
ments performed in triplicates.
Figure 2. Cytopathic effect (CPE) produced by influenza A/WSN/33 virus strain in MDCK cells and its abrogation on treatment with 8g
(1 μM).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1525
Table 6. In Vitro Anti-Influenza Activity of 8g against Various Strains
IC50 (μM ( SD)a
virus strain
8g
4 (Zanamivir)
influenza A/WSN/33 (H1N1)
influenza A/TW/141/04 (H1N1)
influenza A/TW/3446/04 (H3N2)
influenza B/TW/710/05
0.07 ( 0.01
0.09 ( 0.01
0.15 ( 0.01
0.09 ( 0.01
0.09 ( 0.01
0.04 ( 0.01
0.06 ( 0.02
0.35 ( 0.14
0.03 ( 0.01
0.03 ( 0.02
0.02 ( 0.01
0.10 ( 0.01
influenza B/TW/70325/05
influenza B/TW/99/07
a The concentration required for a test compound to reduce the virus-
induced cytopathic effect (CPE) by 50% relative to the virus control was
expressed as IC50. Values are expressed as the mean of at least two
independent determinations, each experiments performed in triplicates.
Figure 3. Reduction of plaque formation by influenza A/WSN/33
in MDCK cells upon treatment with 8g. MDCK cells were infected
with influenza virus A at approximate 125 plaque-forming unit
(PFU)/well. Different concentrations of compound were added at
the stages of viral adsorption and post infection. (A) The effect of 8g
on viral plaque formation. (B) Quantification of viral plaque
formation following treatment with serial dilutions of compound.
Figure 5. Time-of-addition effect of 8g on influenza A/WSN/33
infection in MDCK cells. (A) MDCK cells were inoculated with
influenza A virus at MOI = 0.001. Compound was added at the
indicated times. Viral infection was performed between -1 and 0 h.
After each incubation period, the test medium containing 1 μM of
compound was removed and the cells were incubated with fresh
medium until 12 h post infection (pi). The supernatant was collected
and viral yield was determined by plaque assay. (B) Viral yields
obtained with respect to different exposure period of 8g to the
culture medium during influenza infection.
Figure 4. Reduction of influenza A/WSN/33 viral yields by com-
pound 8g. MDCK cells were infected by 0.0001 multiplicity of
infection (MOI) of A/WSN/33 virus, and the culture supernatants
were collected at 24 h post infection. Various concentrations of 8g
were added to cells when viral adsorption occurred. Viral yields
were measured by plaque assay.
with 100 nM of 8g, during viral adsorption, completely
abolished progeny production, as indicated by the viral yield
(Figure 4). With the confirmation of the anti-influenza virus A
(WSN/33) activity, we were interested to investigate its po-
tential in other strains of the virus. For this purpose, we
selected three strains each of influenza A (two H1N1 and
one H3N2 strain) and B (Table 6). Influenza inhibition
results showed that 8g effectively inhibits all the influenza
virus strains tested similar to clinically used anti-influenza
medication zanamivir (4), with an IC50 in the range of 40-
150 nM. This result suggests that 8g is a broad spectrum anti-
influenza agent similar to zanamivir (4) and could provide
effective coverage against infection over different strains of
influenza.
With the confirmation of the anti-influenza activity, we
were interested to identify the molecular target for the novel
angelicin derivatives. Initially, we carried out time-of-addition
study (Figure 5) to determine the effectiveness of 8g in
decreasing the viral yield with regard to the time at which
compoundwas added tothe culture medium duringthecourse
compound 8g. The cytopathic effect produced by influenza
virus A (WSN/33) on MDCK cells and its abrogation by
treatment with 8g is shown in Figure 2. In addition to the
standard neutralization assay discussed above, to confirm the
anti-influenza activity of 8g, we carried out plaque reduction
assay, which measured the ability of the compound in redu-
cing the number of plaques formed by virus infected MDCK
cells. In the plaque reduction assay, 8g showed over 50%
reduction of plaque formation at a concentration of 20 nM
and complete abrogation of plaque formation at a concentra-
tion of 100 nM (Figure 3). Further, the anti-influenza activity
of8g was confirmedbydeterminingthe viral yield; a reduction
in virus yield, a direct indicator of the ability of the compound
to decrease the production of next generation influenza
(progeny), was observed upon treatment with 8g. Treatment

1526 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Yeh et al.
Figure 6. Inhibition of influenza RNP activity by 8g using reporter
assay. Different concentrations of compound 8g were incubated
with 293 (human embryonal kidney) cells previously transfected
with pPOLI-Fluc and pHW2000 expression vectors for influenza
virus PB1, PB2, PA, and NP proteins. Treatment with 8g decreased
the ratio of Fluc (firefly luciferase) to Rluc (renilla luciferase)
activity, indicating that 8g inhibited RNP complex mediated tran-
scription of pPOLI-Fluc, resulting in decreased Fluc activity with-
out affecting the Rluc activity much. DMSO was included as the
mock control, and Rluc expression was used as transfection control.
Figure 7. Effect of 8g on the production of viral-like RNA synth-
esis. Different concentrations of compound 8g were incubated with
293 cells previously transfected with pHW2000-PB2, PB1, PA, NP
and pPOLI-CAT-RT (lanes 3-8). Total RNA was isolated
for primer extension assay and then analyzed by polyacrylamide
electrophoresis and autoradiography. The RNA species were
marked on the right. No CAT (lane 1) or no PA/NP (lane 2) was
served as a control. 5S RNA is a loading control.
of the infection. It was found that 8g was able to prevent the
progeny formation when added up to 4 h from the time of
infection, however, less effective in preventing the formation
of progeny after this time, as evidenced by increased produc-
tion of progeny at 4-8 time period post infection. These
results suggest that the compound could be acting at early
stages of viral replication cycle, including viral adsorption
and/or early replication phase. This findingsfurther prompted
us to investigate the potential of 8g to acts as hemagglutinin
(HA) inhibitor;HA is a surface glycoproteinofinfluenza virus
involved in viral attachment to the host cell and hence
essential for viral absorption on to the cells. Ability of 8g to
inhibit HA was investigated using hemagglutinination inhibi-
tion assay, where the ability of the compound to prevent the
attachment of virus to guinea pig red blood cells (RBCs) was
measured (Figure 1s, Supporting Information). Compound
8g did not inhibit HA even at a high concentration of 25 μM,
as evidenced by the lack of inhibition of virus attachment to
RBC, ruling out HA as a target for the anti-influenza activity
of this novel angelicin derivatives.
synthesis in 293 cells previously transfected with pcDNA3-
PB2, PB1, PA, NP, and pPOLI-CAT-RT. Transfected cells
were treated with different concentration of 8g and the total
RNA isolated and analyzed through primer extension assay
specific for vRNA (viral RNA) and mRNA through reverse
transcription reaction; the results are shown in Figure 7.
Treatment with 8g decreased the amount of mRNA produc-
tion, indicating that the compound targets viral RNP com-
plex, which drives the reporter gene expression; further, we
have already shown that another potent compound from this
series, 6f, act through interference of viral RNP activity.25
Further, we also evaluated the ability of the potent compound
8g side-by-side with the inactive compound 7d (as negative
control) in plaque formation, viral yield reduction, and viral-
like RNA synthesis assay to show that the anti-influenza
inhibition is restricted to only potent compound 8g (Figures
3s, 4s, and 5s, Supporting Information).
Next, we turned our attention to see if 8g is acting at the
level of viral replication by determining its effect on RNP
(ribonucleoprotein) activity through a plasmid-based reverse
genetics using luciferase reporter assay.23,24 Reporter assay
was performed in human 293 (human embryonal kidney) cells
transfected with pPOLI-Fluc plasmids (contains the firefly
luciferase open reading frame in minus sense) and pHW2000
expression vectors for influenza virus PB1, PB2, PA, and NP
proteins (these viral proteins are essential to amplify and
transcribe the influenza virus-like RNA expressed by pPO-
LI-Fluc into mRNA, resulting in the detection of firefly
luciferase reporter activity), along with pRL-TK as a transfec-
tion control. Transfected cells were then treated with different
concentrations of 8g for 20 h, and the firefly (Fluc) and renilla
(Rluc) luciferase activity measured and the ratio between Fluc
to Rluc activity shown in Figure 6. Treatment with 8g
decreased the amount of Fluc activity without affecting Rluc
activity much (Figure 2s, Supporting Information), indicating
that the 8g treatment decreased the viral-like RNA transcrip-
tion by inhibiting the viral RNP complex, which also drives
the reporter gene expression.
Finally, we have also carried out immunofluorescence
microscopic analysis of MDCK cells infected by influenza
virus using anti-dsRNA (double strand RNA), anti-NP, and
anti-M1 antibodies to visualize the viral RNA and proteins in
the infected MDCK cells. Presence of dsRNA, NP (nucleo-
protein), and M1 (matrix protein) is an indicator of ongoing
viral replication. Treatment of 8g resulted in decreased
amounts of dsRNA, NP, and M1 protein (Figures 6s, 7s,
and 8s, Supporting Information), as evidenced by the de-
creased amount of immunofluorescence signals compared to
the control. Thus the preliminary mechanistic studies using 8g
and 6f25 suggest that they act through interference of viral
RNA synthesis by targeting RNP complex. It should be noted
that there are other agents such as ribavirin, viramidine, and
T-705 reported in the literature to possess anti-influenza
activity, which act as RNA dependent RNA polymerase
inhibitors.3,26,27 Moreover, the novel angelicin derivative 6f
also possesses activity against oseltamivir (3) resistant influ-
enza and other RNA viruses such as enterovirus 71, coxsackie
B, and human rhinovirus; however, the mechanism for broad
antiviral activity needs to be investigated further.25 These
results suggest that the angelicin derivatives described here
In addition to the reporter assay, we carried out primer
extension assay to see the effect of 8g on viral-like mRNA

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1527
are novel anti-influenza agents with mode of action different
from that of the approved influenza therapies in the market.
The above obtained 12 (1.0 equiv) was mixed well with finely
powdered anhydrous aluminum chloride (3.0 equiv) and heated
at 170 ꢀC for 2 h. After cooling, ice and diluted hydrochloric acid
(20 mL) were added and extracted with EtOAc (30 mL ꢀ 3).
The EtOAc layer was washed with diluted hydrochloric acid
(20 mL ꢀ 2) and then with water (20 mL ꢀ 2), followed by satd
NaHCO3(aq) (30 mL ꢀ 2). The combined organic layers were
evaporated to give 13 as a solid product, which was used for next
step without purification.
Conclusion
The threat of a pandemic influenza is real and could be a
global disaster in waiting. Considering the multifaceted pro-
blem of developing vaccines for millions of people, particu-
larly with the genetics of influenza shifting/drifting, there is a
global trend to stockpile effective anti-influenza medication.
However, emergence of resistant strains to this medication
necessitates the development of newer therapeutic alterna-
tives. A novel series of angelicin derivatives identified from the
HTS campaign is optimized through detailed SAR explora-
tion. The results suggest that pharmacophore B consisting of
the angelicin scaffold is very sensitive to structural modifica-
tion and is essential for maintaining the activity, while phar-
macophores A and C are more amenable for structural
modification, with the meta-substituted phenyl/2-thiophene
rings being optimal for activity in this region. The optimized
compound 8g inhibits the activity of both influenza A and B
strains invitro, suggesting thatit couldact asa leadcompound
for further anti-influenza drug development. As preliminary
mechanistic studies point toward viral RNP as a probable
molecular target for these agents, combination therapy with
existing anti-influenza drugs which act through the NA target
could be used to avoid the development of resistant influenza
strains. Moreover, as angelicin derivatives are rarely reported
as antiviral agents, the leads identified here could open up new
opportunities for antiviral drug discovery in general, which
need to be explored further to realize the full potential of this
novel series. Detailed mechanistic studies and further poten-
tial for development are under active pursuit in our labo-
ratories, and the findings will be revealed in due course.
A solution of 13 (1.0 equiv), 2-bromo ketone (R5COCH2Br)
(1.2 equiv), and K2CO3 (3.0 equiv) in CH3CN (5 mL) was
refluxed for 16 h. The reaction mixture was filtered, the solvents
from the filtrate were removed under vacuum, and the crude
product was purified by silica gel column chromatography
(hexane/EtOAc: 3/1 f 1/1) to give the desired products 6-10,
with 15-44% yield over three steps.
8-Benzoyl-4,9-dimethyl-furo[2,3-h]chromen-2-one 6a. Method
A. Yield 31% over three steps. 1H NMR (300 MHz, CDCl3): δ
8.06-8.01 (m, 2H), 7.67 (d, J = 8.7 Hz, 1H), 7.64-7.60 (m, 1H),
7.56-7.51 (m, 3H), 7.45 (d, J = 8.7 Hz, 1H), 6.29 (d, J = 0.9 Hz,
1H), 2.91 (s, 3H), 2.51 (d, J = 0.9 Hz, 3H). LCMS [M þ 1]þ:
319.1.
8-Benzoyl-4-methyl-furo[2,3-h]chromen-2-one 6b (Scheme 2).
To a solution of 7-hydroxy-4-methylcoumarin 11a (2.00 g, 11.30
mmol) in ethanol (5 mL), was added I2 (2.32 g, 9.10 mmol) and
periodic acid (0.53 g, 2.3 mmol) and stirred at room temperature
for 2 h. The reaction mixture was diluted with water, and the
precipitate formed was collected and recrystallized from ethanol
to give light-yellow colored needles of 7-hydroxy-8-iodo-4-
methylcoumarin (14, 2.10 g, 76%). 1H NMR (300 MHz,
CDCl3): δ 10.77 (s, 1H), 7.43 (d, J = 8.7 Hz, 1H), 6.94 (d,
J = 8.7 Hz, 1H), 6.08 (d, J = 1.2 Hz, 1H), 2.41 (d, J = 1.2 Hz,
1H). LCMS [M þ 1]þ: 302.9.
A solution of 14 (1.01 g, 3.30 mmol), 1-phenyl-2-propyn-1-ol
(0.6 mL), CuI (0.01 g, 0.05 mmol), and Et3N (1 mL) in DMF
(5 mL) was stirred at 60 ꢀC for 24 h. The reaction mixture was
quenched by addition of water and extracted with EtOAc
(50 mL ꢀ 3). The combined organic layer was dried over
MgSO4, filtered, and evaporated to give the crude product,
which was purified by column chromatography over silica gel
(hexane/EtOAc: 3/1 f 1/1) to furnish 8-(hydroxy-phenyl-
methyl)-4-methyl-furo[2,3-h]chromen-2-one (15, 0.52 g, 51%).
1H NMR (300 MHz, CDCl3): δ 7.50-7.19 (m, 6H), 6.81 (s, 1H),
6.16 (d, J = 0.9 Hz, 1H), 5.93 (s, 1H), 3.70 (br, 1H), 2.40 (d, J =
0.9 Hz, 3H). LCMS [M þ 1]þ: 307.1.
Experimental Section
General Methods. All commercial chemicals and solvents are
reagent grade and were used without further treatment unless
otherwise noted. All reactions were carried out under an atmo-
sphere of dry nitrogen. Reactions were monitored by TLC using
Merck 60 F254 silica gel glass backed plates (5 ꢀ 10 cm); zones
were detected visually under ultraviolet irradiation (254 nm) or
by spraying with phosphomolybdic acid reagent (Aldrich) fol-
lowed by heating at 80 ꢀC. Flash column chromatography was
done using silica gel (Merck Kieselgel 60, no. 9385, 230-400
mesh ASTM). 1H and 13C NMR spectra were obtained with a
Varian Mercury-300 or Varian Mercury-400 or Bruker DMX-
600 spectrometers. Chemical shifts were recorded in parts per
million (ppm, δ) and were reported relative to the solvent peak
or TMS. High-resolution mass spectra (HRMS) were measured
with a Finnigan (MAT-95XL) electron impact (EI) or using
Finnigan/Thermo Quest MAT 95XL FAB mass spectrometer.
LCMS data were measured on an Agilent MSD-1100 ESI-MS/
MS system. Purity of the final compounds were determined
using a Hitachi 2000 series HPLC system using a C-18 column
(Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm ꢀ 150 mm)
or a phenyl column (Waters XBridge 5 μm, 4.6 mm ꢀ 150 mm)
and were found to be >95% unless otherwise stated.
To a solution of 15 (0.03 g, 0.10 mmol) in acetone (5 mL) was
added Jones reagent at 0 ꢀC and then stirred at room tempera-
ture for 0.5 h. The reaction mixture was evaporated, and the
crude product was purified by column chromatography over
silica gel (hexane/EtOAc: 3/1 f1/1) to give 6b (0.03 g, 95%). 1H
NMR (300 MHz, CDCl3): δ 8.05-8.01 (m, 2H), 7.83 (s, 1H),
7.83-7.68 (m, 2H), 7.66-7.28 (m, 3H), 6.32 (d, J = 0.9 Hz, 1H),
2.53 (s, 3H). LCMS [M þ 1]þ: 305.0.
8-Benzoyl-4-methyl-9-propyl-furo[2,3-h]chromen-2-one 6c.
1
Method A. Yield 32% over three steps. H NMR (300 MHz,
CDCl3): δ 8.06-7.44 (m, 7H), 6.32 (d, J = 1.2 Hz, 1H), 3.37 (t,
J = 5.4 Hz, 2H), 2.52 (d, J = 1.2 Hz, 3H), 1.87 (m, 2H), 1.07 (t,
J = 7.2 Hz, 3H). LCMS [M þ 1]þ: 347.2.
8-Benzoyl-9-hexyl-4-methyl-furo[2,3-h]chromen-2-one 6d.
1
Method A. Yield 35% over three steps. H NMR (300 MHz,
CDCl3): δ 8.06-7.52 (m, 7H), 6.31 (d, J = 1.2 Hz, 1H), 3.38 (t,
J = 7.5 Hz, 2H), 2.52 (d, J = 1.2 Hz, 3H), 1.80 (quintet, J =
7.8 Hz, 2H), 1.62-1.26 (m, 6H), 0.87 (t, J = 6.9 Hz, 3H). LCMS
[M þ 1]þ: 389.2. HPLC purity: 91.9%.
General Synthetic Method for Compounds 6-10 (Method A;
Scheme 1). Appropriately substituted 7-hydroxy-coumarin 11
(1.0 equiv) was dissolved in THF (40 mL), triethylamine
(1.1 equiv) was added, and then the appropriate acyl chloride
(R4COCl; 1.2 equiv) and stirred at room temperature for 16 h.
The solid product (12) formed was filtered, washed with EtOAc,
and the product obtained was used for next step without further
purification.
8-Benzoyl-9-decyl-4-methyl-furo[2,3-h]chromen-2-one 6e.
1
Method A. Yield 39% over three steps. H NMR (300 MHz,
CDCl3): δ 8.06-7.44 (m, 7H), 6.31 (s, 1H), 3.38 (t, J = 7.5 Hz,
2H), 2.51 (s, 3H), 1.80 (quintet, J = 7.5 Hz, 2H), 1.63 (br, 2H),
1.48 (quintet, J = 7.2 Hz, 2H), 1.43 (br, 10H), 0.86 (t, J = 6.3
Hz, 3H). LCMS [M þ 1]þ: 445.2.

1528 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
8-Benzoyl-4-methyl-9-phenyl-furo[2,3-h]chromen-2-one 6f.
Method A. Yield 35% over three steps. H NMR (300 MHz,
Yeh et al.
(300 MHz, CDCl3): δ 7.81-7.74 (m, 3H), 7.60-7.44 (m, 5H),
7.38-7.33 (m, 2H), 7.28-7.22 (m, 1H), 6.23 (d, J = 1.2 Hz, 1H),
2.46 (d, J = 1.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 185.1,
159.2, 156.3, 152.8, 149.7, 148.3, 136.4, 132.6, 133.1, 131.4,
129.5, 129.2, 129.2, 128.2, 127.14, 124.4, 121.6, 116.0, 115.4,
113.7, 108.9, 19.5. HRMS (Mþ): calcd for C25H15BrO4
458.0154, found 458.0144.
1
CDCl3): δ 7.79-7.72 (m, 3H), 7.57 (d, J = 9 Hz, 1H), 7.49-7.42
(m, 3H), 7.43-7.27 (m, 5H), 6.25 (d, J = 1.2 Hz, 1H), 2.50 (d,
J = 1.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 185.5, 159.5,
156.5, 152.8, 150.0, 148.1, 136.6, 132.9, 130.6, 129.7, 129.6,
128.8, 128.1, 127.7, 124.2, 116.4, 115.5, 108.9, 19.6. HRMS
(Mþ): calcd for C25H16O4 380.1049, found 380.1041.
8-Benzoyl-4-methyl-9-naphthalen-1-yl-furo[2,3-h]chromen-2-
one 6g. Method A. Yield 32% over three steps. 1H NMR
(400 MHz, CDCl3): δ 7.84-7.73 (m, 3H), 7.65-7.62 (m, 4H),
7.46-7.25 (m, 5H), 7.07-7.03 (m, 2H), 6.11 (d, J = 0.9 Hz, 1H),
2.44 (d, J = 0.6 Hz, 3H). LCMS [M þ 1]þ: 431.1. HPLC purity:
90.4%.
8-Benzoyl-9-(2-bromo-phenyl)-4-methyl-furo[2,3-h]chromen-2-
one 6q. Method A. Yield 22% over three steps. 1H NMR
(300 MHz, CDCl3): δ 7.81-7.74 (m, 3H), 7.59-7.44 (m, 5H),
7.38-7.33 (m, 2H), 7.28-7.22 (m, 1H), 6.27 (d, J = 1.2 Hz, 1H),
2.51 (d, J = 1.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 185.1,
159.2, 156.3, 152.8, 149.7, 148.3, 136.4, 133.6, 133.1, 131.7,
129.5, 129.2, 129.2, 128.2, 127.1, 124.4, 121.6, 116.0, 115.4,
113.7, 108.9, 19.5. LCMS [M þ 1]þ: 459.0.
8-Benzoyl-4-methyl-9-naphthalen-2-yl-furo[2,3-h]chromen-2-one
6h. Method A. Yield 33% over three steps. 1H NMR (300 MHz,
CDCl3): δ 7.89-7.21 (m, 14H), 6.22 (s, 1H), 2.47 (s, 3H). LCMS
[M þ 1]þ: 431.1.
8-Benzoyl-9-(3,5-dibromo-phenyl)-4-methyl-furo[2,3-h]chromen-
2-one 6r. Method A. Yield 34% over three steps. 1H NMR
(300 MHz, CDCl3): δ 7.84 (d, J = 6.9 Hz, 2H), 7.76 (d, J =
9.0 Hz, 1H), 7.64 (t, J = 1.8 Hz, 1H), 7.53-7.59 (m, 4H), 7.43 (t,
J = 6.0 Hz, 2H), 6.29 (d, J = 1.5 Hz, 1H), 2.52 (d, J = 1.5 Hz,
3H). LCMS [M þ 1]þ: 539.9. HPLC purity: 90.7%.
8-Benzoyl-4-methyl-9-thiophen-2-yl-furo[2,3-h]chromen-2-one
6i. Method A. Yield 26% over three steps. 1H NMR (300 MHz,
CDCl3): δ 7.83-7.29 (m, 10H), 6.26 (d, J = 0.6 Hz, 1H), 2.48 (d,
J = 0.6 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 185.2, 159.5,
156.3, 152.9, 149.7, 148.5, 136.4, 133.1, 131.3, 129.5, 129.2,
128.3, 128.2, 126.8, 124.3, 120.9, 116.0, 115.4, 113.5, 108.9,
19.5. LCMS [M þ 1]þ: 387.0. HPLC purity: 94.5%.
8-Benzoyl-9-(3-methyl-phenyl)-4-methyl-furo[2,3-h]chromen-
2-one 6s. Method A. Yield 26% over three steps. 1H NMR
(300 MHz, CDCl3): δ 7.76-7.72 (m, 3H), 7.57 (d, J = 9.0 Hz,
1H), 7.46-7.41 (m, 1H), 7.33-7.20 (m, 5H), 7.10 (d, J = 7.5 Hz,
1H), 6.25 (d, J = 0.9 Hz, 1H), 2.50 (d, J = 0.9 Hz, 3H), 2.25 (s,
3H). HRMS(Mþ):calcd for C26H18O4 394.1205, found 394.1207.
8-Benzoyl-9-(3-nitro-phenyl)-4-methyl-furo[2,3-h]chromen-2-
one 6t. Method A. Yield 42% over three steps. 1H NMR
(300 MHz, CDCl3): δ 8.25-7.61 (m, 11H), 6.21 (s, 1H), 2.51
(d, J = 1.2 Hz, 3H). LCMS [M þ 1]þ: 426.0.
8-Benzoyl-9-furan-2-yl-4-methyl-furo[2,3-h]chromen-2-one 6j.
1
Method A. Yield 40% over three steps. H NMR (300 MHz,
CDCl3): δ 7.84-7.24 (m, 9H), 6.48-6.46 (m, 1H), 6.28 (s, 1H),
2.49 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 185.5, 159.6, 156.4,
153.0, 149.3, 148.0, 143.3, 142. 8, 136.8, 133.0, 129.2, 128.3,
124.1, 116.9, 115.4, 114.6, 114.4, 113.3, 111.9, 108.9, 19.5.
LCMS [M þ 1]þ: 371.0. HPLC purity: 90.5%.
8-Benzoyl-9-(3-chloro-phenyl)-4-methyl-furo[2,3-h]chromen-2-
one 6u. Method A. Yield 33% over three steps. 1H NMR
(300 MHz, CDCl3): δ 7.83 (d, J = 7.2 Hz, 2H), 7.76 (d, J =
9.0 Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H), 7.56-7.53 (m, 3H), 7.48-
7.37 (m, 4H), 6.28 (s, 1H), 2.52 (s, 3H). LCMS [M þ 1]þ: 415.0.
8-Benzoyl-9-(3-cyano-phenyl)-4-methyl-furo[2,3-h]chromen-2-
one 6v. Method A. Yield 26% over three steps. 1H NMR
(300 MHz, CDCl3): δ 7.88-7.76 (m, 5H), 7.75-7.51 (m, 4H),
7.43-7.38 (m, 2H), 6.27 (d, J = 1.2 Hz, 1H), 2.51 (d, J = 1.2 Hz,
3H). LCMS [M þ 1]þ: 406.1.
8-Benzoyl-4-methyl-9-p-tolyl-furo[2,3-h]chromen-2-one 6k.
1
Method A. Yield 26% over three steps. H NMR (300 MHz,
CDCl3): δ 7.78-7.68 (m, 3H), 7.53-7.45 (m, 2H), 7.42-7.27 (m,
4H), 7.13-7.10 (m, 2H), 6.22 (s, 1H), 2.46 (s, 3H), 2.32 (s, 3H).
13C NMR (75 MHz, CDCl3): δ 185.4, 159.5, 156.3, 152.9, 149.8,
147.9, 138.4, 136.5, 132.7, 130.5, 129.6, 128.7, 128.4, 128.0,
127.8, 126.4, 124.1, 116.2, 115.2, 113.3, 108.8, 21.3, 19.4. LCMS
[M þ 1]þ: 395.2.
8-Benzoyl-9-(4-methoxyphenyl)-4-methyl-2H-furo[2,3-h]chromen-
1
(E)-But-2-enoic Acid 2-Benzoyl-3-phenyl-benzofuran-4-yl Es-
ter 7a (Scheme 3). To a solution of 1-methoxy-3-methoxy-
methoxy-benzene22 (17, 0.20 g, 1.19 mmol) in dry THF (10 mL) at
0 ꢀC was added nBuLi (0.96 mL, 1.6 mol in hexane) dropwise.
After addition was completed, the mixture was stirred at same
temperature for 10 min. Benzoyl chloride (0.17 mL, 1.45 mmol)
was then added dropwise to the solution. The mixture was stirred
for 2 h at room temperature. The reaction was quenched by
addition of water, extracted with EtOAc, dried over MgSO4,
filtered, and concentrated. The residue was diluted with MeOH
(15 mL), and 2 drops of 13N HCl were added and then heated at
reflux temperature for 0.5 h. Then the solvent was removed under
reduced pressure, and water was added to the residue and extracted
with EtOAc. The combined organic layers were dried over MgSO4,
concentrated under reduced pressure and the residue obtained was
purified by column chromatography on silica gel (hexane/EtOAc:
10/1) to give (2-hydroxy-6-methoxy-phenyl)-phenyl-methanone
2-one 6l. H NMR (400 MHz, CDCl3): δ 7.75-7.69 (m, 3H),
7.54 (d, J = 8.8 Hz, 1H), 7.46-7.34 (m, 3H), 7.30-7.26 (m, 2H),
6.84-6.80 (m, 2H), 6.24 (q, J = 1.2 Hz, 1H), 3.78 (s, 3H), 2.48
(d, J = 1.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 185.6, 160.0,
159.6, 156.5, 152.9, 150.1, 148.0, 136.7, 132.8, 132.1, 129.7, 128.7,
128.1, 124.1, 121.6, 116.4, 115.3, 113.5, 113.3, 109.0, 55.2, 19.6.
HRMS calcd for C26H18O5 410.1154, found 410.1153.
8-Benzoyl-4-methyl-9-(4-nitro-phenyl)-furo[2,3-h]chromen-2-
one 6m. Method A. Yield 28% over three steps. 1H NMR
(300 MHz, CDCl3): δ 8.27-8.24 (m, 2H), 7.90-7.80 (m, 2H),
7.77-7.70 (m, 3H), 7.70-7.53 (m, 2H), 7.44-7.27 (m, 2H), 6.28
(d, J = 0.9 Hz, 1H), 2.52 (d, J = 0.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3): δ 184.6, 159.1, 156.2, 152.8, 147.9, 136.9,
136.3, 133.5, 131.5, 130.2, 129.8, 129.5, 128.4, 128.2, 126.4,
124.7, 123.0, 115.7, 113.9, 109.1, 19.5. LCMS [M þ 1]þ: 426.1.
HPLC purity: 91.3%.
8-Benzoyl-9-(4-chloro-phenyl)-4-methyl-furo[2,3-h]chromen-2-
one 6n. Method A. Yield 35% over three steps. 1H NMR
(300 MHz, CDCl3): δ 7.82-7.78 (m, 2H), 7.75 (d, J = 9.3 Hz,
1H), 7.57 (d, J = 8.7 Hz, 1H), 7.54-7.49 (m, 1H), 7.45-7.42 (m,
2H), 7.38-7.30 (m, 4H), 6.27 (d, J = 1.2 Hz, 1H), 2.51 (d, J =
0.9 Hz, 3H). LCMS [M þ 1]þ: 415.1.
1
(18a, 0.20 g, 75%). H NMR (400 MHz, CDCl3): δ 10.72 (s,
1H), 7.61-7.59 (m, 2H), 7.51-7.46 (m, 1H), 7.41-7.35 (m, 3H),
6.65 (dd, J = 0.8, 8.4 Hz, 1H), 6.40 (d, J = 8.4 Hz, 1H), 3.49 (s,
3H). HRMS (FAB) calcd for C14H12O3 228.0786, found 228.0782.
A mixture of 18a (0.30 g, 1.31 mmol), potassium carbonate
(0.40 g, 2.89 mmol), 2-bromoacetophenone (0.31 g, 1.57 mmol),
and 20 mL of acetonitrile was heated at reflux temperature for
16 h. The solution was cooled to room temperature, acetonitrile
removed under reduced pressure, water added to the residue,
and extracted with EtOAc (20 mL ꢀ 3). The combined organic
layers were dried over MgSO4 and concentrated under reduced
pressure. Purification of the residue by column chromatography
8-Benzoyl-9-(4-bromo-phenyl)-4-methyl-furo[2,3-h]chromen-2-
one 6o. Method A. Yield 23% over three steps. 1H NMR
(300 MHz, CDCl3): δ 7.82-7.36 (m, 11H), 6.28 (d, J = 1.2 Hz,
1H), 2.51 (d, J = 1.2 Hz, 3H). HRMS (Mþ): calcd for C25H15-
BrO4 458.0154, found 458.0148.
8-Benzoyl-9-(3-bromo-phenyl)-4-methyl-furo[2,3-h]chromen-
2-one 6p. Method A. Yield 37% over three steps. 1H NMR

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1529
on silica gel (hexane/EtOAc: 10/1) furnished (4-methoxy-3-
phenyl-1-benzofuran-2-yl)(phenyl)methanone (19, 0.31 g, 72%)
as a yellow viscous liquid. 1H NMR (400 MHz, CDCl3): δ
7.79-7.56 (m, 2H), 7.47-7.39 (m, 4H), 7.31-7.23 (m, 6H), 6.70
(d, J = 10.4 Hz, 1H), 3.74 (s, 3H). HRMS calcd for C22H16O3
328.1099, found 328.1097.
(600 MHz, CDCl3): δ 7.95-7.91 (m, 4H), 7.74 (dd, J = 1.2, 9.0
Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.50-7.44 (m, 7H), 7.39-7.32
(m, 3H). 13C NMR (150 MHz, CDCl3): δ 184.8, 153.0, 147.6,
137.4, 132.8, 132.4, 131.5, 131.0, 130.3, 129.8, 129.7, 129.2,
128.7, 128.5, 128.3, 128.0, 127.0, 125.2, 123.2, 122.0, 112.7.
HRMS (Mþ): calcd for C25H16O2 348.3933, found 348.3947.
8-Benzoyl-9-phenyl-furo[2,3-h]chromen-2-one 7d. Method A.
To a solution of 19 (0.25 g, 0.75 mmol) in dry dichloromethane
(15 mL) at 0 ꢀC was added BBr3 (2.25 mL, 1.0 mol in dichloro-
methane) dropwise. After addition was completed, the mixture
was then brought to room temperature and stirred for 12 h. The
reaction was quenched by addition of water, extracted with di-
chloromethane, dried over MgSO4, filtered, and concentrated.
Purification of the residue by column chromatography on silica
gel (hexane/EtOAc: 5/1) furnished (4-hydroxy-3-phenyl-1-
benzofuran-2-yl)(phenyl)methanone (20, 0.19 g, 80%) as a yellow
1
Yield 35% over three steps. H NMR (400 MHz, CDCl3): δ
7.78-7.74 (m, 3H), 7.58-7.41 (m, 5H), 7.32-7.26 (m, 5H), 6.35
(d, J = 9.6 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 185.4,
159.4, 156.6, 150.5, 148.2, 144.0, 136.6, 132.9, 130.6, 129.7, 129.5,
128.8, 128.4, 128.1, 127.8, 127.5, 116.4, 114.9, 114.3, 109.4.
HRMS (Mþ): calcd for C24H14O4 366.0892, found 366.0876.
8-Benzoyl-3-methyl-9-phenyl-furo[2,3-h]chromen-2-one 7e.
1
Method A. Yield 40% over three steps. H NMR (600 MHz,
1
viscous liquid. H NMR (300 MHz, CDCl3): δ 7.87-7.85 (m,
CDCl3): δ 7.76-7.75 (m, 2H), 7.57 (d, J = 1.1 Hz, 1H), 7.51-
7.41 (m, 5H), 7.31-7.26 (m, 5H), 2.15 (s, 3H). 13C NMR
(150 MHz, CDCl3): δ 185.5, 160.9, 155.8, 149.1, 147.9, 139.7,
136.6, 132.8, 130.6, 129.6, 128.7, 128.4, 128.0, 127.7, 126.8,
124.1, 116.1, 114.9, 109.1, 17.7. HRMS (Mþ): calcd for
C25H16O4 380.1049, found 380.1039.
2H), 7.51-7.31 (m, 9H), 7.19 (d, J = 8.4 Hz, 1H), 6.72 (d, J =
7.8 Hz, 1H), 5.31 (br, 1H). HRMS calcd for C21H14O3 314.0943,
found 314.0943.
To a solution of 20 (0.05 g, 0.16 mmol) in dry pyridine (5 mL)
was added crotonic anhydride (0.03 g, 0.20 mmol) and imidazole
(2 mg, 0.03 mmol). The mixture was stirred at room temperature
for 2 h. The reaction was quenched by addition of water,
extracted with EtOAc, dried over MgSO4, filtered, and concen-
trated. Purification of the residue by column chromatography
on silica gel, (hexane/EtOAc: 8/1) furnished 7a (0.05 g, 81%
yield) as a yellow viscous liquid. 1H NMR (400 MHz, CDCl3): δ
7.88-7.86 (m, 2H), 7.56-7.46 (m, 3H), 7.38-7.33 (m, 5H),
7.28-7.24 (m, 2H), 7.07 (dd, J = 1.6, 7.2 Hz, 1H), 6.63 (dq, J =
14.0, 7.0 Hz, 1H), 5.48 (qd, J = 1.8, 14.0 Hz, 1H), 1.74 (dd, J =
1.8, 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 185.2, 164.1,
155.3, 147.6, 146.7, 145.6, 136.9, 132.7, 130.9, 130.1, 130.0,
129.7, 128.3, 128.0, 127.7, 127.5, 121.2, 117.2, 110.2, 17.9.
HRMS calcd for C25H18O4 382.1205, found 382.1196. HPLC
purity: 94.6%.
8-Benzoyl-5-methyl-9-phenyl-furo[2,3-h]chromen-2-one 7f.
1
Method A. Yield 41% over three steps. H NMR (400 MHz,
CDCl3): 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 10.0 Hz,
1H), 7.77-7.75 (m, 2H), 7.74-7.38 (m, 4H), 7.32-7.26 (m, 5H),
6.38 (d, J = 10.0 Hz, 1H), 2.66 (s, 3H). LCMS [M þ 1]þ: 381.7
HPLC purity: 94.2%.
8-Benzoyl-4-ethyl-9-phenyl-furo[2,3-h]chromen-2-one 7g.
Method A. Yield 28% over three steps. H NMR (600 MHz,
1
CDCl3): δ 7.76-7.73 (m, 3H), 7.54 (d, J = 8.9 Hz, 1H), 7.45-
7.42 (m, 3H), 7.30-7.27 (m, 5H), 6.23 (q, J = 2.0 Hz, 1H), 2.85
(qd, J = 7.4, 2.0 Hz, 2H), 1.33 (t, J = 7.4 Hz, 3H). 13C NMR
(150 MHz, CDCl3): δ 185.5, 159.8, 157.8, 156.3, 150.1, 148.1,
136.6, 132.8, 130.6, 129.6, 128.7, 128.0, 127.7, 123.8, 116.5,
114.6, 111.5, 108.9, 25.4, 12.2. HRMS (Mþ): calcd for C26H18-
O4 394.1205, found 394.1205.
Phenyl-(3-phenyl-benzofuran-2-yl)-methanone 7b (Scheme 3).
To a solution of (2-hydroxy-phenyl)-phenyl-methanone (18b)
(0.30 g, 1.51 mmol) in CH3CN (20 mL) was added K2CO3
(0.52 g, 3.80 mmol) and 2-bromo acetophenone (0.36 g, 1.81
mmol) and the mixture refluxed for 16 h. The solvents were
removed under reduced pressure and the residue dissolved in
EtOAc and washed with water. The organic layer was dried over
MgSO4, concentrated, and the residue purified by column
chromatography on silica gel (hexane/EtOAc: 20/1) to give 7b
8-Benzoyl-9-phenyl-4-propyl-furo[2,3-h]chromen-2-one 7h.
Method A. Yield 38% over three steps. H NMR (300 MHz,
1
CDCl3): δ 7.77-7.72 (m, 3H), 7.53-7.41 (m, 4H), 7.31-7.31 (m,
5H), 6.20 (s, 1H), 2.76 (t, J = 7.6 Hz, 2H), 1.76-1.71 (m, 2H), 1.05
(t, J = 7.6 Hz, 3H). LCMS [M þ 1]þ: 409.1. HPLC purity: 94.2%.
8-Benzoyl-4,9-diphenyl-furo[2,3-h]chromen-2-one 7i. Method
A. Yield 18% over three steps. 1H NMR (600 MHz, CDCl3): δ
7.77-7.75 (m, 2H), 7.58 (d, J = 9.0 Hz, 1H), 7.53-7.52 (m, 3H),
7.50-7.47 (m, 3H), 7.50-7.42 (m, 3H), 7.33-7.32 (m, 3H),
7.29-7.27 (m, 2H), 6.31 (s, 1H). 13C NMR (100 MHz, CDCl3): δ
185.5, 159.4, 156.6, 156.3, 150.7, 148.2, 136.6, 135.7, 132.9,
130.7, 129.8, 129.7, 128.9, 128.8, 128.3, 128.1, 127.8, 126.7,
116.6, 114.5, 114.2, 113.6, 108.9. HRMS (Mþ): calcd for
C30H18O4 442.1205, found 442.1206.
1
(0.42 g, 93%). H NMR (400 MHz, CDCl3): δ 7.87-7.85 (m,
2H), 7.38-7.31 (m, 6H), 7.71-7.69 (m, 1H), 7.65-7.62 (m, 1H),
7.54-7.44 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 185.6,
154.5, 147.0, 137.1, 132.6, 130.8, 129.9, 129.7, 129.3, 128.24,
128.20, 128.1, 128.0, 123.8, 122.3, 112.3. HRMS (Mþ): calcd for
C21H14O2 298.0994, found 298.0976.
Phenyl-(1-phenyl-naphtho[2,1-b]furan-2-yl)-methanone 7c
(Scheme 4). To a solution of 2-naphthanol (21) (0.50 g, 3.47
mmol) in dry THF at 0 ꢀC was added NaH (0.18 g, 4.40 mmol)
and MeI (0.26 mL, 4.20 mmol). The mixture was stirred at room
temperature for 12 h. The reaction was quenched by addition of
water, extracted with EtOAc, dried over MgSO4, filtered, and
concentrated to give 2-methoxy-naphthalene (22). This residue
was diluted with dry CH2Cl2 (50 mL) and added AlCl3 (1.38 g,
10.40 mmol) and benzoyl chloride (0.5 mL, 4.26 mmol). After
stirring for 16 h, the reaction was quenched by addition of water,
extracted with CH2Cl2, dried over MgSO4, filtered, and con-
centrated to give (2-hydroxy-naphthalen-1-yl)-phenyl-metha-
none (23). This residue was diluted with CH3CN (50 mL) and
added K2CO3 (0.97 g, 7.00 mmol) and 2-bromo acetophenone
(0.72 g, 3.60 mmol). The reaction mixture was refluxed for 16 h
and then the solvents were removed under reduced pressure. The
residue obtained was dissolved in EtOAc and washed with
water. The organic layer was dried over MgSO4, concentra-
ted, and the residue purified by column chromatography on
silica gel (hexane/EtOAc: 30/1) to give 7c (0.10 g, 8%). 1H NMR
8-Benzoyl-3,4-dimethyl-9-phenyl-furo[2,3-h]chromen-2-one 7j.
Method A. Yield 38% over three steps. H NMR (600 MHz,
1
CDCl3): δ 7.76-7.72 (m, 2H), 7.73 (d, J = 9.0 Hz, 1H), 7.54 (d,
J = 9.0 Hz, 1H), 7.46-7.41 (m, 3H), 7.30-7.26 (m, 5H), 2.46 (s,
3H), 2.17 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 185.6, 160.7,
155.7, 148.1, 148.0, 146.3, 136.7, 132.8, 130.7, 129.8, 129.7,
128.8, 128.7, 128.0, 127.7, 124.2, 120.8, 116.1, 115.9, 108.7,
15.9, 13.4. HRMS (Mþ): calcd for C26H18O4 394.1205, found
394.1194.
16-Benzoyl-15-phenyl-1,2,3,4-tetrahydro-7,17-dioxa-cyclo-
penta[a]phenanthren-6-one 7k. Method A. Yield 25% over three
steps. 1H NMR (600 MHz, CDCl3): δ 7.76 (dd, J = 1.2, 8.4 Hz,
2H), 7.69 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.46-
7.41 (m, 3H), 7.30-7.26 (m, 5H), 2.85-2.83 (m, 2H), 2.54-2.52
(m, 2H), 1.88-1.86 (m, 2H), 1.79-1.78 (m, 2H). 13C NMR
(150 MHz, CDCl3): δ 185.6, 160.4, 155.7, 147.9, 147.3, 136.7,
132.8, 130.1, 129.9, 129.8, 129.7, 128.8, 128.7, 128.0, 127.7, 123.1,
122.4, 116.2, 115.5, 108.6, 25.9, 24.0, 21.5. HRMS (Mþ):
calcd for C28H20O4 420.1263, found 420.1265. HPLC purity:
90.1%.

1530 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Yeh et al.
8-Benzoyl-4-methyl-9-phenyl-2H-thieno[2,3-h]chromen-2-one
7l (Scheme 5). A mixture of 13a (0.10 g, 0.35 mmol), dimethyl-
thiocarbamoyl chloride (0.09 g, 0.71 mmol), sodium hydride
(0.03 g, 0.73 mmol, 60% in mineral oil), 4-dimethylaminopyr-
idine (2 mg, 0.12 mmol), and dry THF (10 mL) was stirred at
room temperature for 24 h. The reaction was quenched with
water and extracted with EtOAc (3 ꢀ 20 mL). The combined
organic layers were dried over MgSO4, concentrated under
reduced pressure, and the residue purified by silica gel column
chromatography (hexane/EtOAc: 2/1) to give dimethyl-thio-
carbamic acid O-(8-benzoyl-4-methyl-2-oxo-2H-chromen-7-yl)
δ 7.57-7.23 (m, 12H), 6.19 (d, J = 0.9 Hz, 1H), 4.15 (s, 2H), 2.25
(d, J = 0.9 Hz, 3H). LCMS [M þ 1]þ: 367.1.
4-Methyl-9-phenyl-furo[2,3-h]chromen-2-one 8c (Scheme 2). A
solution of 7-hydroxy-4-methylcoumarin 11a (2.00 g, 11.30 mmol),
2-bromo acetophenone (2.93 g, 14.70 mmol), and K2CO3 (2.34 g,
16.90 mmol) in CH3CN (20 mL) was refluxed for 16 h. The
reaction mixture was filtered, the solvents from the filtrate were
removed under vacuum, and the crude product 16a (4-methyl-7-(2-
oxo-2-phenyl-ethoxy)-chromen-2-one) obtained was used as such
for the next step without purification.
A mixture of 16a (0.10 g, 0.34 mmol) and phosphoric acid
(5 mL) in xylene (20 mL) was heated at 140 ꢀC for 16 h. The
reaction mixture was cooled to ambient temperature, poured into
crushed ice, and extracted with EtOAc (20 mL ꢀ 3). The organic
layer was pooled, then dried over MgSO4 and evaporated to give
crude product, which was purified over silica gel column chro-
matography (hexane/EtOAc: 1/4) to give compound 8c in 18%
yield for two steps. 1H NMR (300 MHz, CDCl3): δ 7.74-7.70 (m,
3H), 7.57 (d, J = 9.0 Hz, 1H), 7.52-7.39 (m, 4H), 6.26 (d, J =
1.2 Hz, 1H), 2.50 (d, J = 0.9 Hz, 3H). LCMS [M þ 1]þ: 277.1.
4-Methyl-8,9-diphenyl-furo[2,3-h]chromen-2-one 8d (Scheme 2).
Compound 8d was prepared in a manner similar to 8c, except
that 7-hydroxy-4-methylcoumarin 11a and 2-bromo-2-phenyl-
acetophenone were used as the staring materials to give 8d (21%
yield for two steps). 1H NMR (300 MHz, CDCl3): δ 7.65-7.61
(m, 3H), 7.56-7.45 (m, 6H), 7.39-7.29 (m, 3H), 6.25 (d, J =
0.9 Hz, 1H), 2.44 (d, J = 1.2 Hz, 3H). LCMS [M þ 1]þ: 353.1.
8-Acetyl-4-methyl-9-phenyl-furo[2,3-h]chromen-2-one 8e.
1
ester (24, 0.11 g, 84%) as a white solid. H NMR (400 MHz,
CDCl3): δ 7.85-7.83 (m, 2H), 7.64 (d, J = 8.8 Hz, 1H), 7.54-
7.50 (m, 1H), 7.40-7.36 (m, 2H), 7.22 (d, J = 2.0 Hz, 1H), 6.20
(q, J = 1.2 Hz, 1H), 3.18 (s, 3H), 2.96 (s, 3H), 2.42 (d, J = 1.2 Hz,
3H). HRMS calcd for C20H17NO4S 367.0878, found 367.0805.
In a sealed tube, 24 (0.11 g, 0.29 mmol) and mesitylene
(12mL) wereheatedat280 ꢀC for 2 h. The solvent was evaporated
and the residue chromatographed on silica gel (hexane/EtOAc: 1/
1) to afford dimethyl-thiocarbamic acid S-(8-benzoyl-4-methyl-
2-oxo-2H-chromen-7-yl) ester (25, 0.07 g, 65%) as a light-yellow
solid. 1H NMR (400 MHz, CDCl3): δ 7.79-7.77 (m, 2H), 7.69 (d,
J = 8.4 Hz, 1H), 7.57-7.53 (m, 2H), 7.42-7.38 (m, 2H), 6.28 (q,
J = 1.2 Hz, 1H), 2.83 (br, 6H), 2.45 (d, J = 1.2 Hz, 3H). HRMS
calcd for C20H17NO4S 367.0878, found 367.0874.
To a solution of 25 (0.14 g, 0.39 mmol) in dry MeOH (20 mL)
was added sodium methoxide (0.03 g, 0.57 mmol). The mixture
was heated at reflux temperature for 1 h and then cooled to room
temperature and the solvent removed under reduced pressure to
give 8-benzoyl-7-mercapto-4-methyl-chromen-2-one (26). This
was dissolved in acetonitrile (20 mL) and added potassium
carbonate (0.06 g, 0.40 mmol) and 2-bromoacetophenone
(0.15 g, 0.75 mmol) and refluxed for 16 h. The reaction mixture
was cooled to room temperature, removed solvent under re-
duced pressure, water added to the residue, and extracted with
EtOAc (3 ꢀ 20 mL). The combined organic layers were dried
over MgSO4, concentrated under reduced pressure, and the
residue purified by silica gel column chromatography (hexane/
EtOAc: 4/1) to give 7l. (0.06 g, 40% yield over 2 steps) as a light-
yellow solid. 1H NMR (600 MHz, CDCl3): δ 7.78 (d, J = 8.6 Hz,
1H), 7.65 (d, J = 8.6 Hz, 1H), 7.58 (dd, J = 1.0, 7.2 Hz, 2H),
7.34-7.29 (m, 3H), 7.16-7.19 (m, 5H), 6.21 (q, J = 0.8 Hz, 1H),
2.47 (d, J = 0.8 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 191.4,
159.0, 152.6, 151.1, 144.1, 140.4, 139.3, 137.0, 134.2, 132.7,
130.6, 129.5, 128.3, 127.9, 127.4, 126.7, 112.4, 118.5, 116.1,
114.2, 19.5. HRMS calcd for C25H16O3S 396.0820, found
396.0815.
1
Method A. Yield 38% for three steps. H NMR (300 MHz,
CDCl3): δ 7.73-7.51 (m, 7H), 6.21 (s, 1H), 2.48 (s, 3H), 2.34 (d,
J = 0.8 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 188.7, 159.3,
155.9, 152.7, 150.1, 148.2, 130.3, 130.0, 129.2, 128.4, 128.2,
127.9, 124.6, 117.0, 115.2, 113.5, 108.8, 28.5, 19.4. LCMS
[M þ 1]þ: 319.1. HPLC purity: 91.5%.
8-(Furan-2-carbonyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-2-
one 8f. MethodA. Yield35%for three steps. 1H NMR(300 MHz,
CDCl3): δ 7.74 (d, J = 8.7 Hz, 1H), 7.62-7.56 (m, 4H), 7.50-
7.44 (m, 4H), 6.55 (dd, J = 1.8, 1.8 Hz, 1H), 6.25 (d, J = 1.2 Hz,
1H), 2.50 (d, J = 1.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ
171.2, 170.70, 159.4, 156.2, 152.8, 151.0, 149.9, 147.6, 147.1,
130.4, 129.5, 128.8, 127.8, 124.3, 121.2, 116.5, 115.3, 113.6,
112.4, 108.8, 19.6. LCMS [M þ 1]þ: 371.0. HPLC purity: 90.9%.
4-Methyl-9-phenyl-8-(thiophene-2-carbonyl)-furo[2,3-h]chromen-
2-one 8g. Method A. Yield 44% for three steps. 1H NMR
(300 MHz, CDCl3): δ 8.11-8.10 (m, 1H), 7.76-7.71 (m, 2H),
7.63-7.60 (m, 3H), 7.58-7.26 (m, 3H), 7.16-7.14 (m, 1H),
6.25 (d, J = 1.2 Hz, 1H), 2.50 (d, J = 1.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3): δ 175.2, 161.69, 160.37, 155.09, 149.83,
149.60, 142.78, 135.99, 135.26, 134.98, 134.85, 129.59,
129.17, 128.66, 128.55, 126.48, 126.02, 113.56, 113.46,
113.01, 108.77, 107.84, 25.46. HRMS (Mþ): calcd for
8-(Hydroxy-phenyl-methyl)-4-methyl-9-phenyl-furo[2,3-h]-
chromen-2-one 8a (Scheme 6). To a solution of 6f (0.05 g,
0.13 mmol) in THF (5 mL) cooled to 0 ꢀC, NaBH4 (6 mg,
0.16 mmol) was added, and the reaction mixture was stirred for
16 h. Water was added and extracted with EtOAc (20 mL ꢀ 3),
and the combined organic layers washed with brine, dried
over MgSO4, and concentrated to give a crude product, which
was purified over silica gel column chromatography (hexane/
EtOAc: 2/1) to give compound 8a (0.03 g, 60%). 1H NMR (300
MHz, CDCl3): δ 7.58-7.54 (m, 2H), 7.52-7.44 (m, 6H),
7.42-7.28 (m, 4H), 6.18 (d, J = 1.2 Hz, 1H), 5.96 (d, J = 5.4
Hz, 1H), 2.76 (d, J = 5.4 Hz, 1H), 2.44 (d, J = 1.2 Hz, 3H).
LCMS [M þ 1]þ: 383.0.
C
23H14O4S 386.0613, found 386.0609.
4-Methyl-9-phenyl-8-(thiophene-3-carbonyl)-furo[2,3-h]chromen-
2-one 8h. Method A. Yield 39% for three steps. 1H NMR
(400 MHz, CDCl3): δ 8.30-8.29 (m, 1H), 7.73 (d, J = 8.8 Hz,
1H), 7.63-7.62 (m, 1H), 7.58-7.55 (m, 3H), 7.44-7.42 (m, 3H),
7.27-7.25 (m, 1H), 6.23 (d, J = 1.2 Hz, 1H), 2.48 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 177.3, 159.4, 156.0, 153.0, 152.8,
149.8, 148.2, 140.3, 134.8, 133.3, 130.4, 129.7, 129.2, 128.8,
128.5, 128.2, 127.9, 127.8, 127.2, 126.8, 126.6, 126.5, 126.4,
125.6, 124.2, 116.5, 115.3, 113.5, 113.1, 108.8, 19.5. LCMS
[M þ 1]þ: 387.0. HPLC purity: 90.5%.
8-Benzyl-4-methyl-9-phenyl-furo[2,3-h]chromen-2-one 8b
(Scheme 6). To a solution of 6f (0.06 g, 0.15 mmol) in CH2Cl2
cooled to 0 ꢀC, TFA (0.56 mL, 0.73 mmol) was slowly added
with stirring for 5 min. Then NaBH4 (0.03 g, 0.73 mmol) was
slowly added to the reaction mixture and stirred at 0 ꢀC for 16 h.
Water was added, extracted with EtOAc (20 mL ꢀ 3), and the
combined organic layers washed with brine, dried over MgSO4,
and concentrated to give a crude product, which was purified
over silica gel column chromatography (hexane/EtOAc: 2/1) to
give compound 8b (0.04 g, 72%). 1H NMR (300 MHz, CDCl3):
4-Methyl-9-phenyl-8-(pyridine-2-carbonyl)-furo[2,3-h]chromen-
2-one 8i. Method A. Yield 31% for three steps. 1H NMR
(300 MHz, CDCl3): δ 8.25-8.24 (m, 1H), 7.80-7.77 (m, 1H),
7.74-7.67 (m, 2H), 7.58 (d, J = 9.0 Hz, 1H), 7.42-7.38 (m, 2H),
7.25-7.20 (m, 4H), 6.24 (d, J = 1.2 Hz, 1H), 2.49 (d, J = 1.2 Hz,
3H). LCMS [M þ 1]þ: 382.1.
4-Methyl-8-(4-methyl-benzoyl)-9-phenyl-furo[2,3-h]chromen-2-
one 8j. Method A. Yield 25% for three steps. 1H NMR

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1531
(300 MHz, CDCl3): δ 7.73-7.69 (m, 3H), 7.55 (d, J = 8.7 Hz,
1H), 7.51-7.47 (m, 2H), 7.36-7.32 (m, 3H), 7.11 (d, J = 8.1 Hz,
2H), 6.24 (d, J = 0.9 Hz, 1H), 2.49 (d, J = 0.6 Hz, 3H), 2.35 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 185.1, 159.5, 156.3, 152.8,
149.9, 148.3, 143.9, 133.9, 130.6, 129.9, 129.7, 128.8, 128.7,
128.6, 128.2, 127.7, 124.0, 116.3, 115.3, 113.5, 108.9, 21.6,
19.5. LCMS [M þ 1]þ: 395.1. HPLC purity: 93.5%.
8-(3-fluoro-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-2-
one 8t. Method A. Yield 35% for three steps. 1H NMR
(300 MHz, CDCl3): δ 7.77 (d, J = 8.7 Hz, 1H), 7.57 (d, J =
8.7 Hz, 1H), 7.56-7.11 (m, 9H), 6.26 (d, J = 1.2 Hz, 1H), 2.51 (d,
J = 1.2 Hz, 3H). LCMS [M þ 1]þ: 399.0. HPLC purity: 94.0%.
8-(3-Hydroxy-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-
2-one 8u (Scheme 7). To a solution of 8p (1.0 equiv) in DCM
(5 mL) BBr3 (2.0 equiv) was added and stirred at room tem-
perature for 2 h. Ice water was added to the reaction mixture and
extracted with EtOAc (20 mL ꢀ 3). The combined organic layer
was dried over MgSO4 and evaporated to give crude product,
which was purified over silica gel column chromatography
(hexane/EtOAc: 1/3 f 1/1) to give compound 8u in 92% yield
1H NMR (400 MHz, CDCl3): δ 7. 77 (d, J = 8.4 Hz, 1H), 7.58
(dd, J = 8.4, 0.4 Hz, 1H), 7.48-7.46 (m, 2H), 7.34 (t, J = 2.4 Hz,
4H), 7.24-7.23 (m, 1H), 7.16 (t, J = 8 Hz, 1H), 6.94 (ddd, J =
8.0, 2.4, 0.8 Hz, 1H), 6.26 (s, 1H), 2.51 (s, 3H). LCMS [M þ 1]þ:
397.1. HPLC purity: 91.1%.
General Method for the Alkylation of 8u with Alkylbromide
(R1Br) to Give 9a-f (Method B; Scheme 7). A solution of 8u
(1.0 equiv), R1Br (1.5 equiv), K2CO3 (3.0 equiv), and KI
(0.2 equiv) in CH3CN (5 mL) was refluxed for 16 h. Solvent
was removed under vacuum, and water was added and extracted
with EtOAc (20 mL ꢀ 3). The combined organic layer was dried
over MgSO4, concentrated under vacuum to give crude product,
which was purified over silica gel column (hexane/EtOAc: 3/1 f
1/1) to give compounds 9a-f.
8-(4-Methoxy-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-
2-one 8k. Method A. Yield 41% for three steps. 1H NMR
(300 MHz, CDCl3): δ 7.82 (dd, J = 10.2, 2.4 Hz, 2H), 7.70 (dd,
J = 9.0, 1.5 Hz, 1H), 7.56-7.47 (m, 3H), 7.37-7.32 (m, 3H),
6.79 (dd, J = 8.7, 2.7 Hz, 2H), 6.23 (d, J = 0.9 Hz, 1H), 3.81
(s, 3H), 2.48 (d, J = 0.9 Hz, 3H). LCMS [M þ 1]þ: 411.1.
4-Methyl-8-(4-nitro-benzoyl)-9-phenyl-furo[2,3-h]chromen-2-
one 8l. Method A. Yield 15% for three steps. 1H NMR
(300 MHz, CDCl3): δ 8.14-8.09 (m, 2H), 7.89-7.85 (m, 2H),
7.79 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.46-7.43 (m,
2H), 7.36-7.30 (m, 3H), 6.27 (d, J = 0.9 Hz, 1H), 2.52 (d, J =
1.2 Hz, 3H). LCMS [M þ 1]þ: 426.1.
8-(4-Chloro-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-2-
one 8m. Method A. Yield 41% for three steps. 1H NMR
(300 MHz, CDCl3): δ 7.76-7.71 (m, 3H), 7.57 (d, J = 8.7 Hz,
1H), 7.48-7.44 (m, 2H), 7.38-7.32 (m, 3H), 7.29-7.24 (m, 2H),
6.25 (d, J = 1.2 Hz, 1H), 2.50 (d, J = 1.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3): δ 184.1, 159.4, 156.4, 152.8, 150.0, 147.8,
139.2, 134.9, 131.0, 130.6, 129.4, 129.1, 128.9, 128.4, 127.9,
124.4, 116.3, 115.4, 113.6, 108.9, 19.5. LCMS [M þ 1]þ: 415.1.
4-Methyl-8-(3-methyl-benzoyl)-9-phenyl-furo[2,3-h]chromen-2-
one 8n. MethodA.Yield 28% for three steps.1H NMR(300 MHz,
CDCl3): δ 7.75-7.18 (m, 11H), 6.26 (s, 1H), 2.51 (d, J = 0.6 Hz,
3H), 2.25 (s, 3H). LCMS [M þ 1]þ: 395.2. HPLC purity: 91.9%.
4-Methyl-8-(2-methyl-benzoyl)-9-phenyl-furo[2,3-h]chromen-
2-one 8o. Method A. Yield 25% for three steps. 1H NMR
(300 MHz, CDCl3): δ 7.75-6.93 (m, 11H), 6.24 (d, J = 1.2 Hz,
1H), 2.42 (s, 3H), 2.18 (s, 3H). LCMS [M þ 1]þ: 396.5. HPLC
purity: 90.1%.
8-(3-Methoxy-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-
2-one 8p. Method A. Yield 30% for three steps. 1H NMR
(300 MHz, CDCl3): δ 7.73 (d, J = 8.7 Hz, 1H), 7.57 (d, J =
8.7 Hz, 1H), 7.50-7.47 (m, 2H), 7.40-7.28 (m, 5H), 7.20 (t, J =
8.7 Hz, 1H), 7.01-6.97 (m, 1H), 6.24 (d, J = 1.2 Hz, 1H), 3.76 (s,
3H), 2.49 (d, J = 1.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ
185.2, 159.5, 159.2, 156.4, 152.8, 149.9, 148.1, 137.8, 130.6,
129.7, 129.1, 128.8, 127.7, 124.2, 122.5, 119.7, 116.3, 115.3,
113.7, 113.5, 108.9, 55.3, 19.5. HRMS (Mþ): calcd for
C26H18O5 410.1154, found 410.1142.
8-(3-Ethoxy-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-
2-one 9a. Method B. Yield 38%. 1H NMR (300 MHz, CDCl3): δ
7.73 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.50-7.46 (m,
2H), 7.37-7.27 (m, 5H), 7.18 (dd, J = 8.4, 8.4 Hz, 1H), 7.00-
6.96 (m, 1H), 6.25 (d, J = 0.9 Hz, 1H), 3.98 (q, J = 6.9 Hz, 2H),
2.50 (d, J = 1.2 Hz, 3H), 1.40 (t, J = 6.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3): δ 185.3, 159.5, 158.6, 156.4, 152.9, 149.9,
148.1, 137.7, 130.6, 129.7, 129.1, 128.8. 128.7, 127.7, 124.2,
122.4, 120.1, 116.3, 115.3, 114.3, 113.5, 109.0, 63.6, 19.6, 14.7.
LCMS [M þ 1]þ: 425.1.
4-Methyl-9-phenyl-8-(3-propoxy-benzoyl)-furo[2,3-h]chromen-
2-one 9b. Method B. Yield 39%. 1H NMR (300 MHz, CDCl3): δ
7.73 (d, J = 8.7 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.50-7.46 (m,
2H), 7.37-7.26 (m, 5H), 7.21-7.15 (m, 1H), 7.00-6.96 (m, 1H),
6.26 (d, J = 1.2 Hz, 1H), 3.86 (d, J = 6.6 Hz, 2H), 2.50 (d, J =
1.2 Hz, 3H), 1.81-1.72 (m, 2H), 1.02 (t, J = 7.5 Hz, 3H). 13C
NMR (75 MHz, CDCl3): δ 185.4, 159.5, 158.8, 156.4, 152.9, 149.9,
148.1, 137.7, 132.3, 130.6, 130.1, 129.7, 129.2, 129.1, 128.7, 128.6,
128.2, 127.9, 127.7, 124.2, 122.3, 120.1, 120.0, 116.3, 115.3, 114.5,
114.4, 113.502, 109.0, 69.6, 22.4, 19.6, 10.5. LCMS [M þ 1]þ: 439.1.
8-(3-Butoxy-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-
2-one 9c. Method B. Yield 38%. 1H NMR (300 MHz, CDCl3): δ
7.73 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.49-7.46 (m,
2H), 7.37-7.26 (m, 5H), 7.18 (dd, J = 7.8, 7.8 Hz, 1H), 7.00-
6.96 (m, 1H), 6.25 (d, J = 1.2 Hz, 1H), 3.90 (t, J = 6.9 Hz, 2H),
2.60 (d, J = 1.2 Hz, 3H), 1.78-1.70 (m, 2H), 1.51-1.43 (m, 2H),
0.97 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 185.4,
159.5, 158.8, 156.4, 152.9, 149.9, 148.1, 137.7, 130.6, 129.7, 129.1,
128.7, 128.6, 127.7, 124.2, 122.3, 120.1, 116.3, 115.3, 114.4, 113.5,
109.0, 67.8, 31.1, 19.6, 19.2, 13.8. LCMS [M þ 1]þ: 453.2.
4-Methyl-8-(3-pentyloxy-benzoyl)-9-phenyl-furo[2,3-h]chromen-
2-one 9d. Method B. Yield 38%. 1H NMR (300 MHz, CDCl3): δ
7.73 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.49-7.46 (m,
2H), 7.37-7.26 (m, 5H), 7.18 (dd, J = 7.8, 7.8 Hz, 1H), 7.00-
6.96 (m, 1H), 6.25 (d, J = 0.9 Hz, 1H), 3.89 (t, J = 6.6 Hz, 2H),
2.50 (s, 3H), 1.78-1.73 (m, 2H), 1.44-1.36 (m, 4H), 0.93 (d, J =
6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 185.4, 159.5, 158.8,
156.4, 152.9, 149.9, 148.1, 137.7, 130.6, 129.6, 129.0, 128.7,
128.6, 127.7, 124.2, 122.3, 120.1, 116.3, 115.3, 114.3, 113.5,
109.0, 68.1, 28.8, 28.1, 22.4, 19.6, 14.0. LCMS [M þ 1]þ: 467.2.
4-Methyl-8-(3-octyloxy-benzoyl)-9-phenyl-furo[2,3-h]chromen-
2-one 9e. Method B. Yield 37%. 1H NMR (300 MHz, CDCl3):
δ 7.72 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.50-7.27
8-(2-Methoxy-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-
2-one 8q. Method A. Yield 42% for three steps. 1H NMR
(300 MHz, CDCl3): δ 7.70 (d, J = 9.0 Hz, 1H), 7.55 (d, J =
8.7 Hz, 1H), 7.37 (dd, J = 7.2 Hz, 1.8 Hz, 1H), 7.33-7.29 (m,
2H), 7.26-7.17 (m, 4H), 6.86 (td, J = 7.4 Hz, 0.7 Hz, 1H), 6.52
(d, J = 8.4 Hz, 1H), 6.21 (d, J = 1.2 Hz, 1H), 3.57 (s, 3H), 2.48
(d, J = 1.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 185.5,
159.5, 157.3, 156.4, 152.9, 150.1, 149.1, 132.8, 130.3, 130.0,
129.3, 128.5, 128.4, 127.9, 127.1, 124.2, 120.2, 116.6, 115.1,
113.3, 110.5, 109.0, 55.2, 19.5. LCMS [M þ 1]þ: 411.1.
4-Methyl-8-(3-nitro-benzoyl)-9-phenyl-furo[2,3-h]chromen-
2-one 8r. Method A. Yield 41% for three steps. 1H NMR
(300 MHz, CDCl3): δ 8.50 (t, J = 2.0 Hz, 1H), 8.25 (dd, J =
8.1, 1.2 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.7 Hz,
1H), 7.62 (d, J = 9.0 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H),
7.465-7.42 (m, 2H), 7.31-7.26 (m, 3H), 6.27 (s, 1H), 2.52 (d,
J = 0.6 Hz, 3H). LCMS [M þ 1]þ: 426.0.
8-(3-Chloro-benzoyl)-4-methyl-9-phenyl-furo[2,3-h]chromen-
2-one 8s. Method A. Yield 30% for three steps. 1H NMR
(300 MHz, CDCl3): δ 7.64-7.37 (m, 11H), 6.23 (d, J = 1.2 Hz,
1H), 2.50 (d, J = 1.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ
184.0, 159.3, 156.5, 152.8, 150.0, 147.6, 138.1, 134.2, 132.7,
130.5, 129.6, 129.5, 129.4, 129.0, 127.8, 127.6, 124.6, 116.3,
115.4, 113.6, 109.0, 19.5. LCMS [M þ 1]þ: 415.0.

1532 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Yeh et al.
(m, 7H), 7.18 (t, J = 8.1 Hz, 1H), 6.98 (ddd, J = 8.1, 2.7, 0.9 Hz,
1H), 6.24 (d, J = 0.9 Hz, 1H), 3.89 (t, J = 6.6 Hz, 2H), 2.49 (d,
J = 0.9 Hz, 3H), 1.75 (quintet, J = 6.6 Hz, 2H), 1.46-1.29 (m,
10H), 0.87 (t, J = 6.6 Hz, 3H). LCMS [M þ 1]þ: 509.2. HPLC
purity: 94.8%.
147.5, 142.6, 135.3, 134.9, 133.3, 131.9, 131.7, 129.3, 129.0,
128.3, 127.2 124.5, 121.7, 116.4, 115.5, 113.8, 108.8, 19.6. LCMS
[M þ 1]þ: 465.0.
Virus Source. Influenza A/WSN/33 were generated using the
12-plasmid-based reverse genetics system described by Fodor
et al.28 Influenza A/TW/141/02 (H1N1), influenza A/TW/3446/
02 (H3N2), influenza B/TW/710/05, influenza B/TW/99/07, and
influenza B/TW/70325/05 were obtained from the Clinical
Virology Laboratory in Chang Gang Children’s Hospital
(Taipei, Taiwan).
8-[3-(2-Dimethylamino-ethoxy)-benzoyl]-4-methyl-9-phenyl-furo-
[2,3-h]chromen-2-one 9f. Method B. Yield 25%. 1H NMR (300
MHz, CDCl3): δ 7.62 (d, J = 9.0 Hz, 1H), 7.50-7.21 (m, 9H),
7.11 (dd, J = 8.4, 2.1 Hz, 1H), 6.26 (s, 1H), 4.47 (t, J = 4.2 Hz,
2H), 3.43 (t, J = 4.2 Hz, 2H), 2.91 (s, 6H), 2.51 (s, 3H). LCMS
[M þ 1]þ: 468.2. HPLC purity: 91.2%.
Inhibition of Virus Induced CPE on MDCK Cells (IC50
Determination). This assay measured the ability of a test com-
pound to inhibit the cytopathic effect induced by influenza virus
on MDCK cells. The 96-well tissue culture plates were seeded
with 200 μL of MDCK cells at a concentration of 1.1 ꢀ 105 cells/
mL in Dulbecco/Vogt modified Eagle’s minimal essential med-
ium (DMEM) with 10% fetal bovine serum (FBS). The plates
were incubated for 24-30 h at 37 ꢀC and were used at about 90%
confluency. Virus (100 TCID50) mixed with different concentra-
tions of test compounds was added to the cells and incubated at
37 ꢀC for 1 h. After adsorption, the infected cell plates were
overlaid with 50 μL of serum free DMEM (E0: DMEM with
penicillin [100 U/mL], streptomycin [100 μg/mL], L-glutamine
[2 mM], nonessential amino acid mixture [0.1 mM], and trypsin
[2.5 μg/mL]) and 0.1% DMSO. The plate was incubated at 37 ꢀC
for 72 h. At the end of incubation, the plates were fixed by
the addition of 100 μL of 4% formaldehyde for 1 h at room
temperature. After the removal of formaldehyde, the plates were
stained with 0.1% crystal violet for 15 min at room temperature.
The plates were washed and dried, and the density of the well was
measured at 570 nm. The concentration required for a test
compound to reduce the virus-induced cytopathic effect (CPE)
by 50% relative to the virus control was expressed as IC50.
All assays were performed in triplicate and at least twice.
Cytotoxicity Assay (CC50 Determination). The 96-well tissue
culture plates were seeded with 200 μL of MDCK cells at a
concentration of 1.1 ꢀ 105 cells/mL in DMEM with 10% FBS.
The plates were incubated for 24-30 h at 37 ꢀC and were used at
about 90% confluency. Test compounds at various concentra-
tions were added to MDCK cells. The plate was incubated at
37 ꢀC for 72 h. At the end of incubation, the plates were fixed by
the addition of 100 μL of 4% formaldehyde for 1 h at room
temperature. After the removal of formaldehyde, the plates were
stained with 0.1% crystal violet for 15 min at room temperature.
The plates were washed and dried, and the density of the well
was measured at 570 nm. The concentration of a test compound
required to reduce cell viability to 50% of the tested control
culture was expressed as CC50. All experiments were performed
in triplicate and at least twice.
8-[3-(2-Hydroxy-ethoxy)-benzoyl]-4-methyl-9-phenyl-furo[2,3-h]-
chromen-2-one 9g (Scheme 7). Compound 8u (0.05 g, 0.13 mmol)
was reacted with (2-bromo-ethoxy)-tert-butyl-dimethyl-silane
(0.06 g, 0.25 mmol) (R2Br) using method B as for 9a-f to give
the intermediate 8-{3-[2-(tert-butyl-dimethyl-silanyloxy)-ethoxy]-
benzoyl}-4-methyl-9-phenyl-furo[2,3-h]chromen-2-one (45 mg,
64%). 1H NMR (300 MHz, CDCl3): δ 7.73 (d, J = 6.6 Hz, 1H),
7.57 (d, J = 6.6 Hz, 1H), 7.49-6.99 (m, 9H), 6.25 (d, J = 0.9 Hz,
1H), 3.99-3.93 (m, 4H), 2.50 (d, J = 0.9 Hz, 3H), 0.90 (s, 9H),
0.09 (s, 6H). LCMS [M þ 1]þ: 555.3.
The above obtained product (0.035 g, 0.06 mmol) was dis-
solved in THF (5 mL), cooled to 0 ꢀC, and TBAF (0.08 g,
0.32 mmol) was added and stirred for 1.5 h. Water was added
slowly to quench the reaction and extracted with EtOAc
(20 mL ꢀ 3). The combined organic layer was dried over MgSO4
and concentrated under vacuum to give crude product, which
was purified over silica gel column (hexane/EtOAc: 1/2) to give
1
9g (7 mg, 24%). H NMR (300 MHz, CDCl3): δ 7.74 (d, J =
9.0 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.49-7.00 (m, 9H), 6.26 (s,
1H), 4.04 (t, J = 4.5 Hz, 2H), 3.94 (t, J = 3.9 Hz, 2H), 2.50 (s,
3H). LCMS [M þ 1]þ: 441.1.
8-[3-(3-Hydroxy-propoxy)-benzoyl]-4-methyl-9-phenyl-furo-
[2,3-h]chromen-2-one 9h (Scheme 7). Compound 8u (0.05 g,
0.13 mmol) was reacted with (3-bromo-propoxy)-tert-butyl-
dimethyl-silane (0.06 g, 0.25 mmol) (R2Br) using method B as
for 9a-f to give the intermediate 8-{3-[3-(tert-butyl-dimethyl-
silanyloxy)-propoxy]-benzoyl}-4-methyl-9-phenyl-furo[2,3-h]-
chromen-2-one (52 mg, 72%). 1H NMR (300 MHz, CDCl3): δ
7.74 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H), 7.50-6.97 (m,
9H), 6.26 (d, J = 1.2 Hz, 1H), 4.02 (t, J = 6.3 Hz, 2H), 3.78 (t,
J = 6.0 Hz, 2H), 2.51 (d, J = 1.2 Hz, 3H), 1.96 (t, J = 6.0 Hz,
2H), 0.88 (s, 9H), 0.04 (s, 6H). LCMS [M þ 1]þ: 569.3.
The above obtained product (0.04 g, 0.07 mmol) was dis-
solved in THF (5 mL), cooled to 0 ꢀC, and TBAF (0.09 g,
0.35 mmol) was added and stirred for 1.5 h. Water was added
slowly to quench the reaction and extracted with EtOAc
(20 mL ꢀ 3). The combined organic layer was dried over MgSO4
and concentrated under vacuum to give crude product, which
was purified over silica gel column (hexane/EtOAc: 1/2) to give
Inhibition of Virus Induced CPE on MDCK Cells by 8g
(Figure 2). MDCK cells (2.5 ꢀ 105 cells/well) were seeded in a
6 cm tissue culture dish, grown for overnight, and then chal-
lenged with virus (multiplicity of infection [MOI] = 0.01). After
adsorption of virus for 1 h on ice, the cells were washed with
Hanks’ balanced salt solutions (HBSS), after which E0 contain-
ing 8g (1 μM) was added. After incubation for 36 h, the CPE was
evaluated under a microscope.
1
9h (8 mg, 26%). H NMR (300 MHz, CDCl3): δ 7.73 (d, J =
8.7 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.49-6.97 (m, 9H), 6.25
(d, J = 1.2 Hz, 1H), 4.06 (t, J = 6.0 Hz, 2H), 3.85 (t, J = 6.0 Hz,
2H), 2.50 (d, J = 1.2 Hz, 3H), 2.06-1.98 (m, 2H). LCMS
[M þ 1]þ: 455.1.
9-(3-Bromo-phenyl)-8-(3-methoxy-benzoyl)-4-methyl-furo-
[2,3-h]chromen-2-one 10a. Method A. Yield 28% for three steps.
1H NMR (300 MHz, CDCl3): δ 7.73 (d, J = 8.7 Hz, 1H), 7.57-
7.54 (m, 2H), 7.49-7.43 (m, 2H), 7.39-7.35 (m, 1H), 7.29-7.20
(m, 3H), 7.04-7.00 (m, 1H), 6.24 (d, J = 1.5 Hz, 1H), 3.78 (s,
3H), 2.48 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 184.9, 159.3,
159.2, 156.2, 152.8, 149.7, 148.2, 137.6, 133.5, 131.7, 131.6,
129.2, 129.1, 127.1, 124.5, 122.2, 121.5, 119.7, 115.9, 115.4,
113.6, 113.5, 108.9, 55.3, 19.5. LCMS [M þ 1]þ: 489.0.
9-(3-Bromo-phenyl)-4-methyl-8-(thiophene-2-carbonyl)-furo-
[2,3-h]chromen-2-one 10b. Method A. Yield 30% for three steps.
1H NMR (300 MHz, CDCl3): δ 7.73 (d, J = 8.7 Hz, 1H), 7.56-
7.48 (m, 2H), 7.48-7.43 (m, 2H), 7.34-7.17 (m, 3H), 7.01-6.98
(m, 1H), 6.27 (d, J = 0.9 Hz, 1H), 2.50 (d, J = 1.2 Hz, 3H). 13C
NMR (75 MHz, CDCl3): δ 175.0, 159.2, 155.9, 152.6, 149.7,
Plaque Reduction Assay. MDCK cells (5.5 ꢀ 105 cells/well)
were seeded into six-well tissue culture plates and incubated
overnight. The cells were incubated with influenza virus at
approximately 125 PFU/well with or without different concen-
trations of the compound. After adsorption of the virus for 1 h at
37 ꢀC, the viral suspension was removed, and the cells were
washed with HBSS. The cells were then overlain with E0
containing 0.3% agarose with indicated compound concentra-
tion. After incubation for 48 h at 37 ꢀC under 5% CO2, the cells
were fixed with 10% formaldehyde and then stained with 1%
crystal violet. The numbers of plaques were counted and the
antiviral activity of the compound was calculated with respect to
virus control.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1533
Time of Addition Assay. MDCK cells (2.4 ꢀ 105 cells/well)
(6) Yuen, K. Y.; Wong, S. S. Human infection by avian influenza A
H5N1. Hong Kong Med. J. 2005, 11, 189–199.
were seeded in six-well tissue culture plates and incubated
overnight. The cells were challenged with virus (MOI 0.001)
on ice for 1 h. After adsorption of the virus, the viral suspension
was removed and the cells were washed with HBSS, then
replenished with fresh E0 medium (pi = 0). The test medium
containing 1 μM of 8g was added during the periods -1 to 0 h
(adsorption), 0-2, 2-4, 4-6, and 6-8 h. After each incubation
period, the monolayer was washed with HBSS and incubated
with fresh medium until 12 h postinfection (pi). The supernatant
was collected and the viral yield was determined by plaque assay.
Inhibition of Viral RNP Activity Using Reporter Assay. Re-
porter assay was performed in 293 cells transfected with reporter
plasmids and pHW2000 expression vectors for influenza virus
PB1, PB2, PA, and NP proteins. In a 48-well dish, approxi-
mately 5 ꢀ 104 per well of 293 cells were transfected with pPOLI-
Fluc plasmid (0.1 μg), pHW2000-PB1 (0.1 μg), -PB2 (0.1 μg),
-PA (0.1 μg), -NP (0.1 μg), and pRL-TK (5 ng, Promega) as a
transfection control by using calcium phosphate method. Ten-
fold serial dilutions of 8g were added 8 h post-transfection, and
the cells were incubated for further 20 h. Cells were then
harvested for luciferase assay using Dual-Luciferase assay sys-
tem (Promega, Madison, WI) and a VICTOR3 plate reader
(PerkinElmer). Values at each dose were analyzed in triplicate
and graphed with standard deviations. This is an average result
from three experiments and the y-axis is the ratio of Fluc to Rluc
controls, normalized to mock treatment (set arbitrarily to 1.0).
Evaluation of Viral-like RNA Synthesis Inhibition Using Pri-
mer Extension Assay. The production of viral RNAs was
measured by plasmid-based reverse genetics system and primer
extension assay.23 One μg of each pHW2000-PB1, PB2, PA, and
NP and pPOLI-CAT-RT were cotransfected into 293 cells in a
6-well plate. Compound 8g was added at 8 h post transfection.
Total cellular RNA was isolated (RNease Mini kit, Qiagen) 20 h
post transfection and hybridized with [γ-32P] end-labeled pri-
mers at 50 ꢀC for 3 h. This primer extension reaction was carried
out using avian myeloblastosis virus reverse transcriptase
(primer extension system-AMV reverse transcriptase, Promega)
at 42 ꢀC for 2 h. The resulting mixtures were terminated by
denaturing at 90 ꢀC for 10 min and analyzed with 8% poly-
acrylamide gel containing 7 M urea. The expected sizes of
mRNA, cRNA (complementary RNA), and vRNA were
98-106, 89, and 158 bp, respectively.
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Acknowledgment. Financial support from National Health
Research Institutes, National Science Council (95-2745-B-
400-002, NSC-97-2321-B-182-003, and NSC 98-2323-B-400-
003), and National Tsing Hua University, Taiwan, is grate-
fully acknowledged.
Supporting Information Available: Hemagglutination inhibi-
tion assay protocol and results, reporter assay (Fluc) results,
immunofluorescence microscopic analysis (dsRNA, NP, and
M1 protein) protocol and results upon 8g treatment, compara-
tive activity of 8g and 7d in plaque formation, viral yield
reduction, and viral-like RNA synthesis assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
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J. T. BPR2-D2 targeting viral ribonucleoprotein complex-asso-
ciated function inhibits oseltamivir-resistant influenza viruses.
J. Antimicrob. Chemother. 2010, 65, 63–71.
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