The isolation and synthesis of a novel benzofuran compound from Tephrosia purpurea, and the synthesis of several related derivatives, which suppress histamine H1 receptor gene expression

Article abstract of DOI:10.1016/j.bmc.2015.09.049

A novel naturally occurring compound with a benzofuran skeleton was isolated from a plant, Tephrosia purpurea collected in Bangladesh. The chemical synthesis of this compound confirmed its structure, and preliminary biological results showed its suppressi

Full text of DOI:10.1016/j.bmc.2015.09.049

Accepted Manuscript
The isolation and synthesis of a novel benzofuran compound from Tephrosia
purpurea, and the synthesis of several related derivatives, which suppress his-
tamine H1 receptor gene expression
Manik Chandra Shill, Asish Kumar Das, Tomohiro Itou, Sanmoy Karmakar,
Pulok K. Mukherjee, Hiroyuki Mizuguchi, Yoshiki Kashiwada, Hiroyuki Fukui,
Hisao Nemoto
PII:
S0968-0896(15)30074-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.049
BMC 12596
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 September 2015
29 September 2015
30 September 2015
Please cite this article as: Shill, M.C., Das, A.K., Itou, T., Karmakar, S., Mukherjee, P.K., Mizuguchi, H., Kashiwada,
Y., Fukui, H., Nemoto, H., The isolation and synthesis of a novel benzofuran compound from Tephrosia
purpurea, and the synthesis of several related derivatives, which suppress histamine H1 receptor gene expression,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.049
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The isolation and synthesis of a novel benzofuran
compound from Tephrosia purpurea, and the synthesis
of several related derivatives, which suppress histamine
H1 receptor gene expression
Leave this area blank for abstract info.
Manik Chandra Shill, Asish Kumar Das, Tomohiro Itou, Sanmoy Karmakar,
Pulok K. Mukherjee, Hiroyuki Mizuguchi, Yoshiki Kashiwada, Hiroyuki Fukui, Hisao Nemoto*

Bioorganic & Medicinal Chemistry
The isolation and synthesis of a novel benzofuran compound from Tephrosia
purpurea, and the synthesis of several related derivatives, which suppress
histamine H1 receptor gene expression
Manik Chandra Shill,b Asish Kumar Das,c Tomohiro Itou,a Sanmoy Karmakar,d
Pulok K. Mukherjee,d Hiroyuki Mizuguchi,b Yoshiki Kashiwada,e Hiroyuki Fukui,f Hisao Nemoto*a
a Department of Pharmaceutical Chemistry, Institute of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima 770-8505, Japan
bDepartment of Molecular Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, 1-78-1 Sho-machi, Tokushima 770-8505,
Japan
c Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh
d School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India
eDepartment of Parmacognosy, Institute of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima 770-8505, Japan
f Department of Molecular Studies for Incurable Diseases, Institute of Biomedical Sciences, Tokushima University Graduate School, Fujii Memorial Institute of
Medical Sciences, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel naturally occurring compound with a benzofuran skeleton was isolated from a plant,
Tephrosia purpurea collected in Bangladesh. The chemical synthesis of this compound
confirmed its structure, and preliminary biological results showed its suppressive activity
towards histamine H1 gene expression. One isomer and four derivatives were also synthesized,
and their suppression activity was investigated. Although only small quantities of this compound
can be isolated from its natural source, a 10 g scale synthesis was demonstrated by the newly
developed method.
Available online
Keywords:
Histamin
2009 Elsevier Ltd. All rights reserved.
Allergy
benzofuran
Corresponding author: Tel/Fax: +81 88 7284.
Therefore, many researchers, including us, have been attempting
to isolate and determine the structures of key compounds in these
complicated mixtures,8 and examine their therapeutic utility for
allergic diseases, including AR.9,10 tMeds are generally well
known in East Asian countries such as China, Japan and Korea.11
However, Indian subcontinent also directed our attention,
because tMeds from Indian subcontinent may exhibit stronger
anti-allergic activity than that at East Asian tMeds.12
E-mail address: nem@tokushima-u.ac.jp (H. Nemoto)
Introduction
Allergic rhinitis (AR), known as hay fever, affects 5–22% of
the population globally,1 and its causes are gradually being
clarified.2,3 Histamine is a significant key mediator, and acts
mainly through histamine H1 receptor (H1R).2 Determination of
the amino acid sequence of H1R by two of the authors (H. F. and
H. M.),4 has been followed by research into AR therapies,5
including the survey to identify suitable small molecule drugs.
We report the isolation and synthesis of a new compound, 4-
methoxybenzofuran-5-carboxamide (1a, Scheme 1), extracted
from a plant, Tephrosia purpurea. T. purpurea grows from east
India to central Bangladesh. That is the first synthesis of 1a; this
report confirms the chemical structure of 1a and its suppressive
effect on H1R gene expression in allergic model cells.13 The
synthesis of derivatives of 1a and their suppressive effects are
also demonstrated.
Advances in biological and chemical technology have led to
several modern medicines (mMeds) for palliative treatment of
AR.6 Many mMeds are single compounds. Unfortunately,
mMeds often cause clinically negative side effects, and no
conclusive therapeutic agent for treating AR has yet been
identified. In contrast, several traditionally developed medicines
(tMeds) are mildly effective and produce few negative side
effects.7 However, tMeds are generally complicated chemical
mixtures and the scientific basis for their action is often unclear.
1. Results
Isolation and its structural determination of 1a

As shown in scheme 1, a methanolic extract of Tephrosia
purpurea was fractionated using various solvents; the
suppressive activity of each fraction was examined using phorbol
12-myristate 13-acetate (PMA)-induced up-regulation of H1R
gene expression in HeLa cells using our previously established
procedure.10 The chloroform fraction exhibited the highest
activity; this fraction was further fractionated by silica gel
column chromatography, followed by HPLC, resulting in the
isolation of a single molecule. Extensive spectroscopic analysis
identified the compound as 4-methoxybenzofuran-5-carboxamide
(1a).14,15 Details of the structural determination are described
below.
The structure of 1a was elucidated by 2D-NMR analysis. The
olefinic proton signals at H 7.64 (H-2) and 6.98 (H-3) resonated
with the carbon signal at C 144.9 and 105.2, respectively, in the
HSQC spectrum. These resonances showed the HMBC
correlations with the sp2 quaternary carbon at C 158.9 (C-7a) and
118.6 (C-3a) in each case, suggesting the presence of a furan
moiety. In contrast, the following HMBC correlations indicated
the presence of 4,5-disubstituted benzofuran structure; the
aromatic proton signal at H 7.32 (H-7) with C-3a; the carbon
signals at C 117.5 (C-5); the signal at H 8.16 (H-6) with C-7a
and the resonances at C 152.9 (C-4). In addition, the positions of
the carboxamide and the methoxy group were assigned to be C-5
and C-4, respectively, from the HMBC correlation of H-6 with
the carbonyl carbon resonance and of the methoxy signal with C-
4. Based on the spectral analysis described above, the structure of
1a was assigned as 4-methoxybenzofuran-5-carboxamide.
Scheme 1. Extraction and isolation of 1a from Tephrosia
purpurea
Methanolic extract of Tephrosia purpurea
Ethyl acetate
(active)
Water
(inactive)
Synthesis of 1a
Compound 1a was synthesized as shown in Scheme 2. Ether
bond formation between two of the commercially available
compounds, 2-bromoacetaldehyde diethyl acetal and methyl 2,4-
hydroxybenzoate (2), was carried out in anhydrous DMF at
100˚C for 17 h in the presence of anhydrous potassium carbonate
afforded 3 in 59% isolated yield. The phenoxide at the 4-position
was more reactive than that at the 2-position, because the latter
intramolecularly has hydrogen bonds with the neighboring ester
functionality. Treatment of 3 in toluene (0.07 mol/L) with
Amberlyst-15, a polymer supported sulfonic acid, gave a mixture
of benzofuran derivatives 4a and 4b in 80% combined yield. In
contrast, use of a high concentration of 3 in toluene (0.70 mol/L)
provided oligomer-like unidentified mixtures as major products,
Hexane
(inactive)
Methanol/water =
90:10 (active)
Chloroform
(active)
Methanol/water=
50:50 (inactive)
Slica gel column (chloroform:MeOH)~HPLC (Hexane/EtOAc)
H
H
O
N
H
3
4
3a
7a
5
O
H
2
6
4-methoxybenzofuran-5-carboxamide (1a)
H
O
7
H
Table 1. H (500 MHz) and 13C NMR (125 MHz) data for
1
1a
Position
H
Multiplicity &
Coupling (Hz)
C
as expected from
a previously reported preparation of
benzofuran.16 It appeared that the concentration of 3 did not
strongly influence the reaction rate of consumption of 3, probably
because the furan was produced intramolecularly. On the other
hand, over-reactions such as the dimerization of 4 and further
oligomerizations are generally strongly inhibited under dilute
conditions. Indeed, dilute conditions dramatically improved the
chemical yield of 4. Silica gel column chromatography easily
separated 4a, an important synthetic intermediate for 1a, from the
corresponding isomer 4b. The isolated yield of 4a and 4b was
34% and 46%, respectively.
2
7.64
6.98
d, J = 2.2
144.9 (CH)
105.2 (CH)
152.9 (C)
117.5 (C)
128.3 (CH)
107.1 (CH)
158.9 (C)
118.6 (C)
167.6 (C)
61.0 (CH3)
3
dd, J = 0.8, 2.2
4
5
6
8.16
7.32
d, J = 8.8
7
d, J = 0.8, 8.8
7a
3a
C=O
MeO
NH
NH
Scheme 2. Synthesis of 1a
4.19
7.78
5.72
s
br
br
Compound 1a gave a pseudo-molecular ion peak at m/z
214.0498 (calcd for C10H9NO3Na, 214.0480) on ESI-TOF-
HRMS, suggesting the molecular formula is C10H9NO3. The H
Treatment of 4a with iodomethane in the presence of
potassium carbonate, followed by ammonolysis gave 1a in 83%
1
1
overall yield. All the analytical data of H and 13C NMR data
NMR spectrum showed a pair of ortho-coupled aromatic protons
[H 8.16 and 7.32 (each 1H, d, J = 8.8 Hz)], a pair of conjugated
olefinic protons [H 7.64 and 6.98 (each 1H, d, J = 2.2 Hz)], and
from synthetic 1a were identical with that of isolated 1a, as were
the IR and ESI-TOF-HRMS data. The suppressive effect of
synthetic 1a on H1R gene expression (vide infra) in allergic
model cells was reproducibly as high as that of isolated 1a.
1
a methoxyl signal (H 4.19) (Table 1). The H NMR spectrum
also showed two broad D2O exchangeable peaks at 7.78 and
5.72; these coupled with IR absorption bands at 3453 and 1654
Synthesis of the isomer and the related derivatives
cm-1, implied the presence of
a primary carboxamide
functionality. The 13C NMR spectrum exhibited ten carbon
signals, including a methyl, four sp2 methines, four sp2
quaternary carbons, and one carbonyl carbon.
Treatment of 4a with concentrated aqueous ammonia gave 5a
in 87% yield, and 4b was similarly transformed to 5b (82%
yield). Compound 4a was converted to 7a via O-methylation and
hydrolysis steps19) in 80% yield, and 4b was converted to 7b
(89% yield) in the same manner via 6. Finally, the isomer 1b, the

sixth molecule in this small library, was synthesized from 6 in
80% yield via ammonolysis (Scheme 3).
compounds and that benzene ring and all the electronically
negative elements overlap well. The interaction of 1a or 1b with
key proteins/enzymes for allergy is now under investigation.20)
Scheme 3. Synthesis of an isomer 1b and the related
derivatives
Figure 1. Overlapped structures of 1a and 1b.
4. Conclusions
A novel compound, comprising a primary carboxamide on a
benzofuran skeleton was isolated from a plant found in west
India to central Bangladesh. The structure was determined by IR,
ESI-TOF-HRMS, and NMR measurements. Preliminary
examination of H1R mRNA suppression activity confirmed this
compound to hold promise for the treatment of allergic diseases,
including rhinitis. The chemical structure and the activity of the
compound were confirmed by its total synthesis and two novel
molecules designed from the isolated compound exhibited
potential therapeutic utility. Although little of the lead compound
can be isolated from natural source, the synthetic route described
here will allow its synthesis on a scale of 10 g or more with high
reproducibility. Our chemical synthesis and biological research
efforts will continue in order to identify optimal drugs for
treating allergies.
Preliminary investigation of anti-allergic activity
The activity of the six synthesized compounds on PMA-
induced H1R gene expression in HeLa cells was examined,5 and
the results are summarized in Table 2. Isolated 1a suppressed
H1R mRNA up-regulation in HeLa cells with an IC50 of 75.3 μM
(entry 1), and synthesized 1a showed reproducible activity with
an IC50 83.7 μM (entry 2). It is noteworthy that the isomer 1b
suppressed H1R gene expression at a similar level, with an IC50
of 49.14 µM (entry3). Except 5b (entry 5), the other modified
derivatives were inactive.
5. Exprimental Section
IR spectra were recorded on a FT-IR 6200 spectrophotometer.
1H NMR spectra were measured in chloroform-d1 (CDCl3) and
referenced to tetramethylsilane using 400 MHz and 500 MHz
spectrometers unless otherwise noted. 13C NMR were measured
in CDCl3 and referenced to CDCl3 (δ = 77.0) using 125 MHz
spectrometers unless otherwise noted. Column chromatography
was performed on silica gel (N-60). Thin layer chromatography
as performed on pre-coated plates (0.25 mm, silica gel Kieselgel
60F254). All the extract procedures were performed with
commercially available organic solvents without further
purification. All the chemical reactions were performed in oven-
dried glassware equipped with calcium chloride tube. Reaction
mixtures were stirred magnetically. Methanol (MeOH) was
distilled over magnesium. N,N-dimethylformamide (DMF) was
distilled over calcium hydride under reduced pressure. Toluene
was distilled under normal pressure. Acetone was distilled over
anhydrous potassium carbonate (K2CO3).
Table 2. H1R mRNA suppression of the six compounds
Entry Compound
Suppression (IC50) (µM)
75.3
1
2
3
4
5
6
7
Isolated 1a
Synthesized 1a 83.7
1b
5a
5b
7a
7b
49.1
a)
–
84.6
a)
–
a)
–
a) No suppression of H1R mRNA expression was observed.
Phorbol 12-myristate 13-acetate (PMA) were purchased from
Sigma-Aldrich. The pre-developed TaqMan Assay Reagent of
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was from Applied Biosystems (Foster City, CA, USA). Minimal
essential medium (MEM)-α was from Invitrogen (Carlsbad, CA,
USA). RNAiso Plus and the PrimeScript RT reagent Kit were
from Takara Bio Inc. (Kyoto, Japan). Methyl 2, 4-dihydroxy
benzoate and bromoacetaldehyde diethyl acetal were purchased
from Sigma-Aldrich and Tokyo Chemical Company (USA)
respectively. All other chemicals and solvents were of analytical
grade.
3. Discussion
Our interest is to further the synthesis of library molecules
using 1a as a lead molecule. We believe that the primary
carboxamide functionality should be fixed since carboxylic acids
7a and 7b were inactive (entries 6 and 7). In contrast, various
substituents may be introduced on the phenoxy moiety since
demethylated isomer 5b showed the same level of activity as 1a
(entry 5). Figure 1 provides insights into the observed difference
between 1a and 1b. Overrapping 1a and 1b in the orientation
shown in (i) indicates that the relative position of the furan ring
and carboxamide of 1a is exactly the same as that of 1b, whereas
the position of the methoxy group of 1a is quite different from
that of 1b. In contrast, the orientation shown in (ii) suggests that
only the location of the furan plane is different between the two
Plant Material
Aerial parts of Tephrosia purpurea Pers. were collected in
Bangladesh. The plant was identified by Dr. Sarder Nasir Uddin,
National Herbarium Mirpur, Dhaka, Bangladesh (Accession No.

41236). The plant was then shed dried, grounded to obtained
coarse powder material and stored until the present investigation.
4a: FT-IR (KBr): 3414, 3149, 3125, 3029, 2971, 2859, 1868,
1678, 1629, 1471, 1445, 1357, 1284, 1235, 1195, 1170, 1133,
1050, 982, 748 cm–1; 1H NMR (400 MHz, CDCl3) δ 11.47 (s, 1H,
OH), 7.78 (d, J= 8.8 Hz, 1H), 7.57 (d, J=2.0, 1H), 7.04 (d, J=8.8
Hz, 1H), 6.98 (d, J=2.0, 1H), 3.97 (s, 3H); 13C NMR (125 MHz
CDCl3) δ 171.2 (C), 159.6 (C), 157.5 (C), 144.4 (CH), 126.0
(CH), 117.1 (C), 106.0 (C), 104.8 (CH), 103.9 (CH), 52.3 (CH3);
HRESIMS m/z calcd for C10H8NaO4 [M + Na]+ 215.0320, found:
215.0322.
Extraction and isolation of 1a
The powdered plant (4.0 kg) was extracted by maceration at
room temperature using MeOH. The extract was concentrated in
vacuo to afford a residue (118.8 g), which was dissolved in ethyl
acetate (EtOAc), and washed with water. The organic layer was
concentrated in vacuo to afford a residue (65.34 g), which was
dissolved in MeOH:H2O (90:10) and washed with hexane. The
aqueous methanolic layer was concentrated in vacuo to afford a
residue (35.0 g), which was dissolved in chloroform and washed
with MeOH:H2O=50:50. The chloroform layer was concentrated
in vacuo to afford a residue (30 .0 g). Afterward, 16.0 g was
subjected to silica gel column chromatography eluted with
chloroform:MeOH (98.5:1.5, 97:3, 94:6, 90:10, 80:20, 1:1, and
0:1) to afford 24 fractions based on TLC examination. A part
(30.0 mg) of fraction 9 (500.0 mg) was purified by HPLC
(Cosmosil 5 SL, 4.6 mm id × 250 mm length) eluted with
EtOAc:hexane (6:4, flow rate = 1.0 mL/min) in an isocratic
condition to afford 1a (6.0 mg). FT-IR (KBr): 3453, 2360, 1654,
4b: FT-IR (KBr): 3413, 3120, 1663, 1557, 1443, 1414, 1369,
1286, 1258, 1212, 1186, 1143, 1072, 1013, 948, 845, 790 cm–1;
1H NMR (400 MHz, CDCl3) δ 10.92 (s, 1H, OH), 8.13 (s, 1H),
7.54 (d, J=2.4 Hz, 1H), 7.07 (s, 1H), 6.70 (d, J = 2.4 Hz, 1H),
3.98 (s, 3H); 13C (125 MHz, CDCl3): δ 170.9 (C), 159.8 (C),
159.4 (C), 145.5 (CH), 123.1 (CH), 120.3 (C), 109.2 (C), 106.6
(CH), 99.4 (CH), 52.4 (CH3); HRESIMS m/z calcd for C10H7O4
[M – H]+ 191.0350, found: 191.0351.
4-Hydroxybenzofuran-5-carboxamide (5a): To a solution of
4a (90.0 mg, 0.468 mmol) in MeOH (2.0 mL) was added an
aqueous solution of conc. Ammonia (NH3aq) (28%, 10 mL). The
resulting mixture was stirred at 36º C for 24 h, and concentrated
in vacuo. The residue was poured into HClaq (1N, 5.0 mL) and
extracted with EtOAc (50mL × 3). The combined organic layers
were washed with brine, dried over Na2SO4 and concentrated in
vacuo. The residue was purified by silica gel column
chromatography eluted with chloroform/MeOH (9:1) to afford 5a
(72.0 mg, 0.41 mmol, 87% yield). FT-IR (KBr): 3552, 3407,
3179, 1611, 1531, 1471, 1423, 1390, 1309, 1265, 1226, 1201,
1131, 1090, 1049, 1018, 941, 878, 754 cm–1; 1H NMR (500MHz,
DMSO-d6) δ 14.28 (s, 1H, OH), 8.45 (brs, 1H, NH), 7.96 (d, J =
2.0 Hz, 1H), 7.93 (brs, 1H, NH), 7.81 (d, J = 8.5 Hz, 1H), 7.13
(d, J = 8.5 Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H). 13C NMR (125
MHz, DMSO-d6) δ 173.1 (C), 157.8 (C), 157.3 (C), 145.3 (CH),
124.2 (CH), 116.8 (C), 107.4 (C), 104.6 (CH), 102.6 (CH);
HRESIMS m/z calcd for C9H7NO3 [M]+: 177.0426, found:
177.0424.
1
1458, 1420, 1387, 1256, 1066, 978 cm–1, H NMR (500 MHz,
CDCl3) see Table 1; 13C NMR (125 MHz, CDCl3) see Table 1;
HRESIMS m/z calcd for C10H9NNaO3 [M + Na]+ 214.0480,
found: 214.0498.
Synthesis
Methyl 4-hydroxy benzoate 2-acetaldehyde diethyl acetal
(3): To a solution of methyl 2,4-dihhydroxy benzoate (2) (1.00 g,
5.95 mmol) in DMF (10 mL), suspended with anhydrous
potassium carbobate (K2CO3) (1.23 g, 8.93 mmol, 1.5 equiv.)
with vigorously stirring, was added bromoacetaldehyde diethyl
acetal (5.87 g, 29.8 mmol) dropwise over 5 min. The resulting
suspension was heated at 100 ºC with stirring for 17 h. After
being cooled, the suspension was poured into 5% of aqueous
solution of potassium hydrogen sulfate (KHSO4aq, 100 mL) at
0˚C, and extracted with EtOAc (120 mL × 3). The combined
organic layers were washed with brine, dried over anhydrous
sodium sulfate (Na2SO4) and concentrated in vacuo. The residue
was purified by silica gel column chromatography using hexane
and EtOAc (8:2) to afford 3 as a colorless oil (994.0 mg, 3.50
mmol, 59% yield). FT-IR (neat): 3145, 2977, 2884, 1917, 1672,
1624, 1584, 1506, 1442, 1374, 1350, 1257, 1225, 1195, 1178,
6-Hydroxybenzofuran-5-carboxamide (5b): To a solution of
4b (97.6 mg, 0.508 mmol) in MeOH (2.0 mL) was added NH3aq
(28%, 10 mL). The resulting mixture was stirred at 36º C for 5 h,
and concentrated in vacuo. The residue was poured into HClaq
(1N, 5.0 mL) and extracted with EtOAc (50 mL × 3). The
combined organic layers were washed with brine, dried over
Na2SO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography eluted with chloroform/MeOH
(9:1) to afford 5b (74.0 mg, 0.418 mmol, 82% yield). FR-IR
(KBr): 3547, 3422, 3195, 1666, 1638, 1605, 1543, 1466, 1400,
1
1136, 1072, 980, 958, 886, 855, 780, 733, 697, 655 cm–1; H
NMR (400 MHz, CDCl3): δ 10.94 (s, 1H, OH), 7.73 (d, J = 9.2
Hz, 1H), 6.48–6.45 (m, 2H), 4.83 (t, J = 5.2 Hz, 1H), 4.02 (d, J =
5.2 Hz, 2H), 3.91 (s, 3H), 3.76 (dq, J = 6.8, 9.4 Hz, 2H), 3.63 (dq,
J = 6.8, 9.4 Hz, 2H), 1.25 (t, J = 6.8 Hz, CH3 × 2); 13C NMR (125
MHz, CDCl3): δ 170.4 (C), 164.5 (C), 163.7 (C), 131.3 (CH),
107.8 (CH), 105.8 (C), 101.5 (CH), 100.2 (CH), 68.6 (CH2), 62.8
(CH2), 52.0 (CH3), 15.4 (CH3); HRESIMS m/z calcd for
C14H19O6 [M – H] + 283.1182, found: 283.1172.
1
1284, 1236, 1189, 1150, 1114, 1060, 1016, 832 cm–1; H NMR
(400 MHz, DMDO-d6) δ 13.26 (s, 1H, OH), 8.48 (brs, 1H, NH),
8.18 (s, 1H), 7.90 (brs, 1H, NH), 7.90 (d, J = 2.0 Hz, 1H), 7.04 (s,
1H,), 6.90 (d, J = 2.0 Hz, 1H). 13C NMR (125 MHz, DMSO-d6) δ
172.6 (C), 159.6 (C), 157.5 (C), 145.9 (CH), 120.9 (CH), 119.0
(C), 111.2 (C), 106.7 (CH), 98.8 (CH); HRESIMS m/z calcd for
C9H7NNaO3 [M + Na]+: 200.0324, found: 200.0336.
Methyl 4-hydroxybenzofuran-5-carboxylate (4a) and
methyl 6-hydroxybenzofuran -5-carboxylate (4b): A solution
of 3 (389.5 mg, 1.37 mmol) in toluene (20 mL) suspended with
Amberlyst-15® (51.0 mg) was stirred at reflux for 4 h, equipped
with dean-stark apparatus to remove water. After being cooled,
the resulting suspension was filtered, and the residue was washed
with toluene (80 mL). The combined filtrate was concentrated in
vacuo. The residue was purified by silica gel column
chromatography eluted with EtOAc/hexane (1:9) to afford 4a
(89.5 mg, 0.47 mmol; 34% yield) and 4b (121.1 mg, 0.63 mmol,
46% yield), respectively.
Methyl 6-methoxybenzofuran-5-carboxylate (6): A mixture
of 4b (486mg, 2.529 mmol), K2CO3 (1.818g, 13.15 mmol),
iodomethane (2.59g, 25.29 mmol) in acetone (25 mL) was
refluxed for 5 h. After being cooled, the resulting suspension was
filtered. The filtrate was concentrated in vacuo. The residue was
diluted with ether (50 mL), and the resulting suspension was
filtered. The filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (Hexane/EtOAc,
7:3) to afford 6, a pale yellow oil (500 mg, 2.425mmol, 96%
yield). FT-IR (neat): 3107, 2951, 2840, 1720, 1590, 1538, 1475,
1433, 1353, 1298, 1253, 1199, 1177, 1137, 1070, 1008, 899, 834

cm–1; 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.57 (d, J = 2.4
Hz, 1H), 7.10 (s, 1H), 6.73 (d, J = 2.4 Hz), 3.95 (s, 3H), 3.91 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 166.8 (C), 158.0 (C), 157.8
(C), 145.2 (CH), 124.8 (CH), 120.0 (C), 116.6 (C), 106.7 (CH),
95.4 (CH), 56.4 (CH3), 52.1 (CH3); HRESIMS m/z calcd for
C11H10NaO4 [M + Na]+: 229.0477, found: 229.0473.
residue was purified by silica gel chromatography eluted with
chloroform/methanol (9:1) to afford 1a (165.0 mg, 0.863 mmol,
83% yield). FT-IR (KBr): 3459, 3162, 1668, 1605, 1579, 1475,
1461, 1419, 1387, 1348, 1322, 1251, 1233, 1067, 975, 742 cm–1;
1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 8.8 Hz, 1H), 7.77 (brs,
1H, NH), 7.63 (d, J = 2.2 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 6.98
(d, J = 2.2 Hz, 1H), 5.76 (brs, 1H, NH), 4.19 (s, 3H); 13C (125
MHz, CDCl3) δ 167.3 (C), 158.9 (C), 152.8 (C), 145.0 (CH),
128.4 (CH), 118.6 (C), 117.6 (C), 107.2 (CH), 105.3 (CH), 61.2
(CH3); HRESIMS m/z calcd for C10H9NNaO3 [M + Na]+
214.0480, found: 214.0485.
4-Methoxybenzofuran-5-carboxylic acid (7a): A solution of
4a (200.0 mg, 1.04 mmol) in acetone (11 mL) were added
K2CO3 (747.0 mg, 5.41 mmol) and iodomethane (1.47 g, 10.4
mmol), and the resulting suspension was stirred at reflux for 3 h.
After being cooled to room temperature, the resulting suspension
was filtered. The filtrate was concentrated in vacuo, and diluted
with ether (30 mL). The resulting suspension was again filtered,
and the filtrate was concentrated in vacuo. The residue was used
in the next step without further purification. To a solution of the
residue in MeOH (4.0 mL) was added an aqueous solution of
sodium hydroxide (NaOHaq) (3N, 3.0 mL). The mixture was
stirred for 7 h at room temperature, poured into HClaq (2N, 15
mL), extracted with EtOAc (40 mL × 3). The combined organic
layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography eluted with chloroform/methanol (9:1)
to afford 7a (160mg, 0.833 mmol, 80% yield); FT-IR (KBr):
3413, 3138, 2993, 1693, 1617, 1550, 1472, 1427, 1417, 1366,
Synthesis of 4-Methoxybenzofuran 5-carboxamide (1a) on
a large scale from 2: Two times of the large-scale examinations
of the three steps (from 2 to 3, from 3 to 4a, and from 4a to 1a)
confirmed that the overall yield from 2 to 1a was reproducible
(17% ± 5%, 0.59 × 0.34 × 0.83 = 0.17). The following results
were the one of the two examinations. Compound 2 (56.7 g,
336.9 mmol) was converted to 3 (56.5 g, 198.8 mmol) in 59%
yield. Compound 4a (13.0 g, 37.6 mmol) was obtained from 3
(198.8 mmol) in 34% yield along with 4b. Compound 1a (10.72
g, 56.1 mmol) was afforded from 4a (37.6 mmol) in 83% yield.
6-Methoxybenzofuran-5-carboxamide (1b): To a solution of
6 (502 mg, 2.435 mmol) in MeOH (7.0 mL) was added NH3aq
(28%, 30 mL). The mixture was stirred at 35ºC for 12 h,
concentrated in vacuo, poured into HClaq (1N, 15 mL) at 0˚C,
and extracted with EtOAc (100 mL × 3). The combined organic
layers were washed with NaOHaq (1N, 100 mL), brine (150 mL),
dried over Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel chromatography eluted with
chloroform/MeOH (9:1) to afford 1b (373.0 mg, 1.95 mmol, 80%
yield). FR-IR (KBr): 3404, 3179, 1643, 1603, 1538, 1475, 1435,
1
1339, 1285, 1261, 1239, 1176, 1076, 987, 924, 750 cm–1; H
NMR (400 MHz, CDCl3): δ 10.93 (s, 1H), 8.15 (d, J = 8.8 Hz,
1H), 7.69 (d, J = 2.4 Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.02 (d, J
= 2.4 Hz, 1H), 4.34 (s, 3H); 13C NMR (125 MHz, CDCl3): δ
166.0 (C), 159.8 (C), 153.4 (C), 145.6 (CH), 129.4 (CH), 117.8
(C), 113.9 (C), 107.9 (CH) 105.2 (CH), 61.6 (CH3); HRESIMS
m/z calcd for C10H8NaO4 [M + Na]+: 215.0320, found: 215.0321.
1
6-Methoxybenzofuran-5-carboxylic acid (7b): To a solution
of 6 (360.5 mg, 1.75 mmol) in MeOH (5.0 mL) was added
NaOHaq (3N, 3.0 mL). The mixture was stirred for 5 h at room
temperature, poured into HClaq (2N, 15 mL), and extracted with
EtOAc (40 mL × 3). The combined organic layers were washed
with brine (60 mL), dried over Na2SO4 and concentrated in
vacuo. The residue was purified by silica gel column
chromatography eluted with chloroform/methanol (9:1) to afford
7b (300.0 mg, 1.56 mmol, 89% yield). FT-IR (KBr): 3413, 3235,
1690, 1638, 1618, 1581, 1540, 1475, 1427, 1406, 1356, 1270,
1376, 1288, 1201, 1154, 1067, 1003, 905, 826 cm–1; H NMR
(400 MHz, CDCl3) δ 8.50 (s, 1H), 7.59 (d, J = 2.4 Hz , 1H), 7.77
(brs, 1H, NH), 5.79 (brs, 1H, NH), 7.12 (s, 1H), 6.78 (d, J = 2.4
Hz, 1H), 4.02 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 167.4 (C),
157.7 (C), 156.4 (C), 145.3 (CH), 125.7 (CH), 121.0 (C), 117.5
(C), 107.0 (CH), 94.8 (CH), 56.4 (CH3); HRESIMS m/z calcd for
C10H9NNaO3 [M + Na]+: 214.0480, found: 214.0512.
HeLa cell experiments:
HeLa cells were cultured at 37 °C under a humidified ( 5%
CO2, 95% air) atmosphere in MEM-α medium containing 8%
FCS (Sigma-Aldrich) supplemented with 100 IU/ml penicillin
(Sigma-Aldrich) and 50 μg/ml streptomycin (Sigma-Aldrich).
HeLa cells were cultured to 70% confluence in 35-mm dishes
and were serum starved for 24 h at 37 °C before treatment with
100 nM of phorbol-12-myristate-13-acetate (PMA) for 3 h. Cells
were pretreated with TP extract, its different fractions, isolated
compound (1a: 50, 100, 150, 200, and 250 µM) as well as
synthesized compounds (1a: 50, 100, 150, 200, and 250 µM; 1b:
25, 50, 75, 100, and 150 µM; 5a: 50, 100, 200, and 300 µM; 5b:
50, 100, 200, and 300 µM; 7a: 50, 100, 200, and 300 µM; 7a: 50,
100, 200, and 300 µM) for 1 h prior to PMA stimulation.
1
1209, 1188, 1146, 1066, 1003, 901, 828 cm–1; H NMR (400
MHz, CDCl3) δ 10.85 (s, 1H, COOH), 8.49 (s, 1H), 7.64 (d, J =
2.4 Hz, 1H), 7.19 (s, 1H, 6.81 (d, J = 2.4 Hz, 1H), 4.13 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 165.7 (C), 158.4 (C), 156.3 (C),
146.2 (CH), 127.3 (CH), 122.0 (C), 113.9 (C), 107.0 (CH), 95.3
(CH), 57.1 (CH3); HRESIMS m/z calcd for C10H8NaO4 [M +
Na]+: 215.0320, found: 215.0312.
4-Methoxybenzofuran 5-carboxamide (synthetic 1a): A
solution of 4a (200 mg, 1.04 mmol) in acetone (11 mL) were
added K2CO3 (747.4 mg, 5.41 mmol) and iodomethane (1.48 g,
10.43 mol), and the resulting suspension was stirred at reflux for
3 h. After being cooled to room temperature, the resulting
suspension was filtered. The filtrate was concentrated in vacuo,
and diluted with ether (30 mL). The resulting suspension was
again filtered, and the filtrate was concentrated in vacuo. The
residue was used in the next step without further purification. To
a solution of the residue in MeOH (5.0 mL) was added NH3aq
(40 mL, 28%), and the mixture was stirred at 35 ºC for 27 h. The
resulting solution was concentrated in vacuo. To the residue was
added HClaq (1N, 10 mL), extratected with EtOAc (60 mL × 3).
The combined organic layers were washed with a saturated
aqueous solution of sodium hydrogen carbonate (50 mL), brine
(20 mL), dried over Na2SO4 and concentrated in vacuo. The
Real-time quantitative RT-PCR:
After a 3-h treatment with PMA, the cells were harvested with
700 μl of RNAiso Plus (Takara Bio Inc.), mixed with 140 μL of
chloroform, and centrifuged at 17,400 × g for 15 min at 4 °C.
The aqueous phase was collected, and RNA was precipitated by
the addition of isopropyl alcohol. After centrifugation at 17,400 ×
g for 15 min at 4 °C, the resulting RNA pellet was washed with
ice-cold 75% ethanol. Total RNA pellet was resolved in 10 μl of
diethylpyrocarbonate-treated water, and 1 μg of each RNA
samples was used for the reverse transcription reaction. RNA
samples were reverse-transcribed to cDNA using a PrimeScript

Chang, Y. S.; Kim, Y. K.; Cho, S. H.; Min, K. U.; Kim, Y. Y. J
Korean Med Sci. 2008, 23, 232–235.
RT reagent Kit. TaqMan primers and the probe were designed
using Primer Express software (Applied Biosystems). Real-time
PCR was conducted using a GeneAmp 7300 sequence detection
system (Applied Biosystems). The sequences of the primers and
TaqMan probe were as follows: forward primer for human H1R,
10. (a) Nurul, I. M.; Mizuguchi, H.; Shahriar, M.; Venkatesh, P.;
Maeyama, K.; Mukherjee, P. K.; Hattori, M.; Choudhuri, M. S.;
Takeda, N.; Fukui, H. Int Immunopharmacol. 2011, 11, 1766–
1772. (b) Dev, S.; Mizuguchi H.; Das, A. K.; Baba, Y.; Fukui H.
Int Immunopharmacol. 2011, 10, 1504–1509. (c) Venkatesh, P.;
Mukherjee, P. K.; Kumar, N. S.; Bandyopadhyay, A.; Fukui,
H.; Mizuguchi, H.; Islam N. Immunopharmacol. Immunotoxicol.
2010, 32, 272–276. (d) Venkatesh, P.; Mukherjee, P. K.; Kumar,
S. N.; Nema, N. K.; Bandyopadhyay A.; Fukui, H.; Mizuguchi, H.
J. Ethnopharmacol. 2009, 126, 434–436. (e) Das, A.
5′-CAGAGGATCAGATGTTAGGTGATAGC-3′;
reverse
primer for human H1R, 5′-AGCGGAGCCTCTTCCAAGTAA-
3′;
TaqMan
probe,
FAM-
CTTCTCTCGAACGGACTCAGATACCACC-TAMRA.
To
standardize the starting material, the human GAPDH gene
(Applied Biosystems) was used, and data were expressed as the
ratio of H1R mRNA to GAPDH mRNA.
K.; Mizuguchi, H.; Kodama, M.; Dev, S.; Umehara, H.; Kitamura,
Y.; Matsushita, C.; Takeda, N.; Fukui, H. Allergol. Int. 2009, 58,
81–88.
11. (a) Park, H. L.; Lee, H. S.; Shin, B. C.; Liu, J. P.; Shang, Q.;
Yamashita, H.; Lim, B. Traditional Medicine in China, Korea, and
Japan: Evidence-Based Complementary and Alternative Medicine,
2012, 1–9. (b) Nishimura, K.; Plotnikoff, G. A.; Watanabe, K.
JMAJ 2009, 52, 147–149.
12. (a) Dasgupta, S. A history of Indian philosophy, Motilal
Banarsidass Publishers Private Limited, New Delhi, 1975. (b)
Mukherjee, P. K.; Wahile, A. J Ethnopharmacol. 2006, 103, 25–
35. (c) Ninivaggi, F.J. Ayurveda: A Comprehensive Guide to
Traditional Indian medicine for the West Maryland: Rowman and
Littlefield Publisher, Inc., 2008. (d) Kirtikar, K. R.; Basu, B. D.
Indian medicinal plants. Lalit Mohan Basu, Allahabad, India, 2nd
edition, 1956
13. Two of the authors (H. F. and H. M.) have recently established the
standard cell model examination procedure, where H1R gene is
up-regulated in patients with allergic rhinitis and its expression
level strongly correlates with the severity of symptoms.5g We also
have reported that some of the alternative potential molecules
suppressing H1R gene up-regulation alleviate allergic symptoms
in allergy model rats.5c,5e,5h,5i
Acknowledgments
This work was financially supported in part by Grants-in-Aid
for Scientific Research (B) from the Japan Society for the
Promotion of Science (26305004 to H.F.), and by a grant from
the Osaka Medical Research Foundation for Incurable Diseases.
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related paper,18 same authors reported the dimerization reaction of
9. Those results described both in references 17 and 18 confused
us because the condition (including the concentration value) from
8 to 9 was almost same as the condition for dimerization of 9.
Therefore, we assumed that the value of the concentration in
reference 17 was wrong. Accordingly, the step from 3 to 4a/4b
was easily improved by the high dilution condition (0.07 mol/L).
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17. Goel, A.; Dixit, M. Synlett 2004, 1990–1994.
18. Dixit, M.; Sharon, A.; Maulik, P. R.; Goel, A. Synlett 2006, 1497–
1502.
19. Synthesis of 7a was reported via a different route. Foster, R. T.;
Robertson, J. Chem. Soc. 1948, 115–116.
20. A manuscript entitled “A novel benzofuran, 4-
methoxybenzofuran-5-carboxamide, from Tephrosia purpurea
suppressed histamine H1 receptor gene expression through a

protein kinase C-δ-dependent signaling pathway” has been
submitted. Shill, M. C.; Mizuguchi, H.; Karmakar, S.; Kadota, T.;
Mukherjee, P. K.; Kitamura, Y.; Kashiwada, Y.; Nemoto, H.;
Takeda, N.; Fukui. Int. Immunopharmacol.
Supplementary Material
1
Supplementary material for 2D NMR of isolated 1a, for H
and 13C NMR of compounds 1a (isolated and synthesized), 1b, 3,
4a, 4b, 5a, 5b, 6, 7a, and 7b are available.