Synthesis of amide compounds of ferulic acid, and their stimulatory effects on insulin secretion in vitro

Article abstract of DOI:10.1016/S0968-0896(03)00280-3

We prepared amide compounds which were derived from ferulic acid using various amines, and investigated their stimulatory effects on insulin secretion using rat pancreatic RIN-5F cells. Most of these compounds exhibited significant promotion of the insulin-release at a concentration of 10 μM and in particular, the amides having n-butyl, n-pentyl, pyrrolidine, and piperidine groups showed high activity.

Full text of DOI:10.1016/S0968-0896(03)00280-3

Bioorganic & Medicinal Chemistry 11 (2003) 3807–3813
Synthesis of Amide Compounds of Ferulic Acid, and Their
Stimulatory Effects on Insulin Secretion In Vitro
a,
b,y
a
b,{
Eisaku Nomura, * Ayumi Kashiwada, Asao Hosoda, Kozo Nakamura,
c
d
a
Hideko Morishita, Takuo Tsuno and Hisaji Taniguchi
aIndustrial Technology Center of Wakayama Prefecture, 60 Ogura, Wakayama 649-6261, Japan
bJapan Science and Technology Corporation, 4-1-8 Honmachi, Kawaguchi 332-0012, Japan
cFaculty of Education, Wakayama University, 930 Sakaedani, Wakayama 640-8510, Japan
dTsuno Food Industrial Co., Ltd., Katsuragi, Wakayama 649-7194, Japan
Received 1March 2003; accepted 23 April 2003
Abstract—We prepared amide compounds which were derived from ferulic acid using various amines, and investigated their sti-
mulatory effects on insulin secretion using rat pancreatic RIN-5F cells. Most of these compounds exhibited significant promotion of
the insulin-release at a concentration of 10 mM and in particular, the amides having n-butyl, n-pentyl, pyrrolidine, and piperidine
groups showed high activity.
#
2003 Elsevier Ltd. All rights reserved.
Introduction
chlorogenic acid 5 are natural products from green cof-
1
0
fee beans and have various bioactivities. The com-
pound 5 is the ester compound consisting of (À)-quinic
11,12
Ferulic acid 1 is a phenolic compound and some authors
have recently developed a mass production method of 1
from rice bran. Interest in biological activities of 1
and the synthetic compounds derived from 1 has inten-
sified in recent years. For example, alkyl ferulates 2 and
have an anti-carcinogenic potential and in particular,
was more effective cancer chemopreventive agents
than 1. A ferulic acid derivative in which geranyl group
is attached to the phenolic hydroxyl group of ethyl fer-
ulate exhibits colon anti-carcinogenesis in rodent mod-
acid and 4, and inhibits glucose-6-phosphatase to reg-
ulate hepatic glucose production.
1
,2
Recently, some
authors have found that the blood glucose level in STZ-
induced diabetic mice was reduced by administration of
1
3
3
3
1. This finding prompted us to investigate the activity
of ferulic acid and its related compounds on their sti-
mulatory effects on insulin secretion in vitro. We
focused some amide compounds of 1 for an assay sys-
3
tem by the following reasons: (1) Some of the amides
14,15
2À5
els.
A novel polyphenol 6 which was composed of
were isolated from plants
but their biological activ-
two naturally occurring products, ferulic and gallic
acids, was found to have higher activity as an anti-car-
ities were not investigated. (2) Common antidiabetic
agents such as sulfonylureas contains the bond-structure
of amide.
6
cinogen than the original phytochemicals. The com-
pounds 7–9 consisting of ferulic acid and myo-inositol
are also novel polyphenols that have potential of cancer
7À9
chemopreventive agents. Biological activities of nat-
ural occurring compounds related to ferulic acid also
have been investigated. For example, caffeic acid 4 and
*Corresponding author. Tel.: +81-(0)73-477-1271; fax: +81-(0)73-
4
77-2880; e-mail: nomura@wakayama-kg.go.jp
y
Present address: College of Industrial Technology, Nihon University,
1
-2-1 Izumi, Narashino, Chiba 275-8575, Japan.
{
Present address: Faculty of Agriculture, Shinsyu University,
8304 Minami-minaowa, Kami-ina, Nagano 399-4598, Japan.
0
968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00280-3

3808
E. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 3807–3813
In this paper, we describe the synthesis of the amide
compounds of 1 and a preliminary experiment to inves-
tigate the mechanism of antidiabetic properties by insu-
lin secretion in rat pancreatic RIN-5F cells in vitro
incubations using 1 and related compounds including
the amide compounds.
The number of people afflicted by diabetes mellitus is
estimated to be more than 200 million in the world.
Most of the patients are suffering from non-insulin-
dependent diabetes mellitus (NIDDM, type 2 dia-
1
6
betes). Studies on ingredients of plants in the treat-
ment of diabetes have been undertaken and served
1
7
effective dietary adjuncts such as Eucalyptus globules.
On the other hand, common antidiabetic medicines such
as sulfonylureas to the treatment of NIDDM patients
are prescribed carefully with many restrictions due to
various side-effects. Therefore, studies on biological
activities of 1 and their derivatives might serve a new
therapeutic approach as safe hypoglycemic agents which
are synthetic compounds based on natural sources.
Results and Discussion
Chemistry
Various types of ferulic acid derivatives 2–9 are chosen
to examine the activity on insulin secretion. The amide
compounds of ferulic acid were synthesized by con-
densation reactions. The reaction was performed by the
reaction of O-acetyl feruloyl chloride with various
amines, followed by deacetylation. O-Acetyl feruloyl
chloride was prepared by the method reported pre-
6
viously. The condensation was performed in dichloro-
methane in the presence of an alkylamine at room
temperature, and then the removal of acetyl groups was
carried out using sodium methoxide or hydrazine
monohydrate at room temperature to yield correspond-
ing amide compounds 10–17, and 19. The amides 18
and 20 were prepared alternative methods described in
details in the experimental section. The amides 18 and
19 are natural compounds which are isolated from the
fruit of white pepper.
1
4,15
We used the compounds 1–20

E. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 3807–3813
3809
to assess their antidiabetic potential. Tolbutamide 21
that is one of common sulfonylurea antidiabetic agents
was also tested for comparing with the compounds
prepared.
showed significant differences of the activation between
test compounds and the control experiment at the both
concentration of 100 mM and 10 mM. In particular, the
amides, 12, 13, 14, 17, and 18, exhibited 6–8 times insu-
lin secretion, compared with the control experiment.
The structural feature of these amides is an alkyl chain
having 4 or 5 carbons. The other amides, 10, 11, 15, and
16, in which have shorter or longer alkyl chains, showed
moderate activity (3–5 times compared to the control).
The amides, 19 and 20, consisted of tyramine and tyr-
osine, respectively, showed also moderate activity (3–5
times compared to the control). To make clear the
activities of selected compounds including 1 and 21,
dose-response curves were shown in Figure 3. All of
compounds tested showed dose-dependent activities. In
particular, the amide compounds showed higher activ-
ities than 1, but relatively lower than 21. However, the
activities of the amides seem to be comparable to that of
21 due to no significant differences between the amides
tested and 21 as shown in Figure 2.
Biological activity
For ferulic acid 1 and their related compounds 2–10, we
investigated their stimulatory effects on insulin secretion
in vitro in order to estimate their antidiabetic potential.
The rat insulin enzyme-immunoassay system was avail-
able commercially for the evaluation of the activities in
vitro. The activities are estimated from concentrations
of insulin which are released from pancreatic RIN-5F
cells by adding the compounds. The results are shown in
Figure 1. The control experiment without a test com-
pound was carried out in parallel. As shown in Figure
1
1
A, the compounds 1, 5, 7–9, and the amide compound
0 increased the amounts of insulin secretion at a con-
centration of 100 mM comparing with control experi-
ment. On the other hand, alkyl ferulates (2 and 3), 4,
and 6 showed no stimulatory effect. The compounds 1
and 10 showed a marked promotion effect of the insulin
secretion at the concentration of 10 mM as shown in
Figure 1B. Therefore, we then examined the activities
for the amide compounds carrying various alkyl chains
and residues. Tolbutamide 21 was also tested for com-
paring with the compounds prepared. The results are
shown in Figure 2. All of the amide compounds tested
Cell viability in cell culture was determined for selected
test compounds by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The results
are shown in Figure 4. All compounds tested have no
notable cytotoxicity (cell viability of 89–98%). It is
known that an insulin secretion from pancreatic beta
1
8
cells is stimulated by sulfonylureas. The mechanisms
of insulin releasing-action of these drugs have been
Figure 1. Stimulatory effect of ferulic acid and related compounds on
insulin secretion in vitro incubating of RIN-5F cells. Test compounds;
Figure 2. Stimulatory effect of ferulic acid and the amides on insulin
secretion in vitro incubating of RIN-5F cells. Test compounds; 100
mM (A): 10 mM (B). Values are meansÆSD. Significant differences
compared with control group; **p<0.01.
100 mM (A): 10 mM (B). Values are meansÆSD. Significant differences
compared with control group; *p<0.05 and **p6<0.01.

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E. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 3807–3813
demonstrated. Sulfonylureas such as tolbutamide bind
to sulfonylurea receptors, resulting in closure of ATP
Conclusion
+
sensitive K channels in the beta cell plasma membrane
to cause membrane depolarization, calcium influx, and
The amide compounds of ferulic acid were prepared.
Most of these compounds exhibited their stimulatory
abilities on insulin secretion in rat pancreatic RIN-5F
cells. In particular, the amides, 12, 14, 17, and 18 could
be the most promising antidiabete agents among the
compounds evaluated in the present study. Mass pro-
duction method of ferulic acid from rice bran was
1
8
insulin secretion. In addition to these effects, sulfony-
lureas act by direct interaction with the secretory
1
8,19
machinery.
It was found that the amide compounds
we tested stimulated insulin secretion from pancreatic
RIN-5F cells. The mechanisms of these compounds are
not clear. However, the similarity of alkylamide groups
of the ferulic acid derivatives to that of tolbutamide
suggests similar mechanisms of the stimulatory effects in
cells. Various types of sulfonylureas as antidiabetic
agents are known and they have only a bond-structure
of sulfonylurea in common. Therefore, the antidiabetic
activity related to the binding ability toward the sulfo-
nylurea receptors depends on the other groups connect-
ing with the sulfonylurea. In the case of the amides,
important structural factors seem to be the presence of
an appropriate alkyl group and feruloyl group con-
nected with amide. Ferulic acid from rice bran is useful
for a food additive as an antioxidant. The amides such
as 18 and 19 are natural occurring compounds and the
other amides are made up of ferulic acid and simple
amines. Therefore, these compounds would be expected
to be safe hypoglycemic agents with few side effects in
the future diabetes therapy.
1
,2
developed in recent years
and the production was
already on a commercial basis. Therefore, it is valuable
to obtain effective biological active agents as derivatives
from natural compounds for practical use. The present
study represents a potential of the amide compounds as
insulin-secretion stimulatory agents in the first stage of
the assay system. This finding might provide new and
safe orally active agents based on natural occurring
compounds for the treatment of diabetes in the future
therapy.
Experimental
General
Ferulic acid and alkyl ferulates were supplied by Tsuno
Rice Fine Chemicals Co., Ltd. (Wakayama, Japan).
Chlorogenic acid was purchased from Aldrich Chemical
Co. Ltd. Anhydrous CH Cl was purchased from
2
2
Kanto Chemical Co., Inc. (Tokyo, Japan) and used
without further purification. Other solvents and
reagents were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan), and used without fur-
ther purification. The O-acetyl feruloyl chloride was
6
prepared by the literature method. Melting points were
determined by a Yanaco micro melting point apparatus
and are uncorrected. Elemental analyses were per-
1
13
formed on a Perkin–Elmer 2400II. H and C NMR
spectra were recorded on a Varian Unity-plus 400 spec-
trometer using a residual solvent as an internal stan-
dard. FT-IR spectra were obtained on a Shimadzu FT-IR
8200D spectrometer using a diffuse reflectance cell. ESI-
TOF MS spectra were measured using PE Biosystems
Mariner spectrometer.
Figure 3. Dose–response curves for insulin secretion on rat RIN-F5
cells. Test compounds: 1 (*), 12 (^), 14 (&), 17 (&), 21 (*). Plots
are shown in mean values.
Chemicals and cells for bioassay
Rat insulin enzymeimmunoassays (EIA) system (PRN
2567, 96 wells) was obtained from Amersham Pharma-
cia Biotech UK limited (Buckinghamshire, England).
The RIN-5F cell line from the RIN-m rat islet cell line
was purchased from ATCC the Global Bioresource
Center. RPMI 1640 media, fetal bovine serum (FBS),
and Penicillin-Streptomycin were purchased from Gibco
BRL (NY, USA). Other chemicals were purchased from
Wako Pure Chemical Industries, Ltd., unless specified
otherwise.
Assay of insulin secretion activity
RIN-5F cells which were clones derived from rat pan-
creatic beta cells were used to evaluate insulin secretion
activity. The cells at a concentration of 2.0ꢀ10 of the
Figure 4. Cell viability of ferulic acid and the amide compounds. Test
compounds: 100 mM. Values are meansÆSD.
5

E. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 3807–3813
3811
cells/well in 24-well plates were seeded in RPMI 1640
medium (supplemented with 10% FBS) containing
sodium hydrogen carbonate (2 g/L), streptomycin (100
N-Isopropylferuloylamide (11). To a solution of O-acetyl
feruloyl chloride (20 g) and triethylamine (11.9 g) in
anhydrous CH Cl (200 mL) was added dropwise 7.0 g
2
2
ꢁ
mg/mL), and peniciline (100 units/mL) at 37 C under 5%
of iso-propylamine in an ice-bath. The mixture was
stirred for 15 min at room temperature. Acetic acid was
added to the mixture to produce precipitates. The pre-
cipitates were filtered and dissolved in MeOH (400 mL).
A 36.7 g of 28% sodium methoxide in MeOH was
added dropwise to the solution in an ice-bath. After
additional stirring at room temperature for 15 min, the
mixture was neutralized with 10% aqueous sulfuric
acid, concentrated, and extracted with ethyl acetate.
The organic layer was washed with water and dried over
CO atmosphere. After the incubation for 72 h, the
medium in the each well was exchanged 1mL of the fresh
medium and the cells were incubated for another 24 h.
2
The medium in the wells was removed and the cells were
washed with the fresh medium (supplemented with 1%
FBS). A 360 mL of the medium and 40 mL of the test
compound (10 and 100 mM) were pipetted into the all
ꢁ
wells. After incubation at 37 C under a 5% CO for 3
2
h, aliquots in all wells were withdrawn and centrifuged
to separate the cells. The concentration of insulin in the
mediums was determined by EIA system. The activity
was evaluated by an increase of the concentration of
insulin-release comparing with the control experiment
without test compounds. Each experiment was done in
triplicate, and the results are presented as meansÆSD.
Group of data were compared using Student’s t-test.
MgSO . After filtration and removal of the solvent, the
4
product was recrystallized from EtOH to afford 13.1 g
ꢁ
(71%) of prisms: mp=186–188 C; IR (KBr) n=3329,
À1
1
1655, 1518 cm ; H NMR (DMSO-d ) d=1.04 (d, 6H,
6
J=6.6 Hz, CH ), 3.86 (s, 3H, OCH ), 3.86–3.92 (m, 1H,
3
3
CH), 6.36 (d, 1H, J=15.7 Hz, NH), 6.73 (d, 1H, J=8.1
Hz, ArH), 6.92 (dd, 1H, J=8.2, 1.8 Hz, ArH), 7.05 (d,
1H, J=2.0 Hz, ArH), 7.24 (d, 1H, J=15.8 Hz,=CH),
1
3
7.76 (d, 1H, J=8.0 Hz, NH), 9.35 (bs, 1H, OH);
C
NMR (DMSO-d ) d=22.7, 40.5, 55.7, 110.8, 115.8,
6
MTT assay
1
19.6, 121.6, 126.7, 138.8, 148.0, 148.3, 164.5. MS (ESI-
+
Cell viability in cell culture was determined by the 3-
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
TOF) calcd for [C H NO ] 236.13, Found 236.09
13 18 3
+
(
[M+H] . Anal. calcd for C H NO : C, 66.36; H,
13 17 3
mide (MTT) assay. After treatment using selected feru-
lic acid derivatives for 24 h, the cells were further
incubated in a medium containing 0.5 mg/mL of MTT
for 1h the MTT formazan produced by living cells was
dissolved in dimethyl sulfoxide and absorbance at 570
nm was measured on a microplate reader (Spectra &
Rainbow Readers, TECAN).
7.28; N, 5.95. Found: C, 66.17; H, 7.30; N, 5.92.
N-Butylferuloylamide (12). Using the procedure for 11.
The product was recrystallized from EtOH to afford
ꢁ
0.66 g (67%) of prisms: mp=126–128 C; IR (KBr)
À1
1
n=3315, 2963, 2872, 1653, 1587, 1518 cm ; H NMR
(DMSO-d ) d=0.87 (t, 3H, J=7.2 Hz, CH ), 1.25–1.34
6
3
(
m, 2H, CH ), 1.38–1.45 (m, 2H, CH ), 3.12–3.16 (m,
2 2
2
H, CH ), 3.79 (s, 3H, OCH ), 6.42 (d, 1H, J=15.7
2 3
Procedure of synthesis of the amide compounds
Hz,=CH), 6.77 (d, 1H, J=8.2 Hz, ArH), 6.96 (dd, 1H,
J=8.2, 1.9 Hz, ArH), 7.10 (d, 1H, J=1.8 Hz, ArH),
7.28 (d, 1H, J=15.8 Hz,=CH), 7.91 (t, 1H, J=4.9 Hz,
Feruloylamide (10). To a solution of O-acetyl feruloyl
chloride (25 g) in CH Cl (250 mL) was added dropwise
2
2
1
3
1
5 mL of 28% aqueous ammonium solution in an ice-
NH), 9.41(bs, 1H, OH); C NMR (DMSO-d ) d=13.8,
6
bath. The mixture was stirred for 30 min at room tem-
perature to produce precipitates. The precipitates were
filtrated and dissolved in MeOH (400 mL). A 46 g of
19.8, 31.5, 38.4, 55.7, 110.9, 115.8, 119.3, 121.6, 126.6,
138.9, 148.0, 148.4, 165.4. MS (ESI-TOF) calcd for
+
+
[C H NO ] 250.14, found 250.10 [M+H] , Anal.
1
4
20
3
.
calcd for C H NO H O: C, 62.90; H, 7.92; N, 5.24.
14 19 3 2
2
8% sodium methoxide in MeOH was added dropwise
to the mixture in an ice-bath. After additional stirring at
room temperature for 15 min, the mixture was con-
centrated under reduced pressure, and water (100 mL)
was added. The solution was neutralized with 10%
aqueous sulfuric acid and extracted with ethyl acetate.
The organic layer was washed with water and dried over
Found: C, 62.86; H, 7.96; N, 5.25.
N-s-Butylferuloylamide (13). To a solution of 0.5 g of O-
acetyl feruloyl chloride in anhydrous CH Cl (20 mL)
was added 0.32 g of s-butylamine. The mixture was
stirred for 2 h at room temperature under nitrogen. The
reaction mixture was washed with water and the organic
2
2
MgSO . After filtration and removal of the solvent, the
4
product was recrystallized from EtOH to afford 13.1 g
ꢁ
portion was dried over MgSO . After filtration, the
4
(
69%) of prisms: mp=151–153 C; IR (KBr) n=3458,
solution was concentrated and hexane was added to
afford crystals (0.5 g). To a solution of 0.2 g of the
crystals in 10 mL acetonitrile was added hydrazine
monohydrate (38 mg). The mixture was stirred for 2 h
at room temperature. The organic portion was extracted
with ethyl acetate, washed with water and saturated
À1
1
3
344, 1657, 1580, 1512 cm
;
H NMR (DMSO-d6)
d=3.79 (s, 3H, OCH ), 6.41(d, 1H , J=15.6 Hz,=CH),
3
6
(
.77 (d, 1H, J=8.0 Hz, ArH), 6.96 (brs, 1H, NH), 6.97
dd, 1H, J=1.6, 8.0 Hz, ArH), 7.11 (d, 1H, J=1.6 Hz,
ArH), 7.30 (d, 1H, J=15.6 Hz,=CH), 7.39 (brs, 1H,
1
3
NH), 9.42 (s, 1H, OH); C NMR (DMSO-d ) d=55.7,
aqueous NaCl, and dried over MgSO . After the solvent
4
6
1
10.9, 115.8, 119.2, 121.9, 126.5, 139.8, 148.0, 148.5,
+
was evaporated, the product was purified by SiO col-
2
1
Found 194.07 [M+H] . Anal. calcd for C H NO : C,
67.4. MS (ESI-TOF) calcd for [C H NO ] 194.08,
+
umn using CHCl /MeOH (2/1) to afford 0.12 g (73%)
3
1
0
12
3
ꢁ
of solids: mp=130–132 C; IR (KBr) n=3344, 2970,
1
0
11
3
À1
1
6
7
2.17; H, 5.74; N, 7.25; Found: C, 61.95; H, 5.74; N,
.22.
2835, 1655, 1587, 1518 cm ; H NMR (CDCl ) d=0.92
3
(t, 3H, J=7.5 Hz, CH ), 1.16 (d, 3H, J=6.6 Hz, CH ),
3
3

3812
E. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 3807–3813
1
.47–1.54 (m, 2H, CH ), 3.88 (s, 3H, OCH ), 4.00–4.07
2
N-Feruloyl pyrrolidine (17). Following a method similar
to that for 13. Recrystallized from CHCl /Hexane to
afford 0.08 g (60%) of solids: mp=170–172 C; IR
3
(
m, 1H, CH), 5.39 (d, 1H, J=8.1Hz, NH), 5.91(s, 1H ,
OH), 6.21(d, H1 , J=15.6 Hz,=CH), 6.87 (d, 1H,
J=8.2 Hz, ArH), 6.96 (d, 1H, J=1.8 Hz, ArH), 7.02
dd, 1H, J=8.2, 1.8 Hz, ArH), 7.50 (d, 1H, J=15.6
3
ꢁ
(KBr) n=3431, 2963, 2874, 2787, 1641, 1601, 1564
À1
1
(
cm
CH ), 1.87–1.93 (m, 2H, CH ), 3.32–3.39 (m, 2H, CH ),
;
H NMR (DMSO-d ) d=1.76–1.82 (m, 2H,
6
1
3
Hz,=CH); C NMR (CDCl ) d=10.3, 20.5, 29.8, 46.7,
3
2
2
2
5
1
2
5.9, 109.6, 114.7, 118.6, 122.0, 127.4, 140.7, 146.7,
+
47.3, 165.5. MS (ESI-TOF) calcd for [C H NO ]
50.14, Found 250.13 [M+H] . Anal. calcd for
3.59–3.63 (m, 2H, CH ), 3.37 (t, 2H, J=6.9 Hz, CH ),
2 2
3.61(t, 2H, J=6.9 Hz, CH ), 3.81(s, 3H, OCH ), 6.76
2 3
1
4
20
3
+
(d, 1H, J=15.4 Hz,=CH), 6.76 (d, 1H, J=8.2 Hz,
ArH), 7.07 (dd, 1H, J=8.2, 1.8 Hz, ArH), 7.26 (d, 1H,
J=2.0 Hz, ArH), 7.36 (d, 1H, J=15.4 Hz,=CH), 9.42
C H NO : C, 67.45; H, 7.68; N, 5.62. Found: C,
19
1
4
3
6
7.08; H, 7.79; N, 5.53.
1
3
(
brs, 1H, OH); C NMR (DMSO-d ) d=24.1, 25.8,
6
N-Pentylferuloylamide (14). Following a method similar
to that for 13. SiO column using CHCl /MeOH (2/1)
45.8, 46.2, 55.9, 111.5, 115.7, 116.7, 122.4, 126.8, 141.0,
148.0, 148.6, 164.1. MS (ESI-TOF) calcd for
2
3
ꢁ
+
+
afford 0.13 g (76%) of solids: mp=73–76 C; IR (KBr)
1
[C H NO ] 248.13, Found 248.12 [M+H] , Anal.
14 18 3
À1
.
n=3261, 2934, 2866, 1655, 1597, 1518 cm ; H NMR
calcd for C H NO 1/2H O: C, 65.61; H, 7.09; N,
14 17 2
3
(
DMSO-d ) d=0.86 (t, 3H, J=6.8 Hz, CH ), 1.24–1.31
5.46. Found: C, 65.27; H, 6.99; N, 5.55.
6
3
(
2
m, 4H, CH ), 1.39–1.46 (m, 2H, CH ), 3.11–3.14 (m,
H, CH ), 3.79 (s, 3H, OCH ), 6.42 (d, 1H, J=15.8
2 2
N-Feruloyl piperidine (18). To a solution of ferulic acid
(0.39 g) in anhydrous DMF/CH Cl (0.5/3 mL) was
2
3
Hz,=CH), 6.77 (d, 1H, J=8.2 Hz, ArH), 6.96 (dd, 1H,
J=8.2, 1.8 Hz, ArH), 7.10 (d, 1H, J=1.8 Hz, ArH),
2
2
added triethylamine (0.61mL) at room temperature.
Iso-butylchloroformate (0.57 mL) was added dropwise
7
NH), 9.41(brs, H1 , OH);
.29 (d, 1H, J=15.6 Hz,=CH), 7.91 (t, 1H, J=5.5 Hz,
1
3
ꢁ
C NMR (DMSO-d6)
at À15 C under nitrogen. After stirring for 30 min,
d=14.1, 22.1, 28,9, 29.1, 38.8, 55.7, 110.8, 115.8,
19.3, 121.6, 126.6, 138.9, 148.0, 148.4, 165.4. MS
piperidine (1.0 mL) was added dropwise and the tem-
perature rose room temperature in 30 min. After addi-
tional stirring for 1h, 10 % citric acid aqueous solution
(20 mL) was added to reach pH 2. The organic portion
was extracted with ethyl acetate (50 mL ꢀ2) and the
organic layer was washed with 10% citric acid aq solu-
tion, water, and saturated aqueous NaCl, followed by
1
+
(
ESI-TOF) calcd for [C H NO ]
264.16, found
64.15 [M+H] , Anal. calcd for C H NO : C,
1
5
22
3
+
2
6
5
1
5
21
3
8.42; H, 8.04; N, 5.32. Found: C, 68.03; H, 8.08; N,
.36.
N-Hexylferuloylamide (15). Following a method similar
to that for 13. SiO column using CHCl /MeOH (20/1)
drying over MgSO . After filtration and removal of the
4
solvent under reduced pressure, the product was pur-
ified by SiO column using hexane/ethyl acetate (1/0–5/
2
3
to afford 0.18 g (94%) of oil: IR (KBr) n=3283, 2930,
2
À1
1
2
860, 1655, 1591, 1516 cm ; H NMR (CDCl ) d=0.87
1) to afford 0.61 g (91%) of white powder: mp=132–
ꢁ
3
ꢁ
14
1
(
t, 3H, J=6.9 Hz, CH ), 1.27–1.38 (m, 6H, CH ), 1.50–
135 C; (134–135 C ). H NMR (CDCl ) d=1.56–1.68
3
2
3
1
OCH ), 5.52 (t, 2H, J=6.8 Hz, NH), 5.79 (s, 1H, OH),
.57 (m, 2H, CH ), 3.33–3.38 (m, 2H, CH ), 3.90 (s, 3H,
(m, 6H, CH ), 3.56–3.63 (m, 4H, 2ꢀNCH ), 3.90 (s, 3H,
2
2
2
2
CH O), 6.72 (d, 1H, J=15.2 Hz,=CH–), 6.89 (d, 1H,
3
3
6
.21(d, 1H , J=15.6 Hz,=CH), 6.88 (d, 1H, J=8.2 Hz,
J=8.4 Hz, ArH), 6.96 (d, 1H, J=2.0 Hz, ArH), 7.06
(dd, 1H, J=8.4, 1.6 Hz, ArH), 7.56 (d, 1H, J=15.2
ArH), 6.97 (d, 1H, J=1.8 Hz, ArH), 7.04 (dd, 1H,
J=8.24, 1.8 Hz, ArH), 7.51 (d, 1H, J=15.6 Hz,=CH);
1
3
Hz,-CH=), C NMR (CDCl ) d=24.6, 26.2, 55.9,
3
1
3
C NMR (CDCl ) d=14.0, 22.5, 26.6, 29.7, 31.5, 39.8,
109.9, 114.7, 115.0, 121.7, 128.0, 142.5, 146.7, 147.2,
+
3
5
1
2
5.9, 109.6, 114.7, 118.3, 121.9, 127.4, 140.8, 146.7,
+
47.3, 166.1. MS (ESI-TOF) calcd for [C H NO ]
78.18, found 278.17 [M+H] , Anal. calcd for
165.6. MS (ESI-TOF) calcd for [C H NO ] 262.14,
1
5
20
3
+
Found 262.10 [M+H] . Anal. calcd for C H NO :
15 19 3
1
6
24
3
+
C, 68.94; H, 7.33; N, 5.36. Found: C, 68.66; H, 7.27;
N, 5.57.
C H NO : C, 69.29; H, 8.36; N, 5.05. Found: C,
1
6
23
3
6
9.10; H, 8.60; N, 4.85.
N-Feruloyl tyramine (19). Following a method similar to
that for 13. SiO column using CHCl /MeOH (20/1) to
N-Octylferuloylamide (16). Following a method similar
to that for 13. SiO column using CHCl /MeOH (20/1)
2
3
ꢁ
afford 0.17 g (94%) of solids: mp=68–74 C; IR (KBr)
1
2
3
À1
to afford 0.12 g (68%) of oil: IR (KBr) n=3281, 2928,
n=3300, 2939, 2848, 1655, 1591, 1516 cm ; H NMR
(DMSO-d ) d=2.63 (t, 2H, J=7.3 Hz, CH ), 3.29–3.32
À1
1
2
856, 1657, 1591, 1516 cm ; H NMR (CDCl ) d=0.86
3
6
2
(
t, 3H, J=6.9 Hz, CH ), 1.25–1.35 (m, 10H, CH ), 1.50–
(m, 2H, CH ), 3.79 (s, 3H, OCH ), 6.41(d, 1H , J=15.8
3
2
2 3
1
OCH ), 5.58 (t, 1H, J=5.8 Hz, NH), 5.87 (s, 1H, OH),
.57 (m, 2H, CH ), 3.33–3.38 (m, 2H, CH ), 3.89 (s, 3H,
Hz,=CH), 6.66 (d, 1H, J=8.4 Hz, ArH), 6.77 (dd, 1H,
J=8.1Hz, ArH), 6.96 (dd, 2H, J=8.3, 1.7 Hz, ArH),
6.99 (d, J=8.4 Hz, ArH), 7.09 (d, 1H, J=1.7 Hz, ArH),
7.29 (d, 1H, J=15.6 Hz,=CH), 7.97 (t, 1H, J=5.6 Hz,
2
2
3
6
.22 (d, 1H, J=15.6 Hz,=CH), 6.87 (d, 1H, J=8.1Hz,
ArH), 6.96 (d, 1H, J=1.8 Hz, ArH), 7.03 (dd, 1H,
J=8.2, 1.8 Hz, ArH), 7.51 (d, 1H, J=15.4 Hz,=CH);
1
3
NH), 9.17 (brs, 1H, OH), 9.40 (brs, 1H, OH); C NMR
(DMSO-d ) d=34.6, 40.8, 55.7, 110.9 115.3, 115.8,
1
3
C NMR (CDCl ) d=14.1, 22.6, 27.0, 29.2, 29.3, 29.7,
3
6
3
1
1.8, 39.8, 55.9, 109.6,114.7, 118.4, 122.0, 127.4, 140.8,
46.7, 147.3, 166.1. MS (ESI-TOF) calcd for
119.2, 121.7, 126.6, 129.6, 129.7, 139.0, 148.0, 148.4,
+
155.8, 165.5. MS (ESI-TOF) calcd for [C H NO ]
314.14, Found 314.13 [M+H] , Anal. calcd for
.
C H NO 2/3H O: C, 66.44; H, 6.30; N, 4.30. Found:
18 19 4 2
1
8
20
3
+
+
+
[C H NO ] 306.21, found 306.20 [M+H] , Anal.
calcd for C H NO : C, 70.79; H, 8.91; N, 4.59. Found:
1
8
28
3
1
8
27
3
C, 70.42; H, 8.96; N, 4.64.
C, 66.58; H, 6.00; N, 4.61.

E. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 3807–3813
3813
N-Feruloyl tyrosine (20). To a solution of (4-tert-butoxy-
carbonyloxy-3-methoxyphenyl)-2-propenate (0.29 g), 4-
tert-butoxycarbonyl-tyrosine-tert-butyl ester (0.34 g),
and dimetylaminopyridine (12 mg) in anhydrous
2. Taniguchi, H.; Hosoda, A.; Tsuno, T.; Maruta, Y.;
Nomura, E. Anticancer Res. 1999, 19, 3779.
3. Murakami, A.; Kadota, K.; Takahashi, D.; Taniguchi, H.;
Nomura, E.; Hosoda, H.; Tsuno, T.; Maruta, Y.; Ohigashi,
H.; Koshimizu, K. Can. Lett. 2000, 157, 77.
.
CH Cl (2 mL) was added EDC HCl (0.38 g) at room
2
2
4
. Tsuda, H.; Takasuka, N.; Toriyama, Y.; Nomura, E.;
Taniguchi, H. Anticancer Res. 1999, 19, 5 A, 3779.
. Han, B. M.; Park, C. B.; Takasuka, N.; Naito, A.; Sekine,
temperature. After stirring for 6 h, solvent was evapo-
rated and the product was purified by SiO2 column
using hexane/ethyl acetate (10/1–4/1) to afford 0.53 g of
white powder. A 61mg of the powder was dissolved in 1
mL of acetic acid and allowed to stand for 24 h at room
temperature. Water (3 mL) was added to the solution
and the solvent was evaporated under reduced pressure.
Ether was added and allowed to stand in refrigerator to
produce precipitates. The precipitates were filtrated and
5
K.; Nomura, E.; Taniguchi, H.; Tsuno, T.; Tsuda, H. Jpn. J.
Cancer Res. 2001, 92, 404.
6. Nomura, E.; Hosoda, A.; Morishita, H.; Murakami, A.;
Koshimizu, K.; Ohigashi, H.; Taniguchi, H. Bioorg. Med.
Chem. 2002, 10, 1069.
7
. Hosoda, A.; Nomura, E.; Murakami, A.; Koshimizu, K.;
Ohigashi, H.; Mizuno, K.; Taniguchi, H. Bioorg. Med. Chem.
Lett. 2000, 19, 1439.
8. Hosoda, A.; Ozaki, Y.; Kashiwada, A.; Mutoh, M.;
Wakabayashi, K.; Mizuno, K.; Nomura, E.; Taniguchi, H.
Bioorg. Med. Chem. 2002, 10, 1189.
9. Hosoda, A.; Nomura, E.; Murakami, A.; Koshimizu, K.;
Ohigashi, H.; Mizuno, K.; Taniguchi, H. Bioorg. Med. Chem.
2002, 10, 1855.
washed with a small amount of ethyl acetate to afford
ꢁ
2
8 mg of white powder (78%). mp=207–211 C; IR
(
KBr) n=3140, 2940, 2850, 1720, 1632, 1593, 1508
À1
1
cm ; H NMR (DMSO-d ) d=2.84–2.90 (m, 1H, CH),
6
3
OCH ), 6.69 (d, 1H, J=15.9 Hz,=CH), 6.81 (d, 1H,
.14 (dd, 2H, J=14.5, 4.4 Hz, CH ), 3.82 (s, 3H,
2
3
J=8.2 Hz, ArH), 7.08 (d, 2H, J=8.6 Hz, ArH), 7.19
(dd, 1H, J=8.2, 1.9 Hz, ArH), 7.31 (d, 2H, J=8.6 Hz,
ArH), 7.40 (d, 1H, J=8.6 Hz, ArH), 7.72 (d, 1H,
10. Morishita, H.; Onishi, M. Bioactive Natural Products
2001, 25, 919.
11. Schindler, P. W.; Below, P.; Hemmerle, H.; Burger, H.-J.;
Swamy, K. H. S.; Arion, W. J.; Efendic, S.; Herling, A. W.
1
3
J=15.7 Hz,=CH); C NMR (DMSO-d ) d=36.9,
6
Drug Dev. Res. 1998, 44, 34.
1
5
1
6.1, 56.4, 112.1, 114.1, 116.2, 122.2, 124.3, 126.1, 131.0,
35.6, 147.6, 148.6, 149.9, 150.5, 166.0, 169.8. MS (ESI-
2. Hemmerle, H.; Burger, H.-J.; Below, P.; Schubert, G.;
Rippel, R.; Schindler, P. W.; Paulus, E.; Herling, A. W. J.
Med. Chem. 1997, 40, 137.
+
TOF) calcd for [C H NO ] 358.13, found 358.05
1
9
20
3
+
.
M+H] . Anal. calcd for C H NO 1.5H O: C, 59.37;
19 19 6 2
[
13. Morishita, H.; Ohnishi, M.; Tsuno, T.; Hosoda, A.;
Nomura, E.; Taniguchi, H. Res. Adv. Phytochem 2001, 1, 1 .
H, 5.77; N, 3.64. Found: C, 59.04; H, 5.52; N, 4.02.
1
1
4. Nakatani, N.; Inatani, R.; Fuwa, H. Agric. Biol. Chem.
980, 44, 2831.
15. Inatani, R.; Nakatani, N.; Fuwa, H. Agric. Biol. Chem.
Acknowledgements
1
1
1
1
981, 45, 667.
6. Parker, J. C. Drugs of the Future 2001, 26, 687.
7. Gray, A. M.; Flatt, P. R. J. Nutr. 1998, 128, 2319.
8. Flatt, P. R.; Shibier, O.; Szecowka, J.; Berggren, P.-O.
We wish to thank Prof. S. Nanjyo and Dr. H. Sasaki of
Wakayama Medical University for helpful discussion.
Diabete & Metabolisme 1994, 20, 157.
¨
1
9. Eliasson, L.; Renstro
O.; Bertorello, A. M.; Bokvist, K.; Chibalin, A.; Deeney,
J. T.; Flatt, P. R.; Gabel, J.; Gromada, J.; Larsson, O.;
Lindstrom, P.; Rhodes, C. J.; Rorsman, P. Science 1996,
271, 813.
¨
m, E.; Ammala, C.; Berggren, P.-
¨
¨
References and Notes
¨
1. Taniguchi, H.; Nomura, E.; Tsuno, T.; Minami, S.; Kato,
K.; Hayashi, C. JP-2095088. US Patent 5,288,902.
¨