Synthesis of nicotinamide derivatives having a hydroxy-substituted benzene ring and the influence of their structures on the apoptosis-inducing activity against leukemia cells

Article abstract of DOI:10.1002/ddr.20426

With the view of developing novel apoptosis-inducing agents against malignant cells, nicotinamide derivatives containing substituted O-benzoyl-tyrosine, dopamine, and norepinephrine residues were synthesized. Antiproliferative activity measurements using leukemia U937 cells revealed that the benzoyl-tyrosine derivative having two hydroxys at C-2 and C-3 on the benzoyl group, and the one having a hydroxy at C-2 and a methoxy at C-4 proved the most potent among the 9 nicotinamide derivatives. The IC₅₀ values of these compounds were 0.87 and 3.15 μM, which indicates their noteworthy activity compared with epigallocatechin gallate, a catechin component in green tea known for its noticeable activity. The cell death process was confirmed to be the result of apoptosis by agarose gel electrophoresis and dye staining, suggesting the high potential of these compounds as apoptosis-inducing anticancer agents. Drug Dev Res 72: 289-297, 2011.

Full text of DOI:10.1002/ddr.20426

DRUG DEVELOPMENT RESEARCH 72 : 289–297 (2011)
Research Article
Synthesis of Nicotinamide Derivatives Having a Hydroxy-
Substituted Benzene Ring and the Influence of Their
Structures on the Apoptosis-Inducing Activity Against
Leukemia Cells
Ã
Tomoko Yamaguchi,1 Yuriko Matsumura,1 Takuya Ishii,2 Yoshikazu Tokuoka,2
Ã
and Keisuke Kurita1
1Department of Materials and Life Science, Faculty of Science and Technology, Seikei University,
Musashino-shi, Tokyo 180-8633, Japan
2Department of Medical Technology, Faculty of Biomedical Engineering, Toin University
of Yokohama, Aoba-ku, Yokohama-shi, Kanagawa 225-8502, Japan
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Toxicology, Formulation Phases I-III
Technology,
Genomics,
Proteomics
Preclinical
Research
Postmarketing
Phase IV
Drug Delivery,
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
With the view of developing novel apoptosis-inducing agents against malignant cells,
nicotinamide derivatives containing substituted O-benzoyl-tyrosine, dopamine, and norepinephrine
residues were synthesized. Antiproliferative activity measurements using leukemia U937 cells revealed
that the benzoyl-tyrosine derivative having two hydroxys at C-2 and C-3 on the benzoyl group, and the
one having a hydroxy at C-2 and a methoxy at C-4 proved the most potent among the 9 nicotinamide
derivatives. The IC50 values of these compounds were 0.87 and 3.15 mM, which indicates their noteworthy
activity compared with epigallocatechin gallate, a catechin component in green tea known for its
noticeable activity. The cell death process was confirmed to be the result of apoptosis by agarose gel
electrophoresis and dye staining, suggesting the high potential of these compounds as apoptosis-inducing
anticancer agents. Drug Dev Res 72:289–297, 2011.
r 2010 Wiley-Liss, Inc.
Key words: nicotinamide derivatives; anticancer agent; apoptosis-inducing activity; leukemia cells
INTRODUCTION
against malignant cells but have minimal side effects
on normal cells.
Apoptosis, a programmed cell death [Kerr et al.,
1972], is characterized by various morphological
changes, including phosphatidylserine externalization,
cell shrinkage, chromatin condensation, nuclear col-
lapse, and cellular fragmentation into apoptosis bodies,
and the cells are finally eliminated by macrophages
[Wright et al., 1996; Bortner and Cidlowski, 2002;
Grant sponsor: Seikei University.
Ã
Correspondence to: Yuriko Matsumura, Department of
Materials and Life Science, Faculty of Science and Technology,
Seikei University, Musashino-shi, Tokyo 180-8633, Japan.
Ujibe et al., 2005]. Therefore, apoptosis plays a crucial E-mail: matsumura@st.seikei.ac.jp or Keisuke Kurita, Department
of Materials and Life Science, Faculty of Science and Technology,
Seikei University, Musashino-shi, Tokyo 180-8633, Japan.
E-mail: kurita@st.seikei.ac.jp
role in the maintenance of homeostasis in develop-
mental processes and in the elimination of damaged
cells. Certain compounds are known to induce
apoptosis in cancer cells, and their structure–property
Received 14 September 2010; Accepted 8 October 2010
relationships would be key information to develop
new types of anticancer agents that are effective com). DOI: 10.1002/ddr.20426
Published online in Wiley Online Library (wileyonlinelibrary.
ꢀc
2010 Wiley-Liss, Inc.

290
YAMAGUCHI ET AL.
CO2Me
performed with a Mupid-Scope (Advance Co., Ltd.,
O
Tokyo, Japan). Cell staining was examined with a
fluorescence microscope Nikon ECLIPS ETE300.
Thin-layer chromatography (TLC) and column chro-
matography were conducted on Merck Silica Gel 60
N
H
N
F254 and Silica Gel 60
N (spherical, neutral,
1
63–210 mm; Kanto Chemical Co. Inc., Tokyo, Japan).
The elemental analysis was carried out with a Perkin-
Elmer 2400 analyzer series II or Euro Vector EURO-
EA3000-Dual. All chemicals were of reagent grade and
were used without further purification. Anhydrous
solvents containing no stabilizers for synthetic use, such
as THF, CHCl3, CH2Cl2, and dimethyl sulfoxide
(DMSO), were purchased from Wako Pure Chemical
Industries Ltd. (Osaka, Japan). An Annexin
V-Fluorescein Staining Kit containing annexin
V-fluorescein, a binding buffer, and propidium iodide
was obtained from Wako Pure Chemical Industries, Ltd.
HN
OH
O
OH
Fig. 1. Structure of an amide derivative of nicotinamide (1)
[Yamaguchi et al., 2007].
With regard to the apoptosis induction in cancer
cells, green tea polyphenols have been attracting
considerable attention; catechins as exemplified by
(1)-epigallocatechin gallate (EGCG) are particularly
interesting because of their anticancer activity owing to
their apoptosis-inducing ability [Chen et al., 1998; Yang
et al., 1998; Isemura et al., 2000; Katsuno et al., 2001;
Adhami et al., 2003; Hsu et al., 2003; Suzuki et al.,
2004; Ohata et al., 2005]. Dopamine [He and Yuan,
2007] and some nicotinamide derivatives such as
6-aminonicotinamide and N1-methylnicotinamide
[Luo et al., 1998; Ogata et al., 2000; Jaubert et al.,
2006] also induce apoptosis in HL-60 cells and in 293,
K562, and GH3 cells, respectively.
In our previous study, a nicotinamide derivative
of p-aminophenylalanine (1, Fig. 1) was synthesized
and found to exhibit a considerable growth inhibitory
effect on human leukemia U937 cells [Yamaguchi et al.,
2007], higher than that of EGCG. The results
prompted us to study the influence of chemical
structures on the manifestation of apoptosis activity
through structural modifications by (1) replacing the
aminophenylalanine moiety with the tyrosine one to
incorporate an ester linkage as in EGCG, and (2)
diversifying the substitution patterns on the benzoyl
group. Nicotinamides were also derived from dopa-
mine and norepinephrine as excellent candidates. The
resulting compounds were evaluated in terms of
anticancer activity against U937 cells, and the cell
death process was elucidated by agarose gel electro-
phoresis of the DNA and by annexin V staining of the
treated cells.
N-Nicotinoyl-L-Tyrosine Methyl Ester (3)
A solution of 1.45 g (14mmol) of Et3N in 25 ml of
THF was added dropwise at 01C to a solution of 2.86 g
(12 mmol) of L-tyrosine methyl ester hydrochloride and
2.55g (11 mmol) of nicotinic anhydride in 100 ml of
THF; the mixture was stirred at room temperature for
2.5 h. The mixture was filtered, and the filtrate was
evaporated. The residue was added to 200 ml of AcOEt,
and 50ml of H2O was added to the solution. The
organic layer was separated, washed with saturated
NaCl (50 mlꢁ 2), and dried over anhydrous Na2SO4.
The solvent was evaporated to give 3.10g (93%) of the
product (3): mp 123–1241C; IR (KBr):n 3343, 2947,
1742, 1641, 1596, 1524, 1368, 1321, 1223, and 700 cmꢂ1
;
1H-NMR (CDCl3): d 3.13–3.26 [2H, m, CH2 (Tyr)] 3,
3.80 (3H, s, OCH3), 5.06 [1H, dt, J 5 7.8 and 5.4 Hz,
CH (Tyr)], 6.61 (1H, d, J 5 7.8 Hz, NH), 6.76 [2H, d,
J 5 8.5 Hz, 3,5-H (Tyr)], 6.98 [2H, d, J 5 8.5 Hz, 2,6-H
(Tyr)], 7.41 [1H, dd, J 5 4.8 and 7.8 Hz, 5-H (Pyr)], 8.10
[1H, d, J 5 7.8 Hz, 4-H (Pyr)], 8.76 [1H, d, J 5 4.8 Hz,
6-H (Pyr)], and 8.89ppm [1H, s, 2-H (Pyr)]. Anal. Calcd
for C16H16N2O4 ꢃ 0.1 H2O: C, 63.61; H, 5.40; N, 9.27.
Found: C, 63.42; H, 5.46; N, 9.06.
Synthesis of N-Nicotinoyl-O-(2,3-
Dimethoxybenzoyl)-L-Tyrosine Methyl Ester (4a)
Compound 3 (499 mg, 1.7 mmol) and Et3N
(242 mg, 2.4 mmol) were added to 40 ml of CHCl3.
A solution of 446 mg (2.2 mmol) of 2,3-dimethoxyl-
benzoyl chloride in 20 ml of CHCl3 was added
METHODS AND MATERIALS
General
Infrared (IR) spectra were recorded with a dropwise at 01C, and the mixture was stirred at room
JASCO FT/IR-470. 1H-nuclear magnetic resonance temperature for 3 h. The reaction mixture was washed
(NMR) spectra were obtained on a JEOL JNM- with 50 ml of 5% aqueous NaHCO3 and 50 ml of
LA400D NMR spectrometer, with tetramethylsilane saturated aqueous NaCl, and the solution was dried
used as an internal reference. Electrophoresis was over anhydrous Na2SO4. After removal of the solvent,
Drug Dev. Res.

NICOTINAMIDES HAVING APOPTOSIS-INDUCING ACTIVITY
291
the crude product was purified by column chromato- ABX, J 5 5.5 and 13.5 Hz, CH2 (Tyr)], 3.79 (3H, s,
graphy on silica gel with CHCl3/MeOH (20/1) to afford CO2CH3), 3.93 (3H, s, OCH3), 5.10 [1H, dd, J 5 5.5
615 mg (78%) of the product (4a): mp 127–1281C; IR and 13.5 Hz, CH (Tyr)], 6.72 (1H, d, J 5 7.5 Hz, NH),
(KBr): n 3309, 2943, 1739,1200, 1265, 1481, and 7.02–7.06 [2H, m, 3,5-H (Bnz)], 7.14–7.16 [4H, m,
1
747 cmꢂ1; H-NMR (CDCl3): d 3.27 and 3.33 [2H, J 5 7.3 Hz, 2,3,5,6-H (Tyr)], 7.26 [1H, dd, J 5 4.9 and
ABX, J 5 5.3 and 13.9 Hz, CH2 (Tyr)], 3.81 (3H, s, 8.0 Hz, 5-H (Pyr)], 7.53–7.57 [1H, m, 4-H (Bnz)], 7.99
CO2CH3), 3.92 (3H, s, OCH3), 3.95 (3H, s, OCH3), [1H, d, J 5 1.7 Hz, 6-H (Bnz)], 8.03 [1H, d, J 5 7.8 Hz,
5.11 [1H, d, J 5 8.0 Hz, CH (Tyr)], 6.67 (1H, d, 4-H (Pyr)], 8.74 [1H, s, 6-H (Pyr)], and 8.97 ppm [1H, s,
J 5 7.3 Hz, NH), 7.14–7.18 [6H, m, 2,3,5,6-H (Tyr) 2-H (Pyr)]. Anal. Calcd for C24H22N2O6 ꢃ 0.6 H2O: C,
and 5,6-H (Bnz)], 7.39 [1H, dd, J 5 5.1 and 7.7 Hz, 5-H 64.74; H, 5.25; N, 6.29. Found: C, 64.66; H, 4.89; N, 6.17.
(Pyr)], 7.48–7.51 [1H, m, 4-H (Bnz)], 8.05 [1H, d,
N-Nicotinoyl-O-(3-Methoxybenzoyl)-L-Tyrosine
J 5 7.7 Hz, 4-H (Pyr)], 8.74 [1H, s, 6-H (Pyr)], and
8.97 ppm [1H, s, 2-H (Pyr)]. Anal. Calcd for C25H24
Methyl Ester (4e)
N2O7 ꢃ 0.1H2O: C, 64.40; H, 5.23; N, 6.01. Found: C,
Yield 81%; mp 136–1371C; IR (KBr): n 3358,
64.27; H, 5.03; N, 6.02.
2949, 1752, 1638, 1523, 1278, 1088, 1320, and
1
749 cmꢂ1; H-NMR (CDCl3): d 3.28 and 3.35 [2H,
N-Nicotinoyl-O-(2,4-Dimethoxybenzoyl)-L-Tyrosine
ABX, J 5 5.7 and 13.5 Hz, CH2 (Tyr)], 3.81 (3H, s,
CO2CH3), 3.89 (3H, s, OCH3), 5.11 [1H, dd, J 5 5.7
Methyl Ester (4b)
Yield 72%; mp 141–1421C; IR (KBr): n 3290, and 13.5 Hz, CH (Tyr)], 6.65 (1H, d, J 5 7.5 Hz, NH),
3052, 1737, 1714, 1639, 1611, 1333, 1420, and 7.15–7.20 [5H, m, 2,3,5,6-H (Tyr) and 5-H (Bnz)],
701 cmꢂ1 1H-NMR (CDCl3): d 3.25–3.35 [2H, m, 7.39–7.44 [2H, m, 5-H (Pyr) and 2-H (Bnz)], 7.68 [1H,
;
CH2 (Tyr)], 3.80 (3H, s, CO2CH3), 3.80 (3H, s, OCH3), s, 4-H (Bnz)], 7.79 [1H, d, J 5 7.8 Hz, 6-H (Bnz)], 8.06
3.89 (3H, s, OCH3), 5.11 [1H, d, J 5 7.3 Hz, CH (Tyr)], [1H, d, J 5 7.8 Hz, 4-H (Pyr)], 8.75 [1H, s, 6-H (Pyr)],
6.54 (1H, s, NH), 6.53–6.57 [2H, m, 3,5-H (Bnz)], 6.73 and 8.97 ppm [1H, s, 2-H (Pyr)]. Anal. Calcd for
(1H, d, J 5 7.8 Hz, NH), 7.12–7.17 [4H, m, 2,3,5,6-H C24H22N2O6: C, 66.35; H, 5.10; N, 6.45. Found: C,
(Tyr)], 7.43 [1H, dd, J 5 4.9 and 7.8 Hz, 5-H (Pyr)], 66.63; H, 5.16; N, 6.40.
8.07 [1H, d, J 5 8.5 Hz, 6-H (Bnz)], 8.07 [1H, d,
J 5 8.1 Hz, 4-H (Pyr)], 8.75 [1H, s, 6-H (Pyr)], and
9.01 ppm [1H, s, 2-H (Pyr)]. Anal. Calcd for
N-Nicotinoyl-O-(4-Methoxybenzoyl)-L-Tyrosine
Methyl Ester (4f)
C25H24N2O7 ꢃ 0.1H2O: C, 64.40; H, 5.23; N, 6.01.
Yield 88%; mp 188–1891C; IR (KBr): n 3333,
2961, 1740, 1608, 1266, 1075, and 763 cmꢂ1; 1H-NMR
(CDCl3): d 3.27 and 3.34 [2H, ABX, J 5 5.5 and
12.5 Hz, CH (Tyr)], 3.81 (3H, s, CO2CH3), 3.90 (3H, s,
OCH3), 5.11 [1H, dd, J 5 5.5 and 12.5 Hz, CH (Tyr)],
Found: C, 64.29; H, 5.17; N, 6.01.
N-Nicotinoyl-O-(2,5-Dimethoxybenzoyl)-L-Tyrosine
Methyl Ester (4c)
Yield 72%; mp 104–1051C; IR (KBr): n 3258, 6.64 (1H, d, J 5 7.3 Hz, NH), 6.68 [2H, d, J 5 9.0 Hz,
2940, 1720, 1612, 1507, 1438, 1220, 1166, 833, and 3,5-H (Bnz)], 7.14 [2H, d, J 5 8.5 Hz, 3,5-H (Tyr)], 7.18
1
769 cmꢂ1; H-NMR (CDCl3): d 3.27 and 3.33 [2H, [2H, d, J 5 8.5 Hz, 2,6-H (Tyr)], 7.40 [1H, dd, J 5 4.8
ABX, J 5 5.4 and 13.9 Hz, CH2 (Tyr)], 3.80 (3H, s, and 8.0 Hz, 5-H (Pyr)], 8.06 [1H, dd, J 5 8.0 Hz, 4-H
CO2CH3), 3.82 (3H, s, OCH3), 3.90 (3H, s, OCH3), (Pyr)], 8.14 [2H, d, J 5 9.0 Hz, 2,6-H (Bnz)], 8.75 [1H,
5.11 [1H, d, J 5 7.7 Hz, CH (Tyr)], 6.66 (1H, d, d, J 5 4.8 Hz, 6-H (Pyr)], and 8.97 ppm [1H, s, 2-H
J 5 9.2 Hz, NH), 6.98 [2H, d, J 5 9.2 Hz, 3,5-H (Tyr)], (Pyr)]. Anal. Calcd for C24H22N2O6: C, 66.35; H, 5.10;
7.09–7.14 [2H, m, 3,4-H (Bnz)], 7.17 [2H, d, N, 6.45. Found: C, 66.37; H, 5.08; N, 6.42.
J 5 9.2 Hz, 2,6-H (Tyr)], 7.40 [1H, dd, J 5 4.6 and
Synthesis of N-Nicotinoyl-O-(4-Acetoxybenzoyl)-
8.9 Hz, 5-H (Pyr)], 7.51 [1H, d, J 5 3.1 Hz, 6-H (Bnz)],
L-Tyrosine Methyl Ester (4g)
8.05 [1H, d, J 5 8.9 Hz, 4-H (Pyr)], 8.74 [1H, d,
J 5 4.6 Hz, 6-H (Pyr)], and 8.97 ppm [1H, s, 2-H (Pyr)].
To a solution of 407 mg (1.4 mmol) of 3 and
Anal. Calcd for C25H24N2O7: C, 64.65; H, 5.21; N, 206 mg (2.0 mmol) of Et3N in 80 ml of THF was
6.03. Found: C, 64.70; H, 5.10; N, 5.99.
added dropwise a solution of 404 mg (2.0 mmol) of
4-acetoxylbenzoyl chloride in 15 ml of THF at 01C. The
mixture was stirred for 1 h at 01C and for an additional
16 h at room temperature. After removal of the solvent,
N-Nicotinoyl-O-(2-Methoxybenzoyl)-L-Tyrosine
Methyl Ester (4d)
Yield 71%; mp 128–1301C; IR (KBr): n 3319, 50 ml of CHCl3 and 30 ml of H2O were added to the
2952, 1743, 1639, 1490, 1436, 1240, 1038, and residue. The CHCl3 layer was washed with 50 ml of
1
755 cmꢂ1; H-NMR (CDCl3): d 3.26 and 3.32 [2H, saturated NaCl and dried over anhydrous Na2SO4.
Drug Dev. Res.

292
YAMAGUCHI ET AL.
After removal of the solvent, the crude product was and 7.9 Hz, 5-H (Pyr)], 7.93 [1H, d, J 5 8.7 Hz, 6-H
purified by column chromatography on silica gel with (Bnz)], 8.06 [1H, dd, J 5 7.9 Hz, 4-H (Pyr)], 8.73 [1H,
CHCl3/MeOH (20/1) as an eluant to give 545 mg (87%) d, J 5 4.8 Hz, 6-H (Pyr)], 8.95 [1H, s, 2-H (Pyr)] and
of the product (4g): mp 157–1581C; IR (KBr): n 3322, 10.66 ppm (1H, s, OH). Anal. Calcd for C24H22N2O7: C,
2952, 1739, 1640, 1273, 1017, and 699 cmꢂ1; 1H-NMR 63.99; H, 4.92; N, 6.22. Found: C, 63.83; H, 4.91; N 6.08.
(DMSO-d6): d 2.31 (3H, s, OCOCH3), 3.09–3.27 [2H,
N-Nicotinoyl-O-(2,5-Dihydroxybenzoyl)-L-Tyrosine
m, CH2 (Tyr)], 3.78 (3H, s, OCH3), 4.71 [1H, dd,
J 5 5.3 and 13.4 Hz, CH (Tyr)], 7.20 [2H, d, J 5 8.2 Hz,
Methyl Ester (5c)
2,6-H (Bnz)], 7.37 [4H, dd, J 5 8.5 and 13.6 Hz,
Yield 24%; mp 97–981C; IR (KBr): n 3286, 1741,
1H-NMR
2,3,5,6-H (Tyr)], 7.51 [1H, dd, J 5 4.8 and 7.9 Hz, 1640, 1592, 1508, 1312, and 793 cmꢂ1
;
5-H (Pyr)], 8.13–8.16 [3H, m, 3,5-H (Bnz), and NH], (CDCl3): d 3.24 and 3.33 [2H, ABX, J 5 5.5 and
8.71 [1H, d, J 5 4.8 Hz, 4-H (Pyr)], 8.95 [1H, s, 2-H 14.0 Hz, CH2 (Tyr)], 3.81 (3H, s, CO2CH3), 5.11 [1H,
(Pyr)], and 9.14 ppm [1H, d, J 5 7.9 Hz, 6-H (Pyr)]. dd, J 5 5.5 and 13.1 Hz, CH (Tyr)], 6.64 (1H, br, NH),
Anal. Calcd for C25H22N2O7: C, 64.93; H, 4.80; N, 6.92 [2H, d, J 5 9.0 Hz, 3,5-H (Tyr)], 7.07–7.11 [2H, m,
6.06. Found: C, 64.99; H, 4.73; N, 5.96.
3,4-H (Bnz)], 7.19 [2H, d, J 5 9.0 Hz, 2,6-H (Tyr)],
7.38–7.43 [2H, m, 5-H (Pyr) and 6-H (Bnz)], 8.08 [1H,
d, J 5 7.9 Hz, 4-H (Pyr)], 8.75 [1H, d, J 5 4.1 Hz, 6-H
(Pyr)], 8.95 [1H, s, 2-H (Pyr)], and 10.00 ppm (1H, s,
Synthesis of N-Nicotinoyl-O-(2,3-
Dihydroxybenzoyl)-L-Tyrosine Methyl Ester (5a)
To a solution of 106 mg (0.23 mmol) of 4a in 10 ml OH). Anal. Calcd for C23H20N2O7 ꢃ 0.8 H2O: C, 61.28;
of CH2Cl2 was slowly added 0.86 ml (0.86 mmol) of 1 M H, 4.83; N, 6.21. Found: C, 61.35; H, 4.57; N, 6.00.
BBr3 in CH2Cl2 at ꢂ301C under an Ar atmosphere.
N-Nicotinoyl-O-(2-Hydroxy-5-Methoxybenzoyl)-
The reaction mixture was stirred for 72 h at room
L-Tyrosine Methyl Ester (5c0)
temperature. The mixture was cooled to ꢂ301C again,
and 4.0 ml of MeOH was added slowly to quench the
Yield 20%; mp 97–981C; IR (KBr): n 3274, 1744,
1H-NMR
reaction. After evaporation of the solvent, the residue 1662, 1592, 1508, 1348, and 773 cmꢂ1
;
was adjusted to pH 7 with 1 M NaOH, and the mixture (CDCl3): d 3.26 and 3.36 [2H, ABX, J 5 5.5 and
was extracted with CHCl3 (40 ml ꢁ 3). The combined 14.0 Hz, CH2 (Tyr)], 3.81 (3H, s, CO2CH3), 3.82 (3H,
organic layers were dried over anhydrous MgSO4, and s, OCH3), 5.11 [1H, dd, J 5 5.5 and 13.1 Hz, CH (Tyr)],
after removal of the solvent, the crude product was 6.69 (1H, d, J 5 7.3 Hz, NH), 6.98 [2H, d, J 5 9.0 Hz,
purified by column chromatography on silica gel with 3,5-H (Tyr)], 7.14–7.18 [2H, m, 3,4-H (Bnz)], 7.23 [2H,
CHCl3/MeOH (6/1) to afford 44 mg (16%) of the d, J 5 9.0 Hz, 2,6-H (Tyr)], 7.41 [1H, dd, J 5 4.1 and
product (5a): mp 129–1301C; IR (KBr): n 3320, 2951, 7.9 Hz, 5-H (Pyr)], 7.47 [1H, d, J 5 3.1 Hz, 6-H (Bnz)],
1747, 1682, 1593, 1532, 1369, 1299, and 741 cmꢂ1
;
8.08 [1H, d, J 5 7.9 Hz, 4-H (Pyr)], 8.75 [1H, d,
1H-NMR (CDCl3): d 3.27 and 3.36 [2H, ABX, J 5 5.4 J 5 4.1 Hz, 6-H (Pyr)], 8.95 [1H, s, 2-H (Pyr)]
and 13.9 Hz, CH2 (Tyr)], 3.81 (3H, s, CO2CH3), 5.11 and 10.08 ppm (1H, s, OH). Anal. Calcd for
[1H, dd, J 5 5.3 and 12.4 Hz, CH (Tyr)], 6.66 (1H, d, C24H22N2O7 ꢃ 0.5 H2O: C, 62.74; H, 5.05; N, 6.10.
J 5 7.3 Hz, NH), 7.14–7.23 [6H, m, 4,6-H (Bnz), and Found: C, 62.88; H, 4.96; N, 5.82.
2,3,5,6-H (Tyr)], 7.41 [1H, m, 5-H (Pyr)], 7.59 [1H, d,
N-Nicotinoyl-O-(2-Hydroxybenzoyl)-L-Tyrosine
J 5 7.5 Hz, 5-H (Bnz)], 8.07 [1H, d, J 5 7.5 Hz, 4-H
(Pyr)], 8.75 [1H, m, 6-H (Pyr)], and 8.96 [1H, s, 2-H
Methyl Ester (5d)
(Pyr)] and 10.5 ppm (1H, br, OH). Anal. Calcd for
Yield 19%; mp 129–1311C; IR (KBr): n 3333,
C23H20N2O7 ꢃ 1.2 H2O: C, 60.31; H, 4.93; N, 6.12. 3218, 2948, 1749, 1484, and 762 cmꢂ1
;
1H-NMR
Found: C, 60.23; H, 4.75; N, 5.88.
(CDCl3): d 3.28 and 3.36 [2H, ABX, J 5 5.6 and
13.5 Hz, CH2 (Tyr)], 3.81 (3H, s, CO2CH3), 5.11 [1H,
dd, J 5 5.6 and 13.5 Hz, CH (Tyr)], 6.72 (1H, s, NH),
6.96 [1H, m, 5-H (Bnz)], 7.04 [1H, d, J 5 8.3 Hz, 3-H
N-Nicotinoyl-O-(2-Hydroxy-4-Methxybenzoyl)-
L-Tyrosine Methyl Ester (5b)
Yield 59%; mp 148–1491C; IR (KBr): n 3322, (Bnz)], 7.14–7.16 [2H, m, 2,6-H (Tyr)], 7.21–7.23 [2H,
1742, 1671, 1589, 1531, 1357, and 773 cmꢂ1; 1H-NMR m, 3,5-H (Tyr)], 7.43 [1H, dd, J 5 4.8 and 8.0 Hz, 5-H
(CDCl3): d 3.24 and 3.33 [2H, ABX, J 5 5.6 and (Pyr)], 7.52–7.57 [1H, m, 4-H (Bnz)], 8.05 [1H, d,
13.5 Hz, CH2 (Tyr)], 3.80 (3H, s, CO2CH3), 3.85 (3H, J 5 8.0 Hz, 6-H (Bnz)], 8.10 [1H, d, J 5 8.0 Hz, 4-H
s, OCH3), 5.10 [1H, dd, J 5 5.6 and 13.5 Hz, CH (Tyr)], (Pyr)], 8.75 [1H, s, 6-H (Pyr)], 8.99 [1H, s, 2-H (Pyr)],
6.48–6.53 [2H, m, 3,5-H (Bnz)], 6.81 (1H, d, and 10.5 ppm (1H, br, OH). Anal. Calcd for
J 5 7.3 Hz, NH), 7.13 [2H, d, J 5 8.5 Hz, 3,5-H (Tyr)], C23H20N2O6 ꢃ 0.4H2O: C, 64.60; H, 4.90; N, 6.55.
7.20 [2H, d, J 5 8.5 Hz, 2,6-H (Tyr)], 7.38 [1H, dd, 4.8 Found: C, 64.66; H, 4.98; N, 6.35.
Drug Dev. Res.

NICOTINAMIDES HAVING APOPTOSIS-INDUCING ACTIVITY
293
N-Nicotinoyl-O-(3-Hydroxybenzoyl)-L-Tyrosine
product (8): mp 197–1981C; IR (KBr): n 3480, 3312,
3074, 1636, 1539, 1200, 1371, 1292, and 703 cmꢂ1
Methyl Ester (5e)
;
1H-NMR (CD3OD): d 2.75 (2H, m, CH2), 3.55 (2H, m,
NH-CH2), 6.54–6.56 (1H, m, 50-H,), 6.67–6.68 (2H, s,
20,60-H), 7.51 [1H, dd, J 5 4.8 and 7.9 Hz, 5-H (Pyr)],
8.18 [1H, d, J 5 7.9 Hz, 4-H (Pyr)], 8.64–8.66 [1H, m,
6-H (Pyr)], and 8.80 ppm [1H, s, 2-H (Pyr)]. Anal.
Calcd for C14H14N2O3: C, 65.11; H, 5.46; N, 10.85.
Found: C, 65.24; H, 5.55; N, 10.83.
Yield 22%; mp 163–1641C; IR (KBr): n 3319,
1739, 1642, 1269, 1165, and 700 cmꢂ1 1H-NMR
;
(CDCl3): d 3.17 and 3.26 [2H, ABX, J 5 5.8 and
13.9 Hz, CH2 (Tyr)], 376 (3H, s, CO2CH3), 5.07 [1H,
dd, J 5 5.8 and 13.9 Hz, CH (Tyr)], 7.05–7.11 [4H, m,
2,3,5,6-H (Tyr)], 7.13 [1H, s, 4-H (Bnz)], 7.15 [1H, s,
2-H (Bnz)], 7.31 [1H, t, J 5 7.8 Hz, 5-H (Bnz)], 7.38
[1H, dd, J 5 4.8 and 7.6 Hz, 5-H (Pyr)], 7.62 (1H, s,
NH), 7.65 [1H, d, J 5 7.5 Hz, 6-H (Bnz)], 8.10 [1H, d,
J 5 7.6 Hz, 4-H (Pyr)], 8.69 [1H, s, 6-H (Pyr)], and
8.97 ppm [1H, s, 2-H (Pyr)]. Anal. Calcd for
C23H20N2O6: C, 65.71; H, 4.79; N, 6.66. Found: C,
65.82; H, 4.83; N, 6.31.
Synthesis of N-nicotinoylnorepinephrine (9)
Norepinephrine (352 mg, 2.1 mmol) and nicotinic
anhydride (508 mg, 2.2 mmol) were dissolved in a
mixture of 60 ml of THF and 20 ml of MeOH, and the
solution was stirred for 20 h at room temperature. After
evaporation of the solvent, the crude product was
recrystallized from EtOH to give 208 mg (37%) of the
product (9): mp 1951C (decomp.); IR (KBr): n 3295,
Synthesis of N-Nicotinoyl-O-(4-Hydroxybenzoyl)-
L-Tyrosine Methyl Ester (5g)
1640, 1543, 1368, 1291, and 704 cmꢂ1 1H-NMR
;
A mixture of 287 mg (0.62 mmol) of 4g in 20 ml of
MeOH/THF (1/1) and 86 mg (0.62 mmol) of K2CO3
was stirred for 1 h at room temperature. An additional
30 mg (0.22 mmol) of K2CO3 was added, and the
mixture was further stirred for 30 min. After removal of
the solvent, 20 ml each of AcOEt and H2O were added
to the residue. The resulting solid was collected by
suction filtration to give 166 mg (64%) of the product
(5g): mp 2201C (decomp.); IR (KBr): n 3057, 3319,
2948, 1739, 1642, 1269, 1068, and 700 cmꢂ1; 1H-NMR
(DMSO-d6): d 3.06–3.25 [2H, m, CH2 (Tyr)], 3.65 (3H,
s, CO2CH3), 4.66–4.72 [1H, m, CH (Tyr)], 6.89 [2H, d,
J 5 8.6 Hz, 2-H and 6-H (Bnz)], 7.13 [2H, d,
J 5 8.5 Hz, 2,6-H (Tyr)], 7.35 [2H, d, J 5 8.5 Hz,
3,5-H (Tyr)], 7.50 [1H, dd, J 5 4.9 and 8.0 Hz, 5-H
(Pyr)], 7.93 [2H, d, J 5 8.6 Hz, 3,5-H (Bnz)], 8.12 [1H,
d, J 5 8.0 Hz, 4-H (Pyr)], 8.70 [1H, d, J 5 4.9 Hz, 6-H
(Pyr)], 8.92 [1H, s, 2-H (Pyr)], and 9.13 ppm (1H, d,
J 5 8.0 Hz, OH). Anal. Calcd for C23H20N2O6: C,
65.71; H, 4.79; N, 6.66. Found: C, 65.82; H, 4.83;
N, 6.31.
(CDCl3): d 3.53–3.55 (2H, m, CH2), 4.74 (1H, dd,
J 5 5.6 and 7.3 Hz, CH), 6.72–6.73 (2H, m, 50,60-H),
6.85 (1H, s, 20-H), 7.52 [1H, dd, J 5 4.8 and 7.8 Hz,
5-H (Pyr)], 8.19 [1H, m, 4-H (Pyr)], 8.66 [1H, d,
J 5 4.8 Hz, 6-H (Pyr)], and 8.82 ppm [1H, s, 2-H (Pyr)].
Anal. Calcd for C14H14N2O4: C, 61.31; H, 5.14; N,
10.21. Found: C, 61.43; H, 5.11; N, 10.19.
Measurement of Antiproliferative Activity Against
U937 Cells
U937 cells were seeded in each well of a 12-well
culture plate (0.8 ꢁ 105 cells/well) with 760 ml of an
RPMI 1640 medium containing 5% penicillin-strepto-
mycin and 10% fetal bovine serum (FBS). A compound
synthesized above was dissolved in DMSO to prepare a
10 mM solution, which was added to each well so that
the final concentration of the compound would be
88 mM. After incubation at 371C under an atmosphere
of 5% CO2–95% air for 48 h, the number of viable cells
was determined by staining with trypan blue.
Synthesis of N-Nicotinoyldopamine (8)
DNA Fragmentation Analysis by Electrophoresis
Dopamine hydrochloride (541 mg, 2.8 mmol) was
U937 cells (2.4 ꢁ 105 cells) were suspended in
added to a solution of 538 mg (2.7 mmol) of nicotinic 20 ml of an RPMI 1640 medium containing 5%
anhydride in 100 ml of THF. A solution of 304 mg penicillin-streptomycin and 10% FBS, and a DMSO
(3.0 mmol) of Et3N in 20 ml of THF was added solution of a nicotinamide compound was added to
dropwise at 01C. After 20 h stirring at room tempera- make the final concentration of the compound 99 mM.
ture, the resulting salt was removed by filtration. The cells were incubated in a similar manner and
Evaporation of the filtrate gave a solid, to which 200 ml centrifuged at 15,000 rpm for 10 min. The cells were
of AcOEt and 20 ml of H2O were added. The AcOEt washed with cooled phosphate buffer saline (PBS)
layer was separated, washed with 50 ml of saturated several times, centrifuged again, and lysed in 200 ml of a
aqueous NaCl, and dried over anhydrous Na2SO4. lysis buffer (1 ml of 1 M Tris-HCl buffer of pH 7.4,
After removal of the solvent, the crude product was 0.2 ml of 0.5 M EDTA, and 0.5 ml of 10% Triton
purified by column chromatography on silica gel with X-100). After lysed cells were held at 41C for 10 min,
CHCl3/MeOH (20/1) to afford 120 mg (20%) of the the supernatant was incubated with 2 ml of RNase A
Drug Dev. Res.

294
YAMAGUCHI ET AL.
(10 mg/ml in Tris-EDTA buffer) at 501C for 30 min and various benzoyl groups (4a–f) in satisfactory yields
then with 2 ml of proteinase K (10 mg/ml in distilled (72–87%). Generation of hydroxy groups from the
water) for 45 min at 501C. The solution was mixed with methoxy groups of 4 was carried out with BBr3 to
20 ml of 5 M NaCl and 120 ml of 2-propanol, and the synthesize the target compounds (5) (Fig. 2).
mixture was incubated at ꢂ201C for 24 h. It was
Deprotection of 4a, 4d, and 4e was effected
centrifuged at 15,000 rpm for 20 min. The precipitated smoothly to give the corresponding hydroxy-containing
DNA was dissolved in 5 ml of Tris-EDTA buffer and compounds, 5a, 5d, and 5e. Compound 4c, however,
subjected to electrophoresis with 2% agarose gel using gave a mixture of monohydroxy and dihydroxy deriva-
Tris-acetate-EDTA buffer at 50 V. The DNA fragmenta- tives 5c and 5c0 in comparable yields, implying that the
tion pattern was visualized with a UV transilluminator. demethylation at C-5 was somewhat sluggish compared
with that at C-2. In the deprotection of 4b and 4f, the
C-4 methoxy was found to be very resistive, and several
Annexin V and PI Staining
U937 cells (2.0 ꢁ 105 cells) were suspended in
attempts to generate a hydroxy were unsuccessful.
4950 ml of an RPMI 1640 medium containing 5%
Compound 4b thus gave 5b having a hydroxy only at
penicillin-streptomycin and 10% FBS and treated with
50 ml of a 5 mM DMSO solution of a nicotinamide
compound whose final concentration was 50 mM. They
were incubated similarly, washed with cooled PBS,
C-2, but in the reaction of 4f, only 3 was identified in
the reaction mixture. In order to prepare a derivative
having a hydroxy at C-4, therefore, acetyl protection
was applied to make possible facile deprotection.
collected by centrifugation, and lysed in annexin
Acylation of 3 was performed with p-acetoxybenzoyl
V-fluorescein (2 ml), a binding buffer (100 ml), and
chloride, and the product (4g) was treated with
propidium iodide (2 ml). The suspension was incubated
for 15 min at room temperature, and the cells were
observed by fluorescence microscopy.
aqueous potassium carbonate, giving rise to the
p-hydroxy derivative (5g) (Fig. 3).
Transformations of 4a–e to 5a–e were not
quantitative owing to the difficulty in demethylation.
Besides small scales of the reactions, some side
reactions were responsible for lowering the yields.
RESULTS AND DISCUSSION
Synthesis of Nicotinamide Derivatives
The major side product was 3 as confirmed by TLC
during the deprotection reaction, and this is most likely
interpreted as a result of ester cleavage with a strong
Lewis acid like BBr3 that proceeds easily even at low
temperatures [Yazawa et al., 1974]. Compared with
L-Tyrosine methyl ester hydrochloride (2) was
nicotinoylated with nicotinic anhydride in the presence
of Et3N to give N-nicotinoyl-L-tyrosine methyl ester (3).
Subsequent acylation with substituted benzoyl chlor-
ides resulted in the formation of nicotinamides having
O
O
CO2Me
CO2Me
O
O
ClH3N
N
H
N
N
N
Et3N, dry THF
2
3
OH
OH
COCl
R1
CO2Me
O
N
4a: R1=R2=OMe, R3=R4=H
4b: R1=R3=OMe, R2=R4=H
4c: R1=R4=OMe, R2=R3=H
4d: R1=OMe, R2=R3=R4=H
4e: R2=OMe, R1=R3=R4=H
4f : R3=OMe, R1=R2=R4=H
H
R4
R2
N
R4
R1
R3
R3
R2
4
Et3N, dry CHCl3
O
O
CO2Me
O
N
H
BBr3
5a : R1=R2=OH, R3=R4=H
5b : R1=OH, R2=R4=H, R3=OMe
5c : R1=R4=OH, R2=R3=H
5c': R1=OH, R2=R3=H, R4=OMe
5d : R1=OH, R2=R3=R4=H
5e : R2=OH, R1=R3=R4=H
N
R4
dry CH2Cl2
R3
R2
5
O
O
R1
Fig. 2. Synthesis of compounds 5a–e.
Drug Dev. Res.

NICOTINAMIDES HAVING APOPTOSIS-INDUCING ACTIVITY
295
COCl
CO2Me
CO2Me
O
CO2Me
O
O
N
N
N
H
H
H
K2CO3
N
N
OAc
N
OAc
OH
THF, MeOH
3
Et3N, dry CHCl3
OH
O
O
O
4g
5g
O
Fig. 3. Synthesis of compound 5g.
O
O
O
ClH3N
O
N
H
N
N
N
OH
6
OH
8
Et3N, dry THF
OH
OH
O
O
O
O
OH
OH
H2N
N
N
N
H
N
dry THF
OH
OH
9
7
OH
OH
Fig. 4. Synthesis of compounds 8 and 9.
demethylation of 4f, deacetylation of 4g was quite easy,
and 5g was obtained in a satisfactory yield.
Dopamine (6)- and norepinephrine (7)-contain-
ing nicotinamide derivatives (8 and 9) were synthesized
by nicotinoylation of dopamine and norepinephrine
(Fig. 4), and the yields were not high. The low yields
appeared to be the result of the limited solubility of
dopamine and norepinephrine in the reaction solvent.
Apoptosis-Inducing Activity of the Nicotinamide
Derivatives
Fig. 5. Viability percentage of U937 cells treated with nicotinamide
Ã
derivatives. Po0.05 vs. control of difference between indicated data
using a t-test. The values are expressed as means7SD, n 5 3.
EGCG is known to induce apoptosis through the
activation of caspase-3 and caspase-9, induction of
proapoptotic Bax, Bak, and Bcl-XS, and inhibition of 371C in an atmosphere of 5% CO2–95% air, the
antiapoptotic Bcl-2 and Bcl-XL [Shankar et al., 2007]. viabilities of the cells were measured.
Although structurally dissimilar to EGCG, 6-aminoni-
The influence of the products on the viability
cotinamide was also reported to induce apoptosis via along with that of EGCG is shown in Figure 5, where
the caspase pathway. Compounds 5, 8, and 9 the values of the products with only one hydroxy on the
synthesized here have both the phenol and nicotina- benzoyl group (5d, 5e, and 5g) indicated that 5e was
mide moieties, and it is thus considered of interest to the most effective antiproliferative agent, suggesting
explore their potential of apoptosis induction.
the importance of the position C-3 to accommodate a
As a primary screening test, antiproliferative hydroxy.
activities of 5, 8, and 9 were evaluated by adding their
Although both the hydroquinone- and catechol-
DMSO solution to U937 cells in an RPMI-1640 containing remoxipeide metabolites, NCQ344 and
medium containing 5% penicillin-streptomycin and NCQ436, were reported to promote apoptosis in
10% FBS, final concentration of a nicotinamide HL-60 cells [Inayat-Hussain et al., 2000], our results
derivative being 88 mM. After incubation for 24 h at showed that catechol derivative 5a was more potent
Drug Dev. Res.

296
YAMAGUCHI ET AL.
than hydroquinone derivative 5c. The activity of ester derivatives [Nesterenko et al., 2003], rhinacanthin-N
derivative 5a appeared to be comparable with that of [Siripomg et al., 2006], and antraquinone derivatives
the corresponding amide derivative 1 in our previous [Shi et al., 2008]. The remarkable activity of 5b as
report [Yamaguchi et al., 2007]. These results support evident in Figure 5 confirmed the high potential of
the importance of the catechol-type structure with C-2 such compounds having a methoxy and a hydroxy on an
and C-3 hydroxy groups on the benzene ring to aromatic ring. The difference in the activity of 8 and 9
effectively suppress the growth of U937 cells.
When the compounds having a hydroxy at C-2 secondary hydroxy group appeared to lower the activity.
and a methoxy at C-3 (5b) or C-4 (5c0) were compared,
IC50 values were then determined with the four
was substantial, and the introduction of an additional
5b was far more active than 5c0. As a compound with a most potent compounds, 5a, 5b, 5e, and 8 as a
methoxy at C-4, p-methoxychalcone exhibited antipro- measure to explicate the effective concentrations in
liferative activity against A375 cells [Henmi et al., view of actual clinical chemotherapy. Judging from the
2009]. Apoptosis in various human cell lines was also IC50 values in Table 1, 5a and 5b were especially
observed with some compounds having a methoxy and noteworthy and even more potent than 1 and EGCG.
a hydroxy on an aromatic ring; aromatic amine
To elucidate the death process of cells, either
apoptosis or necrosis, the state of DNA of U937 cells
after treatment was examined by agarose gel electro-
phoresis. Since internucleosomal DNA fragmentation
is a distinctive biochemical feature of the apoptotic
process, it will provide a suitable proof of apoptosis.
The results of electrophoresis for 5a, 5b, 5e, and 8
together with those of references are shown in Figure 6.
A typical DNA ladder pattern due to apoptosis was
observed for the cells treated with 5a and 5b, as in the
case with EGCG, but not for the cells treated with 5e
and 8 under the experimental conditions. These results
indicate that 5a and 5b evidently induced apoptosis in
U937 cells, but as for 5e and 8, actual triggers for the
cell death were not specified.
TABLE 1. IC50 Values Against U937 Cellsy
Compound
IC50 (mM)
EGCG
5a
5b
5e
8
15.6572.39
3.1570.70
0.8770.52
20.8372.30
56.4374.95
Ã
Ã
Ã
yValues are expressed as means7SD, n 5 3; Po0.05 vs. EGCG.
M
1
2
3
4
5
6
7
To confirm further that the cell death was really
associated with apoptosis on treatment with 5a and 5b,
staining of externalized phosphatidylserine with annex-
in V and propidium iodide (PI) was carried out.
Annexin V, a calcium-dependent phospholipid-binding
protein, shows a strong affinity for phosphatidylserine
that is externalized onto the cell surface in the early
stage of apoptosis cell death, resulting in green
fluorescence [Engeland et al., 1998; Kim et al., 2006;
Lee et al., 2006]. Necrosis, on the other hand, increases
permeability of cell membranes, which allows staining
of the cell nucleus by PI to express red fluorescence.
Accordingly, fluorescence microscopy after staining will
be a reliable tool to distinguish between apoptosis and
necrosis. As shown in Figure 7, only green fluorescence
Fig. 6. Agarose gel electrophoresis of DNA fragmentation induced in
U937 cells by nicotinamide derivatives visualized under UV light with
ethidium bromide staining: M, DNA marker; ]1, control; ]2, DMSO;
]3, EGCG; ]4, compound 8; ]5, compound 5e; ]6, compound 5a; and
]7, compound 5b.
1
2
3
Fig. 7. Annexin V and PI staining of U937 cells treated with 5a and 5b: ]1, DMSO; ]2, compound 5a; and ]3, compound 5b. [Color figure can
be viewed in the online issue which is available at wileyonlinelibrary.com]
Drug Dev. Res.

NICOTINAMIDES HAVING APOPTOSIS-INDUCING ACTIVITY
297
was observed for the cells treated with 5a and 5b, Katsuno Y, Koyama Y, Saeki K, Sazuka M, Ookawa K, Isemura M.
2001. Apoptosis-inducing activity of a driselase digest fraction of
green tea residue. Biosci Biotechnol Biochem 65:198–201.
indicating that the cells were annexin V-positive and
PI-negative. The results obtained by agarose gel electro-
phoresis and annexin V staining thus conclude that 5a
and 5b induced apoptosis in U937 cells effectively.
Kerr JFR, Wyllie AH, Currie AR. 1972. Apoptosis: a basic biological
phenomenon with wide rang implications in tissue kinetics. Br J
Cancer 26:239–257.
Kim DS, Kim SY, Jeong YM, Jeon SE, Kim MK, Kwon SB, Na JI,
Park KC. 2006. Light-activated indole-3-acetic acid induces apoptosis
in G361 human melanoma cells. Biol Pharm Bull 29:2404–2409.
CONCLUSION
Of 9 compounds synthesized and evaluated in this
study, 5a and 5b were confirmed to exhibit the highest
antiproliferative activities, and they proved promising
as a new type of apoptosis inducing agents. The results
obtained in this study would be useful as basic
information for designing intelligent molecular archi-
tectures to express improved apoptosis-inducing activ-
ity against malignant cells, and our efforts will be
concentrated further on the in-depth study of the
structure-property relationship to develop potent antic-
ancer agents for chemotherapy.
Lee DH, Park T, Kim HW. 2006. Induction of apoptosis by
disturbing mitochondrial-membrane potential and cleaving PARP
in Jurkat T cells through treatment with acetoxyscirpenol
mycotoxins. Biol Pharm Bull 29:648–654.
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induces apoptosis through an oxidation-involved SAPK/JNK
activation pathway. J Biol Chem 273:3756–3764.
Nesterenko V, Putt KS, Hergenrother PJ. 2003. Identification from a
combinatorial library of a small molecule that selectively induces
apoptosis in cancer cells. J Am Chem Soc 125:14672–14673.
Ogata S, Takeuchi M, Fujita H, Shibata K, Okumura K, Taguchi H.
2000. Apoptosis induced by nicotinamide-related compounds and
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ACKNOWLEDGMENTS
This work was supported in part by a Grant for
Scientific Research from Seikei University.
Ohata M, Koyama Y, Suzuki T, Hayakawa S, Saeki K, Nakamura Y,
Isemura M. 2005. Effects of tea constituents on cell cycle
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