Novel ninhydrin adduct of catechin with potent antioxidative activity

Article abstract of DOI:10.1016/j.tetlet.2009.09.149

The reaction of ninhydrin with (+)-catechin in the presence of TMSOTf resulted in condensation product 1, which consists of a 2:1 mixture of epimers at the C-2 position. The antioxidative radical-scavenging activity of 1 against the galvinoxyl radical, ac

Full text of DOI:10.1016/j.tetlet.2009.09.149

Tetrahedron Letters 50 (2009) 6989–6992
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Novel ninhydrin adduct of catechin with potent antioxidative activity
a
Kiyoshi Fukuhara a, , Akiko Ohno , Ikuo Nakanishi b,c, Kohei Imai d, Asao Nakamura d, Kazunori Anzai b,
*
Naoki Miyata a,e, Haruhiro Okuda a
a Division of Organic Chemistry, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan
b Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555, Japan
c Graduate School of Engineering, Osaka University, SORST, Japan Science and Technology Agency, Suita, Osaka 565-0871, Japan
d Department of Applied Chemistry, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama-shi, Saitama 337-8570, Japan
e Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction of ninhydrin with (+)-catechin in the presence of TMSOTf resulted in condensation product
1, which consists of a 2:1 mixture of epimers at the C-2 position. The antioxidative radical-scavenging
activity of 1 against the galvinoxyl radical, acting as an oxyl radical, was significantly enhanced compared
to (+)-catechin. Our results offer a new method for chemical modification of a natural phenolic
antioxidant.
Received 23 August 2009
Revised 24 September 2009
Accepted 25 September 2009
Available online 2 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Catechin
Antioxidant
Ninhydrin
Galvinoxyl radical
Radical scavenging
Polyphenolic compounds based on catechin structure are ma-
jor components in green tea and have been reported to possess
catechin showed antioxidative activity over five times stronger
than that of (+)-catechin.4 Furthermore, fixation of catechin struc-
ture led to a dramatic increase in the inhibitory activity of gluco-
sidase, an antiviral target.5 Also, planar catechin strongly
inhibited the syncytium formation in BHK cells adapted to New-
castle disease virus.6 These results imply that the structure of (+)-
catechin is important to exert various types of biological effects in
addition to its antioxidative ability. Therefore, chemical modifica-
tion of (+)-catechin is a promising approach for developing che-
mopreventive agents.
a
wide range of pharmacological properties with numerous
health-promoting effects.1 Recently, we have focused on the
chemical modification of (+)-catechin to improve its pharmaco-
logical properties with the aim of developing clinically useful
chemopreventive agents.2 Treatment of (+)-catechin with tri-
methylsilyl trifluoromethane sulfonate in the presence of a ke-
tone by way of an Oxa-Pictet-Spengler reaction enabled the
structure of catechin to be planar (Scheme 1).3 Synthesized planar
OH
OH
OH
OH
O
HO
O
HO
O
R1
R2
R1
R2
TMSOTf
O
OH
OH
planar catechin
OH
(+)-catechin
Scheme 1. Fixation of (+)-catechin structure to be planar.
* Corresponding author. Tel.: +81 3 3700 1141; fax: +81 3 3707 6950.
E-mail address: fukuhara@nihs.go.jp (K. Fukuhara).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.09.149

6990
K. Fukuhara et al. / Tetrahedron Letters 50 (2009) 6989–6992
OH
H
O
OH
OH
HO
H
H
HO
O
O
O
8
2
3
O
OH
OH
H
OH
1a
(+)-catechin
TMSOTf
OH
O
OH
17
OH
HO
H
16
H
HO
O
O
H
H
OH
: NOE correlation
1b
Scheme 2. The TMSOTf-mediated reaction of ninhydrin with (+)-catechin.
We are also interested in the unique reactivity of ninhydrin
tion of condensation products with various aromatic substrates in
the presence of acid under mild conditions.8 It has also been re-
ported that ninhydrin reacts with phenol at the ortho position
OH group in high yield.9 In this regard, carboxylic acid can be
toward aromatic compounds.7 The C-2 position of ninhydrin is
reactive toward nitrogen-, sulfur-, and oxygen-based nucleophiles.
The electrophilic character of the C-2 position allows for the forma-
O
O
O
O
+
H+
− H2O
+
OH
OH2
OH
OH
OH
O
O
(+)-Catechin
O
O
O
OH
OH
OH
+
+
HO
HO
OH
OH
OH
O
− H+
HO
H+
OH
O
O
HO
O
HO
HO
O
8
3
OH
OH
OH
OH
OH
OH
2
− H+
OH
OH
O
OH
O
O
OH
OH
OH
O
HO
OH
OH
HO
HO
−
HO
O
HO
O
H+
− H+
HO
H+
O
2
O
O
O
− H+
OH
OH
OH
1a
3
1b
Scheme 3. Possible reaction mechanism of ninhydrin and (+)-catechin.

K. Fukuhara et al. / Tetrahedron Letters 50 (2009) 6989–6992
6991
introduced into the ortho position of phenol by heating p-toluidine
as well as by the Kolbe–Schmitt reaction.10 This unique reactivity
led us to examine the usefulness of the ninhydrin reaction for
chemical modification of natural polyphenols aiming to synthesize
new types of chemopreventive agents. Herein, we describe the
synthesis and radical-scavenging ability of unique ninhydrin ad-
ducts of (+)-catechin. The possible reaction mechanism of ninhy-
drin with (+)-catechin is also discussed.
As expected, the ninhydrin adduct of (+)-catechin could be
readily prepared by modifying the previously described synthesis
of phenol–ninhydrin adducts.9 The choice of acid proved to be crit-
ical, with TMSOTf giving the best results. The optimized reaction
condition was defined as (+)-catechin, 1.0 equiv of ninhydrin and
1.2 equiv of TMSOTf in THF at ꢀ10 °C under argon for 2 h. The reac-
tion mixture was purified by silica-gel column chromatography
under acidic conditions and 1 was obtained as a pale yellow pow-
der at a yield of 78%.11 The analysis by 1H NMR of 1 in DMSO-d6
exhibited a spectrum showing disappearance of the 8-H and 3-
OH signals while other protons assigned to catechin remained,
indicating that the cycloaddition of ninhydrin proceeded at these
positions to afford cycloadduct 1. It should be noted that 1 was iso-
lated as an inseparable 2:1 mixture of diastereoisomer where the
C-2 position in the catechin-type conformation (1a) was epimer-
ized (Scheme 2). The 1H NMR revealed the diagnostic C-2 proton
in catechin type (1a) and epi type (1b) at 4.67 ppm and
4.77 ppm, respectively. The structures of these diastereoisomers
were elucidated by combinations of MS, 1H, 13C, COSY, HMBC,
HSQC, and NOESY. Analysis of the NOESY data revealed key corre-
lations between the 17-OH and 16-H in the 1b, whereas no such
correlation was found in 1a, thereby confirming the stereochemis-
try at C-2 in these diastereoisomers.
According to the results obtained, a tentative mechanistic inter-
pretation to explain the formation of cycloadduct 1 was proposed
(Scheme 3). Following the formation of oxonium ion in the pres-
ence of TMSOTf, a regioselective electrophilic substitution of ortho
to the hydroxyl group could proceed at C-8 to form intermediate 2.
Intramolecular cyclization involving nucleophilic reaction of hy-
droxyl at C-3 with carbonyl carbon of ninhydrin could further take
place into intermediate 2 leading to the formation of 1a. This pro-
cess is similar to the acid-catalyzed reaction of catechin and ketone
for the synthesis of planar catechin. It has been reported that epi-
merization of C-2 in (+)-epigallocatechin gallate, a typical tea cat-
echin, could occur in thermal processes by
a mechanism
involving a quinone methide intermediate.12 1H NMR analysis of
1 in DMSO-d6 showed that the ratio of diastereoisomer gradually
changed with time, attaining a 1:1 equilibrium ratio within 1–
2 days at 25 °C. Therefore, in analogy with epigallocatechin gallate,
the formation of diastereoisomer 1b would result in a thermally
driven epimerization of 1a via quinone methide intermediate 3.
To evaluate the ability of 1 to function as an antioxidative agent,
radical-scavenging activity was investigated by the hydrogen-
transfer reaction using galvinoxyl radical (GÅ) as an oxyl radical
species.13 The hydrogen abstraction from 1 by GÅ in deaerated ace-
tonitrile was monitored by the decrease of absorbance at 428 nm
O
O •
OH
O •
O
O
OH
OH
OH
OH
HO
HO
G •
HO
O
HO
O
O
O
OH
OH
1
O
OH
0.25
0.2
0.15
k
0.1
0.05
0
0
1
2
3
4
5
10-4 [catechin], M
Figure 1. Plot of the pseudo-first-order rate constants (kobs) versus [catechin] for the radical-scavenging reaction of 1 (d) or (+)-catechin (j) toward GÅ (1.1 ꢁ 10ꢀ5 M) in
deaerated acetonitrile at 298 K.

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K. Fukuhara et al. / Tetrahedron Letters 50 (2009) 6989–6992
due to GÅ obeying pseudo-first-order kinetics, when the concentra-
tion of 1 was maintained at >10-fold excess of the GÅ concentration.
From the linear plot of the observed pseudo-first-order rate con-
stant (kobs) versus 1, we determined that the second-order rate
6. Hakamata, W.; Muroi, M.; Nishio, T.; Oku, T.; Takatsuki, A.; Osada, H.;
Fukuhara, K.; Okuda, H.; Kurihara, M. J. Appl. Glycosci. 2006, 53, 149–154.
7. Joullie, M. M.; Thompson, T. R.; Nemeroff, N. H. Tetrahedron 1991, 47, 8791–
8830.
8. Klumpp, D. A.; Fredrick, S.; Lau, S.; Jin, K. K.; Bau, R.; Prakash, G. K. S.; Olah, G. A.
J. Org. Chem. 1999, 64, 5152–5155.
9. Prabhakar, K. R.; Veerapur, V. P.; Bansal, P.; Vipan, K. P.; Reddy, K. M.; Barik, A.;
Reddy, B. K. D.; Reddanna, P.; Priyadarsini, K. I.; Unnikrishnan, M. K. Bioorg.
Med. Chem. 2006, 14, 7113–7120.
10. Schmitt, G.; Dinh An, N.; Poupelin, J. P.; Vebrel, J.; Laude, B. Synthesis 1984,
1984, 758–759.
11. Experimental procedure for the preparation of 1: To a solution of (+)-catechin
(2.88 g, 10 mmol) in dry THF (100 ml), ninhydrin (1.78 g, 10 mmol) in dry
THF(20 ml) was added. After stirring for a few minutes at ꢀ10 °C, trimethylsilyl
trifluoromethanesulfonate (TMSOTf, 2.17 mL, 12 mmol) was added. After
stirring for 2 h, the mixture was poured into water, and extracted with ethyl
acetate (3 ꢁ 150 mL). The organic layer was washed with brine, dried over
Na2SO4,and filtered and the solvent was evaporated. The resultant solid was
purified by column chromatography on silica gel (20:1 ethyl acetate–
methanol) to give 3.18 g (78%) of 1 as a pale yellow powder; HRMS (EI):
calcd for C24H18O9 450.0951, found 450.0955; Compound 1a: 1H NMR (DMSO-
d6, 600 MHz) d: 2.35 (1H, dd, J = 16.4 and 7.25 Hz), 2.46 (1H, dd, J = 16.4 and
5.15 Hz), 3.1–3.4 (2H, br s), 3.76 (1H, ddd, J = 7.25, 5.15 and 6.60 Hz), 4.67 (1H,
d, J = 6.60 Hz), 5.81 (1H, s), 6.61 (1H, dd, J = 8.2 and 1.9 Hz), 6.66 (1H, d,
J = 8.2 Hz), 6.70 (1H, d, J = 1.9 Hz), 7.56–7.68 (1H, br s), 7.59 (1H, t, J = 7.7 Hz),
7.71 (1H, d, J = 7.7 Hz), 7.80 (1H, t, J = 7.7 Hz), 7.83 (1H, d, J = 7.7 Hz), 8.68 (1H,
br s), 8.83 (1H, br s), 9.60 (1H, s); 13C NMR (DMSO, 150 MHz) d: 27.2, 27.2, 65.9,
80.3, 89.3, 100.8, 102.7, 110.1, 114.4, 114.9, 117.5, 122.7, 124.8, 130.6, 130.7,
134.5, 135.7, 144.6, 144.7, 148.0, 153.1, 156.6, 158.5, 197.3; Compound 1b: 1H
NMR (DMSO, 600 MHz) d: 2.33 (2H, m), 3.1–3.4 (2H, br s), 3.94 (1H, m), 4.77
(1H, d, J = 5.23 Hz), 5.81 (1H, s), 6.67 (1H, d, J = 7.9 Hz), 6.70 (1H, dd, J = 7.9 and
2.0 Hz), 6.77 (1H, d, J = 2.0 Hz), 7.56–7.68 (1H, br s), 7.58 (1H, t, J = 7.7 Hz), 7.66
(1H, d, J = 7.7 Hz), 7.80 (1H, t, J = 7.7 Hz), 7.84 (1H, d, J = 7.7 Hz), 8.68 (1H, br s),
8.83 (1H, br s), 9.55 (1H, s); 13C NMR (DMSO, 150 MHz) d: 25.6, 25.6, 65.3, 79.7,
89.2, 100.6, 102.5, 110.1, 113.9, 115.1, 117.4, 122.7, 124.8, 130.4, 130.7, 134.4,
135.7, 144.5, 144.6, 148.0, 153.0, 156.6, 158.5, 197.1.
constant (k) for hydrogen abstraction of
1
by GÅ was
5.5 ꢁ 10 Mꢀ1 sꢀ1 (Fig. 1). The k values for (+)-catechin were deter-
mined in the same manner to be 2.6 ꢁ 10 Mꢀ1 sꢀ1, showing that
the radical-scavenging activity of 1 is about two times greater than
that of (+)-catechin. Because the radical-scavenging of (+)-catechin
is attributed to a one-electron transfer reaction from catechol
structure to GÅ, chemical modification of the catechol ring is effec-
tive for improving the k value of (+)-catechin. Therefore, the en-
hanced radical-scavenging property of 1 where the catechol
structure remains unchanged is probably caused by the effect of
the hydroxyl group derived from ninhydrin.
In summary, ninhydrin and (+)-catechin undergo a reaction in
the presence of TMSOTf to form cyclization product 1. The reaction
is an electrophilic substitution of ortho to the hydroxyl group of
(+)-catechin with subsequent nucleophilic reaction at the carbonyl
carbon of ninhydrin. Despite the catechol ring not being chemically
modified by the ninhydrin reaction, the scavenging rate constant of
GÅ by 1 is increased as compared to (+)-catechin. Because of the
structural originality distinct from natural (+)-catechin, various
types of biological activities might be expected for 1. We believe
that chemical modification of natural phenolic antioxidant by use
of ninhydrin has enormous potential for the development of phar-
macologically important chemicals.
12. Wang, R.; Zhou, W.; Jiang, X. J. Agric. Food Chem. 2008, 56, 2694–2701.
13. Experimental procedure for analyzing the radical scavenging of 1: Since the
phenoxyl radical of 1 generated in the reaction of 1 with radicals readily reacts
with molecular oxygen, the reactions were carried out under strictly deaerated
conditions. A continuous flow of argon gas was bubbled through an acetonitrile
solution (3.0 mL) containing galvinoxyl radical (GÅ, 1.1 ꢁ 10ꢀ5 M) in a square
quartz cuvette (10 mm id) with a glass tube neck for 10 min. Air was prevented
from leaking into the neck of the cuvette with a rubber septum. Typically, an
aliquot of 1 (5.4 ꢁ 10ꢀ2 M), which was also in deaerated acetonitrile, was
added to the cuvette with a microsyringe. This led to a reaction of 1 with GÅ.
UV–vis spectral changes associated with the reaction were monitored using an
Agilent 8453 photodiode array spectrophotometer. The rates of the GOÅ-
scavenging reactions of 1 were determined by monitoring the absorbance
Acknowledgment
This work was supported by a Grant-in-Aid for Scientific Re-
search (B) (No. 20390038) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
References and notes
1. Rice-Evance, C.; Packer, L. Flavonoids in Health and Disease (Antioxidants in
Health and Disease); Marcel Dekker: New York, 2003.
2. Fukuhara, K. Genes Environ. 2006, 28, 41–47.
change at 428 nm due to GÅ
(
e
= 1.32 ꢁ 105 Mꢀ1 cmꢀ1) using a stopped-flow
technique on a UNISOKU RSP-1000-02NM spectrophotometer. The pseudo-
first-order rate constants (kobs) were determined by a least-squares curve fit
using an Apple Macintosh personal computer. The first-order plots of
ln(A ꢀ A1) versus time (A and A1 are denoted as the absorbance at the
reaction time and the final absorbance, respectively) were linear until three or
more half-lives with the correlation coefficient q >0.999.
3. Fukuhara, K.; Nakanishi, I.; Kansui, H.; Sugiyama, E.; Kimura, M.; Shimada, T.;
Urano, S.; Yamaguchi, K.; Miyata, N. J. Am. Chem. Soc. 2002, 124, 5952–5953.
4. Nakanishi, I.; Ohkubo, K.; Miyazaki, K.; Hakamata, W.; Urano, S.; Ozawa, T.;
Okuda, H.; Fukuzumi, S.; Ikota, N.; Fukuhara, K. Chem. Res. Toxicol. 2004, 17, 26–
31.
5. Hakamata, W.; Nakanishi, I.; Masuda, Y.; Shimizu, T.; Higuchi, H.; Nakamura,
Y.; Saito, S.; Urano, S.; Oku, T.; Ozawa, T.; Ikota, N.; Miyata, N.; Okuda, H.;
Fukuhara, K. J. Am. Chem. Soc. 2006, 128, 6524–6525.