Syntheses of procyanidin B2 and B3 gallate derivatives using equimolar condensation mediated by Yb(OTf)3 and their antitumor activities

Article abstract of DOI:10.1016/j.bmcl.2013.06.061

Synthesis of procyanidin B2 and B3 gallate derivatives, 3-O-gallate, 3″-O-gallate, and 3,3″-di-O-gallate, were synthesized using equimolar condensation mediated by Yb(OTf)₃. Synthesized compounds showed significant antitumor effects against human prostate PC-3 cell lines. Their activities were weaker than well-known EGCG and prodelphinidin B3.

Full text of DOI:10.1016/j.bmcl.2013.06.061

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4935–4939
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses of procyanidin B2 and B3 gallate derivatives using
equimolar condensation mediated by Yb(OTf)3 and their antitumor
activities
Manato Suda a, Miyuki Katoh a, Kazuya Toda b, Kiriko Matsumoto b, Koichiro Kawaguchi b,
Sei-ichi Kawahara c, Yasunao Hattori d, Hiroshi Fujii b, , Hidefumi Makabe a,
⇑
⇑
a Graduate School of Agriculture, Sciences of Functional Foods, Shinshu University, 8304 Minami-Minowa Kami-Ina, Nagano 399-4598, Japan
b Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minami-Minowa Kami-Ina, Nagano 399-4598, Japan
c St. Cousair Co., Ltd, 1260 Imogawa, Kamiminochi, Nagano 389-1201, Japan
d Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Synthesis of procyanidin B2 and B3 gallate derivatives, 3-O-gallate, 300-O-gallate, and 3,300-di-O-gallate,
were synthesized using equimolar condensation mediated by Yb(OTf)3. Synthesized compounds showed
significant antitumor effects against human prostate PC-3 cell lines. Their activities were weaker than
well-known EGCG and prodelphinidin B3.
Article history:
Received 31 May 2013
Revised 18 June 2013
Accepted 21 June 2013
Available online 29 June 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Polyphenol
Condensation
Lewis acid
Procyanidin gallate
Antitumor
Procyanidin gallates are paid attention due to their significant
biological activities.1 For example, inhibitory activity of DNA poly-
merase2–4 and antitumor activity were reported.5 As to the procy-
anidin B2 and B3 gallates, a number of isolation and biological
studies have been reported; procyanidin B2 3-O-gallate (1),5–10
B2 300-O-gallate (2),6–9,11–17 B2 3,300-O-di-gallate (3),12,13 and B3 3-
O-gallte (4).18,19 Furthermore, many procyanidin gallates including
various oligomer have been reported. However, it is difficult to iso-
late procyanidin gallates in pure state from the nature, the struc-
tural-activity relationship study (SAR study) of these compounds
have not been clarified yet. Thus, synthetic studies on procyanidin
gallates are very important to obtain them in pure state for the bio-
logical study. By far, the report of synthesis of procyanidin gallates
are quite limited.2–4,20,21 The reported syntheses of procyanidin
gallates was accomplished using Lewis acid-mediated condensa-
tion of catechin and/or epicatechin derived nucleophiles and elec-
trophiles. The disadvantage of this reaction is that using an excess
amount of nucleophile was necessary to avoid polymerization. So
far, we have developed an efficient synthesis of procyanidin dimers
through equimolar condensation of catechin nucleophiles with
electrophiles using Yb(OTf)3 as a Lewis acid.22–25 Herein we dem-
onstrate equimolar condensation of a catechin and/or a epicatechin
nucleophile containing gallate moiety with a catechin and/or epi-
catechin derived electrophile and synthesis of procyanidin B2
and B3 gallates (Fig. 1).
As to the synthesis of procyanidin B2 gallates (1–3), the nucle-
ophile 7, 8 and the electrophile 9, 10 were prepared by the re-
ported procedure.2–4,22,23 Equimolar condensation between
nucleophile 7 and electrophile 9 using Yb(OTf)3 as a Lewis acid
to afford 11 in 53% yield. The benzylated procyanidin B2 300-O-gal-
late (12) and procyanidin B2 3,300-O-gallate (13) were also obtained
from the condensation between nucleophile 8 and electrophile 10,
nucleophile 8 and electrophile 9 in 22% and 43% yields, respec-
tively. The 1H and 13C NMR data of the all condensed products
11–13 were identical with the reported values.3
Similarly, related compounds of the procyanidin B3 gallates
were synthesized. The nucleophile 14, 15 and the electrophile 16,
17 were prepared by the reported procedure.2–4,22,23 Equimolar
condensation between nucleophile 14 and electrophile 16 using
Yb(OTf)3 as a Lewis acid to afford 18 in 68% yield. The benzylated
procyanidin B2 300-O-gallate (19) and procyanidin B2 3,300-O-di-gal-
late (20) were also obtained from the condensation between nucle-
ophile 15 and electrophile 17, nucleophile 15 and electrophile 16
⇑
Corresponding authors. Tel./fax: +81 265 77 1629 (H.F.); tel.: +81 265 77 1630;
fax: +81 265 77 1700 (H.M.).
E-mail addresses: hfujii@shinshu-u.ac.jp (H. Fujii), makabeh@shinshu-u.ac.jp
(H. Makabe).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.061

4936
M. Suda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4935–4939
OH
OH
OH
OH
However, severe broadening of the peak was observed in the 1H
NMR spectra of 1–6 in our measurement. Thus we switched to
use methanol-d4 for measurement of NMR. As to the HRMS data
of 1–6, we obtained satisfied results. We transfromed 1–6 into per-
acetate 24–29 to confirm the structures. The 1H and 13C NMR spec-
tral data of peracetate 24–26 were in good agreement with the
reported values.3 Peracetate 27–29 also gave satisfied 1H and 13C
NMR data (Scheme 3).
HO
O
HO
O
OR1
O
OR1
O
OH
OH
OH
OH
OH
HO
OH
HO
OR2
OR2
Our interest was focused on examining the antitumor activities
of the newly synthesized procyanidin B2 and B3 gallates. The syn-
thesis of procyanidin B2 3-O-gallate (1), procyanidin B2 300-O-gal-
late (2), procyanidin B2 3,300-O-gallate (3), procyanidin B3 3-O-
gallate (4), procyanidin B3 300-O-gallate (5), and procyanidin B2
3,300-O-gallate (6) allowed us to obtain sufficient quantities of puri-
fied compounds to screen against PC-3 prostate cancer cell lines to-
gether with procyanidin B2 (PCB2), B3 (PCB3) and prodelphinidin
B3 (PDB3) which were prepared by us previously (Fig. 2).22–25,27
Results were obtained by cell count measurement. Epigallocate-
chin gallate (EGCG) was used as a positive control. As shown in Fig-
ure 3, procyanidin B2 3-O-gallate (PCB2 3-OG, 1), procyanidin B2
300-O-gallate (PCB2 300-OG, 2), procyanidin B2 3,300-O-di-gallate
(PCB2 3,300-ODG, 3), procyanidin B3 3-O-gallate (PCB3 3-OG, 4),
procyanidin B3 300-O-gallate (PCB3 300-OG, 5), and procyanidin B2
3,300-O-di-gallate (PCB3 3,300-ODG, 6) exhibited significant cytotoxic
activity. The activities of procyanidin B2 3,300-O-di-gallate (3) and
procyanidin B2 3,300-O-di-gallate (6) were stronger than those of
mono-substituted gallate compounds. However, a comparison of
the potencies of 3 and 6 with prodelphinidin B3 showed that the
cytotoxic effects were a little bit weaker. This finding suggests that
esterified pyrogallol moiety shows weaker activity compared to
the compounds such as PDB3 (Fig. 3).
OH
OH
1: R1 = G (galloyl), R2 = H
2: R1 = H, R2 = G
4: R1 = G, R2 = H
5: R1 = H, R2 = G
6: R1 = R2 = G
3: R1 = R2 = G
Figure 1. The structures of procyanidin B3 and B2 gallates (1–6).
in 28% and 68% yields, respectively. The 1H and 13C NMR data of the
all condensed products 18–20 were identical with the reported
values (Scheme 1).2,4
Because the yields of condensation between 8 and 10 (22%), 15
and 17 (28%) were low, we investigated the esterification of benzy-
lated procyanidin B2 (21) and B3 (22) with benzylated gallic acid
(23). In the case of the esterification between 21 and 23 using EDCI,
the yield of desired product 12 was only 18% along with 11 (25%)
and 13 (15%). On the other hand, esterification between 22 and
23 using DCC gave 19 in 65% yield. This method improved the syn-
thesis of 19 compared to Yb(OTf)3 mediated equimolar coupling
(Scheme 2).
Deprotection of the benzyl ethers of 11–13 and 18–20 and sub-
sequent lyophilization afforded 1–6 in good yield. We confirmed
that lyophilized 1–6 was pure by HPLC analysis.26 The specific rota-
tion value of synthetic 1–6 were similar to those reported values.2,3
We found that low concentration was necessary for measurements
because of low degree of solubility when acetone was used as a sol-
vent. Thus, we supplied the data of optical rotation values using
MeOH as a solvent in the Supplementary data. The 1H and 13C
NMR data were reported in the literature using acetone-d6.11,18,19
After treatment of cells with PCB2 3-OG (1), PCB2 300-OG (2),
PCB2 3,300-ODG (3), PCB3 3-OG (4), PCB3 300-OG (5), and PCB3
3,300-ODG (6) for 48 h, the cell proliferation was determined by cell
count as shown in Supplementary data. The values were repre-
sented as the rate of inhibition of cell proliferation by the treated
sample compared to the untreated control (vehicle). Values are
OBn
BnO
Yb(OTf)3
O
OBn
OBn
OBn
OBn
OR1
O
BnO
O
BnO
O
OBn
OBn
OBn
OBn
BnO
+
OR1
OR1
OBn
OBn OEE
OR2
R1= H: 7
R1 = G: 8
OBn
9: R1= G
10: R1 = H
11: R1 = G, R2 = H (53%)
12: R1 = H, R2 = G (22%)
13: R1 = R2= G (43%)
OBn
BnO
Yb(OTf)3
O
OBn
OBn
OBn
OBn
OBn
OR1
BnO
O
BnO
O
OBn
OBn
OBn
BnO
+
O
OR1
OR2
OBn
OBn OEE
OR2
14: R1 = H
16: R1 = G
17: R1 = H
OBn
15: R1 = G
18: R1 = G, R2= H (68%)
19: R1 = H, R2 = G (28%)
20: R1 = R2 = G (68%)
Scheme 1. Synthesis of benzylated procyanidin B2 and B3 gallates via Yb(OTf)3 mediated equimolar condensation.

M. Suda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4935–4939
4937
OBn
OBn
OBn
OBn
BnO
O
BnO
O
OH
O
HO2C
OBn
OBn
OBn
OBn
EDCI, DMAP
18%
OBn
BnO
OH
O
+
OBn
OBn
OBn
BnO
OBn
O
23
OBn
OBn
OBn
OH
O
OBn
21
12
OBn
OBn
OBn
OBn
OBn
BnO
O
BnO
O
HO2C
OBn
OBn
OH
O
DCC, DMAP
65%
+
OBn
OBn
OH
O
OBn
OBn
OBn
BnO
OBn
BnO
OBn
O
23
OH
OBn
OBn
OBn
OBn
O
22
19
OBn
Scheme 2. Esterification of benzylated procyanidin B2 (21) and B3 (22) with benzylated gallic acid (23).
OAc
OAc
OBn
OBn
OH
OH
AcO
O
BnO
O
HO
O
OR1
OR1
O
OR1
O
OAc
OAc
OBn
OBn
OH
OH
Ac2O, DMAP
H2, Pd(OH)2
OBn
BnO
OH
HO
OAc
AcO
O
OR2
OR2
OR2
OAc
OBn
OH
11: R1 = G, R2 = H
12: R1 = H, R2 = G
13: R1 = R2 = G
1: R1 = G, R2 = H (quant.)
2: R1 = H, R2 = G (quant.)
3: R1 = R2 = G (quant.)
24: R1 = G, R2 = Ac (31%)
25: R1 = Ac, R2= G (36%)
26: R1 = R2 = G (28%)
OBn
OBn
OH
OAc
BnO
O
HO
O
AcO
O
OAc
OH
OR1
O
OR1
OR1
OAc
OBn
OBn
OH
OH
Ac2O, DMAP
H2, Pd(OH)2
OBn
BnO
OH
HO
OAc
AcO
O
O
OAc
OR2
OR2
OR2
OBn
OH
OAc
18: R1 = G, R2 = H
19: R1 = H, R2 = G
20: R1 = R2 = G
4: R1= G, R2 = H (quant.)
5: R1 = H, R2 = G (quant.)
6: R1 = R2 = G (quant.)
27: R1 = G, R2 = Ac (56%)
28: R1 = Ac, R2 = G (42%)
29: R1 = R2 = G (10%)
Scheme 3. Synthesis of 1–6 and their peracetate 24–29.
means SDs. for three independent experiments. Asterisks indi-
cated a significant difference between the control- and test-com-
pound-treated cells, as analyzed by Student’s test (p <0.001).
In summary, syntheses of procyanidin B2 3-O-gallate (1), procy-
anidin B2 300-O-gallate (2), procyanidin B2 3,300-O-di-gallate (3),
procyanidin B3 3-O-gallate (4), procyanidin B3 300-O-gallate (5),
and procyanidin B2 3,300-O-di-gallate (6) were achieved via equi-
molar condensation of a catechin and/or a epicatechin nucleophile
containing gallate moiety with a catechin and/or epicatechin de-
rived electrophile. As to the synthesis of 5, esterification between
benzylated procyanidin B2 (22) and benzylated gallic acid (23)
using DCC was effective. The antitumor activities of 1–6 suggested
that these compounds showed significant cytotoxic activity but
weaker than EGCG and prodelphinidin B3.

4938
M. Suda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4935–4939
OH
OH
OH
OH
OH
OH
OH
HO
O
HO
O
HO
O
OH
O
OH
O
OH
OH
OH
OH
O
OH
HO
OH
HO
OH
OH
OH
O
OH
OH
OH
OH
OH
procyanidin B2 (PCB2)
epigallocatechin-gallate (EGCG)
procyanidin B3 (PCB3)
OH
OH
OH
OH
HO
O
HO
O
HO
O
OH
OH
OH
OR1
O
OR1
O
OH
O
OH
OH
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
OR2
OR2
OH
OH
OH
OH
1: R1 = G, R2 = H
2: R1 = H, R2 = G
3: R1 = R2 = G
4: R1 = G, R2 = H
5: R1 = H, R2 = G
6: R1 = R2 = G
prodelphinidin B3 (PDB3)
Figure 2. The structures of test compounds for PC-3 prostate anticancer activity.
Figure 3. Effects of various concentrations of test compounds on cell proliferation.
3. Saito, A.; Mizushina, Y.; Ikawa, H.; Yoshida, H.; Doi, Y.; Tanaka, A.; Nakajima, N.
Bioorg. Med. Chem. 2005, 13, 2759.
Acknowledgments
4. Sakuda, H.; Saito, A.; Mizushina, Y.; Yoshida, H.; Tanaka, A.; Nakajima, N.
Heterocycles 2006, 67, 175.
5. Agarwal, C.; Veluri, R.; Kaur, M.; Chou, S.-C.; Thompson, J. A.; Agarwal, R.
Carcinogenesis 2007, 28, 1478.
6. Monagas, M.; Gomez-Cordoves, C.; Bartolome, B.; Laureano, O.; Ricardo-da-
Silva, J. M. J. Agric. Food. Chem. 2003, 51, 6475.
7. Jordao, A. M.; Ricardo-Da-Silva, J. M.; Laureano, O. Vitis 2001, 40, 17.
8. Sun, B.; Belchior, G. P.; Ricardo-da-Silvab, J. M.; Spranger, M. I. J. Chromatogr. A
1999, 841, 115.
9. Dallas, C.; Ricardo-Da-Silva, J. M.; Laureano, O. Vitis 1995, 34, 51.
10. Ricardo-Da-Silva, J. M.; Rigaud, J.; Cheynier, V.; Cheminat, A.; Moutounet, M.
Phytochemistry 1991, 30, 1259.
11. Nonaka, G.-I.; Nishioka, I.; Nagasawa, T.; Oura, H. Chem. Pharm. Bull. 1981, 29,
2862.
12. Nonaka, G.-I.; Kawahara, O.; Nishioka, I. Chem. Pharm. Bull. 1983, 31, 3906.
13. Anke, J.; Petereit, F.; Engelhardt, C.; Hensel, A. Nat. Prod. Res. 2008, 22,
1237.
14. Trotin, F.; Moumou, Y.; Vasseur, J. Phytochemistry 1993, 32, 929.
15. Moumou, Y.; Trotin, F.; Vasseur, J.; Vermeersch, G.; Guyon, R.; Dubois, J.;
Pinkas, M. Planta Med. 1992, 58, 516.
This work was supported in part by Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Culture, Sports,
and Technology of Japan (22570112 to H.F.) and by Grant from
Uehara Memorial Foundation (to H.F.). We also thank Professor
Dr. Toshiyuki Kan for providing EGCG.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
06.061.
References and notes
1. Harbone, J. B.; Baxter, H. The Handbook of Natural Flavonoids; John Wiley &
Sons: NY, 1999.
2. Saito, A.; Emoto, M.; Tanaka, A.; Doi, Y.; Shoji, K.; Mizushina, Y.; Ikawa, H.;
Yoshida, H.; Matsuura, N.; Nakajima, N. Tetrahedron 2004, 60, 12043.
16. Liang, X.; Xiao, P. Planta Med. 1989, 55, 402.
17. Boukharta, M.; Girardin, M.; Metche, M. J. Chromatogr. 1988, 455, 406.

M. Suda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4935–4939
4939
18. Tanaka, T.; Nonaka, G.-I.; Nishioka, I. Phytochemistry 1983, 22, 2575.
19. Cho, Y.-J. Agric. Chem. Biotechnol. 2000, 43, 273.
25. Katoh, M.; Oizumi, Y.; Mohri, Y.; Hirota, M.; Makabe, H. Lett. Org. Chem. 2012, 9,
233.
20. Tückmantel, W.; Kozikowski, A. P.; Romanczyk, L. J., Jr. J. Am. Chem. Soc. 1999,
121, 12073.
21. Kozikowski, A. P.; Tückmantel, W.; Hu, Y. J. Org. Chem. 2001, 66, 1287.
22. Mohri, Y.; Sagehashi, M.; Yamada, T.; Hattori, Y.; Morimura, K.; Kamo, T.;
Hirota, M.; Makabe, H. Tetrahedron Lett. 2007, 48, 5891.
23. Mohri, Y.; Sagehashi, M.; Yamada, T.; Hattori, Y.; Morimura, K.; Hamauzu, Y.;
Kamo, T.; Hirota, M.; Makabe, H. Heterocycles 2009, 79, 549.
24. Oizumi, Y.; Mohri, Y.; Hirota, M.; Makabe, H. J. Org. Chem. 2010, 75, 4884.
26. HPLC measurement condition: column; InertSustain C18 250 ꢀ 4.6 mm
Waters, eluent 0.2% CH3COOH–CH3OH, flow rate: 0.8 mL/min, detection: UV
280 nm, retention time; procyanidin B2 3-O-gallate (1) 16.58 min, procyanidin
B2 300-O-gallate, (2) 18.14 min, procyanidin B2 3,300-O-gallate, (3) 20.50 min,
procyanidin B3 3-O-gallate, (4) 17.87 min, procyanidin B3 300-O-gallate, (5)
25.18 min, procyanidin B2 3,300-O-gallate, (6) 24.86 min.
27. Fujii, W.; Toda, K.; Kawaguchi, K.; Kawahara, S.-i.; Hattori, Y.; Fujii, H.; Makabe,
H. Tetrahedron 2013, 69, 3543.