Inhibitory Activity of Synthesized Acetylated Procyanidin B1 Analogs against HeLa S3 Cells Proliferation

Article abstract of DOI:10.3390/molecules19021775

Proanthocyanidins, also known as condensed tannins and/or oligomeric flavonoids, occur in many edible plants and have various interesting biological activities. Previously, we reported a synthetic method for the preparation of various procyanidins in pure form and described their biological activities. Here, we describe the synthesis of procyanidin B1 acetylated analogs and discuss their inhibition activities against HeLa S3 cell proliferation. Surprisingly, the lower-unit acetylated procyanidin B₁ strongly inhibited the proliferation of HeLa S3 cells. This molecule showed much stronger inhibitory activity than did epigallocatechin-3-O-gallate (EGCG), green tea polyphenol, and dimeric compounds that included EGCG as a unit. This result suggests that the phenolic hydroxyl groups of the upper-units in flavan-3-ols are important for their inhibitory activity against cancer cell proliferation and that a hydrophobic lower unit dimer enhances this activity.

Full text of DOI:10.3390/molecules19021775

Molecules 2014, 19, 1775-1785; doi:10.3390/molecules19021775
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Inhibitory Activity of Synthesized Acetylated Procyanidin B1
Analogs against HeLa S3 Cells Proliferation
Syuhei Okamoto 1, Sayaka Ishihara 1, Taisuke Okamoto 1, Syoma Doi 1, Kota Harui 1,
Yusuke Higashino 1, Takashi Kawasaki 2, Noriyuki Nakajima 3,* and Akiko Saito 1,*
1
Graduate School of Engineering, Osaka Electro-communication University (OECU),
18-8 Hatsu-cho, Neyagawa-shi, Osaka 572-8530, Japan; E-Mails: me12a003@oecu.jp (S.O.);
me12a002@oecu.jp (S.I.); me12a004@oecu.jp (T.O.); me12a008@oecu.jp (S.D.);
me13a012@oecu.jp (K.H.); me13a013@oecu.jp (Y.H.)
2
Department of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu,
Shiga 525-8577, Japan; E-Mail: kawa0227@fc.ritsumei.ac.jp
3
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University,
5180 Kurokawa, Imizu, Toyama 939-0398, Japan
* Authors to whom correspondence should be addressed; E-Mails: nori@pu-toyama.ac.jp (N.N.);
a-saito@isc.osakac.ac.jp (A.S.); Tel.: +81-766-56-7500 (N.N.); Fax: +81-766-56-2498 (N.N.);
Tel.: +81-72-824-1131 (A.S.); Fax: +81-72-824-0014 (A.S.).
Received: 19 November 2013; in revised form: 22 January 2014 / Accepted: 28 January 2014 /
Published: 4 February 2014
Abstract: Proanthocyanidins, also known as condensed tannins and/or oligomeric
flavonoids, occur in many edible plants and have various interesting biological activities.
Previously, we reported a synthetic method for the preparation of various procyanidins in
pure form and described their biological activities. Here, we describe the synthesis of
procyanidin B1 acetylated analogs and discuss their inhibition activities against HeLa S3
cell proliferation. Surprisingly, the lower-unit acetylated procyanidin B1 strongly inhibited
the proliferation of HeLa S3 cells. This molecule showed much stronger inhibitory activity
than did epigallocatechin-3-O-gallate (EGCG), green tea polyphenol, and dimeric compounds
that included EGCG as a unit. This result suggests that the phenolic hydroxyl groups of the
upper-units in flavan-3-ols are important for their inhibitory activity against cancer cell
proliferation and that a hydrophobic lower unit dimer enhances this activity.

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Keywords: condensed tannins; oligomeric flavonoid; synthesis; cancer cells proliferation;
inhibitory activity
1. Introduction
There is currently a great interest in research involving compounds having strong anti-oxidation
activities and superior radical scavenging abilities. Food and the ingredients that can eliminate active
oxygen and free radicals have recently received increased attention. Polyphenols occur in various
plants and are consumed regularly by eating vegetables and fruits. Many plants considered to be health
foods have high levels of polyphenols, which are widely believed to have a beneficial impact on
health. Scientific investigations of polyphenolic compounds have become increasingly important
because of the various strong biological activities of these substances. For example, the flavan-3-ol
EGCG (1), the major polyphenol in green tea, has been the focus of intense research interest because
of its protective effect against a variety of cancers, such as lung, prostate, and breast [1]. In addition,
surface plasmon resonance has shown the 67-kDa laminin receptor, which is widely expressed at high
levels in cancer cell membranes, to be one of the receptors of EGCG [2]. This finding is evidence
supporting the selective cytotoxic activity of EGCG against cancer cells.
Proanthocyanidins, which are condensed tannins or oligomeric flavonoids [3,4] are known to be
extremely strong antioxidants. Research on these compounds has become increasingly important
because of their various strong biological activities. However, in many cases, proanthocyanidins are
obtained as a mixture of various analogs, which makes purification of each compound difficult. After
an elegant contribution by Kozikowski et al. [5–7], a large number of syntheses of oligomeric catechin
and epicatechin derivatives were reported [8–15]. We have also developed and reported a simple,
versatile, and stereoselective methods for synthesizing procyanidin oligomers using (−)-epicatechin (2)
and (+)-catechin (3) (Figure 1) [16–28]. In this report, we describe our synthetic methodologies for
syntheses of the procyanidin B1 analogs 8 and 14. We also report the surprising enhancement of the
HeLa S3 cell proliferation inhibitory activity of a hydrophobic procyanidin B1 analogue in which the
lower-unit was acetylated.
Figure 1. The structure of (−)-epigallocatechin gallate (1), (−)-epicatechin (2), (+)-catechin
(3), procyanidin B1 (4) and procyanidin B1 peracetate (15).

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2. Results and Discussion
Our synthetic methodologies are easily applicable to various procyanidin oligomers such as the
3-O-substituted and partially-modified oligomers. The key step in our recently developed procyanidin
synthesis method is the coupling reaction between electrophilic C-4-hydroxyl peracetates and
nucleophilic compounds. As shown in Scheme I, the electrophile 5 derived from epicatechin (2) was
condensed with the nucleophile 6 derived from catechin (3) in the presence of SnCl4 as the Lewis acid,
which produced the procyanidin B1 derivative (7) in 33% yield and with excellent stereoselectivity.
Benzyl group hydrogenation of 7 was performed using H2/Pd(OH)2, which gave the upper-unit
acetylated procyanidin B1 analogue 8 in good yield (Scheme 1).
Scheme 1. Synthesis of 8 (upper unit of procyanidin B1 is acetylated).
Another general methodology for synthesizing procyanidin oligomers is shown in Schemes 2 and 3.
The key step in this method is the coupling reaction between a nucleophile and a 2-ethoxyethoxy
derivative of monomeric flavan-3-ol as an electrophile using a Lewis acid activator, such as TiCl4,
SnCl4, or trimethylsilyl trifluoromethanesulfonate (TMSOTf). These condensation reactions proceeded
smoothly and afforded good yields of 4–8 condensed oligomers with good stereoselectivity. The
electrophile 9 derived from epicatechin (2) was condensed with the TBDMS protected nucleophile 10 [29]
in the presence of SnCl4 as a catalyst, to produce the dimeric compound 11 in moderate yield.
Deprotection of TBDMS groups by tetra-n-butylammonium fluoride (TBAF) followed by acetylation
of phenolic groups and aliphatic hydroxyl groups gave 13. Benzyl group hydrogenation of 13 gave the
lower-unit acetylated procyanidin B1 analog (14). Procyanidin B1 peracetate 15 was also prepared
according to a previously reported method for biological assay [28].
Scheme 2. Synthesis of 14 (lower unit of procyanidin B1 is acetylated).

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Furthermore, we synthesized other procyanidin B1 analogs that had high numbers of hydroxyl
groups in their lower-units. Using the same procedure as Scheme 2, the condensation reaction between
the electrophile 9 and two nucleophiles 16 or 17 produced dimeric products 18 or 19 in 85 and 52%
yields, respectively. They were hydrogenated by a general procedure that gave 20 or 21 in 87% and
56% yields, respectively.
Scheme 3. Synthesis of procyanidin B1 analogs.
The inhibitory activities of the synthetic procyanidin B1 analogs against HeLa S3 cell proliferation
are shown in Figure 2. The inhibitory activities of epicatechin (2) and procyanidin B1 (4) were not
measurable because they decomposed under the cell proliferation measurement conditions (data not
shown). Compounds 2 and 4 decomposed in the medium (D-MEM) without cells and were not
converted into any compounds detectable at 450 nm. The reason for this decomposition is not clear,
but we think this stability of the compounds is associated with their various activities and SARs, so
studies of the decomposed compounds and elucidation of the decomposition mechanism(s) are now
underway. No inhibitory effect were observed for EGCG (1), the upper-unit acetylated procyanidin B1
8, and epicatechin-(4-8)-epigallocatechin (20). It is noteworthy that the lower-unit acetylated
procyanidin B1 14 inhibited proliferation of HeLa S3 cells quite strongly.
Figure 2. Inhibitory activity of synthetic procyanidin B1 analogs against HeLa S3 cell
proliferation. HeLa S3 was incubated with final 100 M of each compound for 24 h. All
error bars represent standard deviations of the mean. *** p < 0.001, ** p < 0.005 vs.
DMSO-treated groups.

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On the basis of these results, the upper unit of the dimeric procyanidins are considered to be the
more critical part for the inhibitory activity. In addition, compounds 14 and 15, which have
hydrophobic lower-units, showed strong inhibitory activity against cell proliferation. These results
suggest that the biological activities of proanthocyanidins depend not only on the number of phenolic
hydroxyl groups but also on various factors such as their structures, hydrophobic properties, and
hydrophilic properties. As was the case for epicatehin-(4-8)-epigallocatechin gallate (21), moderate
inhibitory activity was observed. This activity is believed to be because of the influence of the gallic
acid moiety.
3. Experimental
3.1. General
All commercially available chemicals for chemical synthesis were used without further purification.
All reactions were performed under an argon atmosphere and monitored using thin-layer
chromatography (TLC) with 0.25-mm pre-coated silica gel plates Merck 60F254 Art 5715. An
ATAGO AP-300 spectrometer was used to measure optical rotation 1H-NMR spectra were recorded on
a Agilent Inova 500 Spectrometer (500 MHz) and an Agilent DD2 NMR Spectrometer (400 MHz). A
JEOL JMS-AX500 mass spectrometer was used to acquire fast atom bombardment (FAB) mass
spectra. The human cervical adenocarcinoma cell line, HeLa-S3 (RCB0191), was provided by the
RIKEN BRC through the National Bio-Resource Project of the MEXT, Saitama, Japan. Synthesized
compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at –25 °C.
3.2. Synthesis
[4,8]-2,3-cis-3,4-trans:2'',3''-trans-3,5,7,3',4',3''-Hexa-O-acetyl-(−)-epicatechin-(+)-catechin (8) A
solution of 7 [28] (13.3 mg, 0.011 mmol) in THF/MeOH/H2O (20:1:1, 11 mL) was hydrogenated over
20% Pd(OH)2/C (2 mg) for 12 h at RT. Filtration and concentration afforded a pale brown solid, which
was purified with preparative TLC to give 8.7 mg (0.011 mmol, 94%) of 8 as an amorphous solid.
Because of NMR spectra broadening, compound 8 was acetylated according to a general procedure
and then identified [28].
[4,8]-2,3-cis-3,4-trans-2'',3''-trans-5,7,3',4'-Tetra-O-benzyl-5'',7'',3''',4'''-tetra-O-TBDMS-(−)-epicatechin-
(+)-catechin (11) To a solution of 9 (342 mg, 0.46 mmol) and 10 (1.35 g, 1.84 mmol) in CH2Cl2
(100 mL) SnCl4 (0.69 mL, 0.69 mmol, 1.0 M solution in CH2Cl2) was added dropwise in the presence
of MS3Å at 0 °C. After stirring for 5 min, the pale yellow reaction mixture was quenched with
saturated NaHCO3. The mixture was extracted with CHCl3 and the organic phase was washed with
water and brine, and then dried (Na2SO4). Filtration, concentration, and preparative silica gel TLC
purification (hexane/EtOAc, 4:1) afforded 246 mg (0.18 mmol, 39%) of 11 as a colorless amorphous
substance: 2D4 +56.4 (c 1.93, CHCl3); H-NMR (500 MHz, CDCl3, 0.8 : 0.2 mixture of rotational
1
isomers) major isomer: 7.46-6.94 (18.4H, m), 6,71 (0.8H, d, J = 8.5 Hz), 6.49 (0.8H, d, J = 2.2 Hz),
6.39 (0.8H, dd, J = 2.2, 8.5 Hz), 6.12 (0.8H, s), 6.01 (0.8H, d, J = 2.2 Hz), 5.64 (0.8H, d, J = 2.2 Hz),
5.26–4.56 (0.8H, m), 3.96–3.95 (0.8H, m), 3.83-3.80 (0.8H, m), 3.65 (0.8H, d, J = 8.5 Hz), 3.67 (0.8H, dd,

Molecules 2014, 19
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J = 6.0, 16.5 Hz), 2.52 (0.8H, dd, J = 8.9, 16.5 Hz), 1.70 (0.8H, d, J = 6.5 Hz), 1.33 (0.8H, d, J = 3.0 Hz),
1.04 (7.2H, s), 0.97 (7.2H, s), 0.95 (7.2H, s), 0.86 (7.2H, s), 0.30 (3H, s), 0.29 (3H, s), 0.26 (3H, s),
0.19 (3H, s), 0.16 (3H, s), 0.15 (3H, s), 0.04 (3H, s), 0.02 (3H, s); minor isomer: 7.46–6.94 (4.8H, m),
6.78 (0.2H, d, J = 2.2 Hz), 6.65 (0.2H, d, J = 2.2 Hz), 6.29 (0.2H, d, J = 2.2 Hz), 6.20 (0.2H, d, J = 2.2 Hz),
5.96 (0.2H, s), 5.33–4.54 (2H, m), 3.95–3.98 (0.2H, m), 3.89–3.88 (0.2H, m), 3.63–3.61 (0.2H, m),
3.57–3.50 (0.2H, m), 3.09–3.04 (0.2H, m), 2.62 (0.2H, dd, J = 9.4, 16.5 Hz), 1.65–1.50 (0.2H, m),
13
1.33–1.25 (0.2H, m), 1.07-0.75 (7.2H, m), 0.30–0.01 (4.8H, m); C-NMR (125 MHz, CDCl3, 0.8: 0.2
mixture of rotational isomers) major isomer: 158.0, 157.0, 155.4, 154.8, 152.9, 152.7, 149.0, 148.5,
147.0, 146.6, 137.54, 137.52, 137.48, 137.37, 132.9, 130.0, 128.7-126.8 (C×7), 120.8, 119.8, 119.3,
115.1, 113.8, 113.4, 106.2, 104.4, 102.2, 94.1, 93.0, 81.2, 75.3, 72.1, 71.50, 71.49, 69.7, 69.0, 68.4,
36.1, 28.9, 25.95, 25.91, 25.89, 25.81, 18.42, 18.40, 18.31, 18.29, −3.7–4.6 (C×4); minor isomer:
158.53, 158.46, 155.2, 154.7, 153.6, 152.4, 148.7, 148.1, 147.10, 147.07, 137.9, 137.7, 137.6, 137.3,
132.6, 130.8, 128.7–126.8 (C×7), 120.1, 119.1, 118.9, 115.0, 114.2, 112.0, 104.8, 104.5, 102.2, 95.0,
93.6, 81.3, 76.0, 72.5, 71.0, 70.6, 70.2, 69.6, 68.8, 36.7, 28.5, 26.4, 25.1–25.8 (Cx3), 19.1-18.0 (C×4),
−3.7–4.2 (C×4); FABMS (m/z) 1398 (31), 1397 (43), 1396 ([M+H]+, 53), 838 (28), 837 (38), 836 (57),
725 (26), 722 (19), 721 (28), 654 (55), 653 (100); FABHRMS calcd for C82H107O12Si4 [M+H]+,
1395.6840; found: 1395.6913.
[4,8]-2,3-cis-3,4-trans-2'',3''-trans-3,3'',5'',7'',3''',4'''-Hexa-O-acetyl-,5,7,3'4'-tetra-O-benzy-(−)-
epicatechin-(+)-catechin (13) A solution of 11 (246 mg, 0.18 mmol) in THF (50 mL) was added
dropwise to TBAF (1.44 mL, 1.44 mmol, 1M solution in THF) in the presence of AcOH (0.08 mL,
1.44 mmol) at 0 °C. Concentration and preparative silica gel TLC purification (hexane/EtOAc, 1:2)
afforded 52 mg (0.055 mmol, 31%) of 12 as a colorless amorphous substance. Compound 12 was
immediately dissolved in dry pyridine (2 mL) and acetic anhydride (40 mg, 0.39 mmol) was added in
the presence of cat. DMAP. The mixture was extracted with CHCl3 and the organic phase was washed
with water and brine, and then dried (Na2SO4). Filtration, concentration, and preparative silica gel TLC
purification (hexane/EtOAc, 3:1) afforded 63 mg (0.053 mmol, 96%) of 13 as a colorless amorphous
substance: 2D4 + 61.9 (c 0.63, CHCl3); 1H-NMR (500 MHz, CDCl3) 7.47–6.83 (22H, m), 7.08 (1H, d,
J = 2.2 Hz), 7.01 (1H, d, J = 8.5 Hz), 6.96 (1H, d, H = 2.2 Hz), 6.81 (1H, dd, J = 2.2, 8.5 Hz), 6.46
(1H, s), 6.33 (1H, d, J = 2.2 Hz), 6.25 (1H, d, J = 2.2 Hz), 5.27 (2H, s), 5.26 (2H, s), 5.11–4.79 (9H, m),
4.95 (1H, d, J = 8.0 Hz), 4.77 (1H, d, J = 10.0 Hz), 4.71 (1H, d, J = 10.0 Hz), 4.47 (1H, br s), 2.99 (1H,
dd, J = 5.5, 16.5 Hz), 2.60 (1H, dd, J = 8.5, 16.5 Hz), 2.33 (3H, s), 2.24 (3H, s), 2.22 (3H, s), 1.93 (3H,
13
s), 1.57 (3H, 2), 1.52 (3H, s); C-NMR (125 MHz, CDCl3) 170, 168.9, 168.8, 168.7, 168.0, 167.9,
159.0, 158.0, 155.2, 152.2, 149.0, 148.7, 147.8, 147.5, 141.9, 141.6, 137.30, 137.28, 137.0, 136.6,
136.1, 131.1, 128.6-127.4 (Cx13), 124.6, 123.3, 122.3, 119.8, 119.1, 114.8, 113.5, 110.5, 102.4, 94.7,
93.6, 77.8, 77.2, 74.9, 71.5, 71.3, 70.2, 70.0, 68.3, 34.0, 25.1, 20.89, 20.87, 20.7, 20.6, 20.3, 19.9.
FABMS (m/z) 1192 (32), 1191 ([M+H]+, 44), 1133 (17), 1132 (41), 1131 (66), 1130 (53), 1042 (37),
1041 (62), 1040 (82), 950 (18), 949 (28), 605 (23), 604 (45), 603 (100); FABHRMS calcd for
C70H63O18 [M+H]+, 1191.4014; found: 1191.3949.
[4,8]-2,3-cis-3,4-trans-2'',3''-trans-3,3'',5'',7'',3''',4'''-Hexaacetyl-(−)-epicatechin-(+)-catechin (14) A
solution of 13 (63 mg, 0.053 mmol) in THF/MeOH/H2O (11 mL, 20:1:1) was hydrogenated over 20%

Molecules 2014, 19
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Pd(OH)2/C (2 mg) for 6 h at RT Filtration and concentration afforded a pale brown solid, which was
acetylated according to a general procedure to give 27 mg (0.033 mmol, 62%) of 14 as an amorphous
solid: 2D4 0 (c 0.012, CHCl3); H-NMR (500 MHz, CDCl3, 0.7:0.3 mixture of rotational isomers)
1
major isomer: 7.25–6.41 (4.2H, m), 6.12–6.10 (1.4H, m), 6.12 (0.7H, s), 5.19–5.13 (1.4H, m), 5.02
(0.7H, d, H = 10.0 Hz), 4.70 (0.7H, t, J = 10.0 Hz), 4.43 (0.7H, br s), 2.92 (0.7H, dd, J = 5.0,
16.0 Hz), 2.67 (0.7H, dd, J = 10.0, 16.0 Hz), 2.33 (2.1H, s), 2.23 (2.1H, s), 2.21 (2.1H. s), 1.92 (2.1H.
s), 1.71 (2.1H, s), 1.62 (2.1H, s); minor isomer: 7.25–6.08 (2.7H, m), 5.19–4.68 (1.5H, m), 4.47 (0.3H,
br s), 2.85–2.81 (0.3H, m), 2.75–2.61 (0.3H, m), 2.32 (1.8H, s), 2.01 (0.9H, s), 1.90 (0.9H, s), 1.69
(0.9H, s), 1.65(0.9H, s); 13C-NMR (125 MHz, CDCl3) major isomer: 176.1, 174.3, 173.4, 173.2, 173.0,
173.4, 162.2, 161.4, 159.4, 159.3, 151.9, 151.7, 148.7 (×2), 140.8, 133.4, 132.9, 131.9-131.4 (×7),
123.0, 122.9, 121.6, 118.4, 117.3 (×2), 114.6, 109.0, 104.7, 99.4, 98.6, 78.5, 76.3, 73.7, 72.4, 38.0,
27.8, 23.4, 23.12, 23.05 (×2), 22.8, 22.6. Minor isomer was not identified. FABMS (m/z) 832 (3.6),
831 ([M+H]+, 9.1), 605 (27), 604 (45), 603 (100); FABHRMS calcd for C42H39O18 [M+H]+, 831.2136;
found: 831.2116.
[4,8]-2,3-cis-3,4-trans-2'',3''-cis-5,7,3',4',5'',7'',2''',3''',4'''-Nona-O-benzyl-(−)-epicatechin-(−)-epi-
gallocatechin (18) To a solution of 9 (39 mg, 0.053 mmol) and 16 (160 g, 0.21 mmol) in CH2Cl2
(100 mL) TMSOTf (0.13 mL, 0.064 mmol, 0.5 M solution in CH2Cl2) was added dropwise at −20 °C.
After stirring for 5 min, the pale yellow reaction mixture was quenched with saturated NaHCO3. The
mixture was extracted with CHCl3 and the organic phase was washed with water and brine, and then
dried (Na2SO4). Filtration, concentration, and preparative silica gel TLC purification (hexane/EtOAc,
3:1) afforded 63 mg (0.045 mmol, 85%) of 18 as a colorless amorphous substance: 2D4 0 (c 0.030,
1
CHCl3); H NMR (500 MHz, CDCl3, 0.7:0.3 mixture of rotational isomers) major isomer: 7.51-6.80
(32.9H, m), 6.45 (1.4H, s), 6.40 (0.7H, s), 6.07 (0.7H, d, J = 2.0 Hz), 5.75 (0.7H, d, J = 2.0 Hz), 5.61
(0.7H, s), 5.19–4.56 (12.6H, m), 4.71 (0.7H, d, J = 11.0 Hz), 4.56 (0.7H, d, J = 11.0 Hz), 4.12 (0.7H, br
s), 4.04 (0.7H, s), 3.92 (0.7H, d, J = 4.5 Hz), 3.04 (0.7H, d, J = 17.5 Hz), 2.95 (0.7H, dd, J = 5.0,
17.5 Hz), 1.85 (0.7H, br s), 1.60 (0.7H, br s); minor isomer: 7.51–6.80 (13.8H, m), 6.34 (0.3H, s), 6.29
(0.3H, d, J = 2.5 Hz), 6.24 (0.3H, s), 6.12 (0.3H, d, J = 2.2 Hz), 5.19–3.95 (6.0H, m), 4.68 (0.3H, d,
J = 12.0 Hz), 4.42 (0.3H, d, J = 12.0 Hz), 3.20–3.12 (0.3H, m), 3.05–3.00 (0.3H, m), 1.80–1.40 (0.6H,
13
m); C-NMR (125 MHz, CDCl3, 0.7:0.3 mixture of rotational isomers) major isomer: 158.3, 157.0,
156.6, 156.9, 155.2, 154.4, 152.9, 152.5, 149.3, 148.8, 138.4–136.9 (C×11), 133.3, 132.7, 128.7–126.6
(C×21), 119.8, 115.2, 113.5, 111.3, 105.4, 104.4, 102.4, 93.8, 93.3, 91.7, 79.2, 75.7, 75.2, 72.5, 71.4,
71.3, 71.1, 70.5, 70.1, 70.0, 69.1, 66.4, 35.8, 28.6; minor isomer: 158.4, 156.9, 156.7, 155.6, 155.2,
153.1, 152.8, 149.1, 148.7, 138.4–136.9 (C×12), 133.6, 132.5, 128.7–126.6 (C×21), 120.1, 115.1,
114.2, 111.7, 105.6, 104.5, 101.8, 94.5, 93.5, 93.1, 78.3, 75.6, 75.2, 72.2, 71.6, 71.4, 71.3, 70.6, 70.1,
69.9, 69.6, 65.2, 36.1, 28.9.
[4,8]-2,3-cis-3,4-trans-2'',3''-cis-5,7,3',4',5'',7'',2''',3''',4'''-Nona-O-benzyl-(−)-epicatechin-(−)-epi-
gallocatechin-3''-O-(tri-O-benzyl)gallate (19). To a solution of 9 (284 mg, 0.38 mmol) and 17 (1.79 g,
1.52 mmol) in CH2Cl2 (100 mL) was added dropwise TMSOTf (0.46 mL, 0.46 mmol, 1.0 M solution
in CH2Cl2) at −20 °C. After stirring for 5 min, the pale yellow reaction mixture was quenched with sat.
NaHCO3. The mixture was extracted with CHCl3 and the organic phase was washed with water and

Molecules 2014, 19
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brine, and dried (Na2SO4). Filtration, concentration, and preparative silica gel TLC purification
(hexane/EtOAc, 3:1) afforded 359 mg (0.20 mmol, 52%) of 19 as a colorless amorphous solid: D24
1
−4.6 (c 1.64, CHCl3); H-NMR (500 MHz, CDCl3) major isomer: 7.45–6.05 (67H, m), 5.63–5.62
(3H, m), 5.51 (1H, d, J = 6.0 Hz), 5.30–4.51 (26H, m), 4.14–4.10 (2H, m), 3.20 (1H, dd, J = 6.0, 18.5 Hz),
3.04 (1H, d, J = 18.5 Hz), 1.78 (1H, d, J = 6.0 Hz); 13C-NMR (125 MHz, CDCl3) 165.3, 158.4, 156.9,
156.2, 155.4, 154.8, 152.8, 152.5, 149.3, 148.9, 143.4, 138.1–136.5 (C×7), 132.8, 132.3, 128.7–125.7
(C×26), 119.7, 114.5, 113.6, 111.9, 109.9, 106.0, 105.8, 104.2, 102.6, 93.8, 93.4, 91.8, 78.4, 75.9,
75.3, 75.1, 75.1, 71.8, 71.4, 71.2, 70.8, 70.7, 70.2, 69.2, 68.9, 35.9, 26.9.
[4,8]-2,3-cis-3,4-trans-2'',3''-cis-(−)-Epicatechin-(−)-epigallocatechin (20). A solution of 18 (63 mg,
0.045 mmol) in THF/MeOH/H2O (20:1:1, 22 mL) was hydrogenated over 20% Pd(OH)2/C (2 mg) for
12 h at RT. Filtration and concentration afforded a pale brown solid, which was purified using HPLC
to give 38 mg (0.039 mmol, 87%) of 20 as an amorphous solid. 2D4 −12.5 (c 0.016, CH3OH); 1H NMR
(400 MHz, CD3OD, −40 °C) 6.82 (1H, br s), 6.87 (1H, d, J = 8.5 Hz), 6.59 (1H, br d, J = 8.5 Hz), 6.59
(2H, s), 5.86 (1H, s), 6.10–5.15 (2H, m), 5.03 (1H, br s), 4.86 (1H, br s), 4.60 (1H, br s), 4.25 (1H, br
s), 3.79 (1H, br s), 2.92 (1H, br d, J = 16.0 Hz), 2.79 (1H, d, J = 16.0 Hz); FABMS (m/z) 596 (29), 595
([M+H]+, 27), 581 (33), 561 (18), 537 (26), 493 (26), 329 (100); FABHRMS calcd for C30H27O13
[M+H]+, 595.1452; found: 595.1402.
[4,8]-2,3-cis-3,4-trans-2’’,3’’-cis-(−)-Epicatechin-(−)-epigallocatechin-3''-O-gallate (21) A solution
of 19 (164 mg, 0.090 mmol) in THF/MeOH/H2O (20:1:1, 22 mL) was hydrogenated over 20%
Pd(OH)2/C (2 mg) for 12 h at RT. Filtration and concentration afforded a pale brown solid, which was
purified using HPLC to give 38 mg (0.051 mmol, 56%) of 21 as an amorphous solid. 2D4 0 (c 0.025,
1
CH3OH); H NMR (400 MHz, CD3OD, −40°C) 7.01 (2H, s), 6.89 (1H, br s), 6.87 (1H, br s), 6.80
(6.60 (2H, m), 6.58 (2H, s), 6.13 (1H, br s), 5.89 (1H, br s), 5.55 (1H, br s), 5.14 (1H, br s), 5.12 (1H,
br s), 4.78 (1H, br s), 4.54 (1H, br s), 3.84 (1H, br s), 3.14–2.72 (2H, m); FABMS (m/z) 749 (18), 748
(25), 747 (27), 746 ([M+H]+, 17), 719 (17), 603 (21), 545 (23), 503 (20), 487 (23), 329 (100);
FABHRMS calcd for C37H31O17 [M+H]+, 747.1561; found: 747.1519.
3.3. Inhibitory Activity of Cell Proliferation
HeLa S3 cells were seeded into a 96-well flat-bottomed tissue culture plate (Iwaki) in a 37 °C
incubator equilibrated with a 5% CO2: 95% humidified air atmosphere. D-MEM (Dulbecco's Modified
Eagle’s Medium; Gibco® (Life technologies, Grand Island, NY, USA) supplemented with 5% fetal calf
serum and 1% Pen-Strep; InvitrogenTM (Life technologies, Grand Island, NY, USA). After 24 h of
incubation, synthesized compounds in DMSO were added (final 100 M) and incubated for 24 h. A Cell
Count Reagent SF colorimetric assay (Nacalai Tesque, Kyoto, Japan) was performed to evaluate the
inhibitory activity of cell proliferation. In brief, 10 µL of Cell Count Reagent SF was added to each
well, and incubation was performd for 2 h at 37 °C. Following which, viable cells were assessed using
a microplate reader (Filter Max F5 multi-mode microplate reader; Molecular Devices, Downingtown,
PA, USA) to measure the OD at 450 nm. Cells cultured with DMSO treatment served as the control.

Molecules 2014, 19
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4. Conclusions
In conclusion, we have used our synthetic methodologies to synthesize various procyanidin B1
analogs to clarify the biological activities of the compounds. Investigation of cell proliferation
inhibitory activity against HeLa S3 cells showed that free-phenolic hydroxyl groups were important for
the activity and that lower-unit hydrophobicity enhanced this activity. Biological and chemical
mechanistic studies are currently being conducted.
Acknowledgments
This work was supported by BRAIN: Program for Promotion of Basic and Applied Research for
Innovations in Bio-oriented Industry (to A. Saito), JSPS KAKENHI Grant Number 23780123 (Grant-
in-Aid for Young Scientist (B) to A. Saito) and Grant Number 24590014 (Grants-in-Aid for Scientific
Research (C) to N. Nakajima).
Conflicts of Interest
The authors declare no conflict of interest.
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