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

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.


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.

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 SnCl 4 as the Lewis acid, which produced the procyanidin B 1 derivative (7) in 33% yield and with excellent stereoselectivity. Benzyl group hydrogenation of 7 was performed using H 2 /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 TiCl 4 , SnCl 4 , 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 SnCl 4 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]. Furthermore, we synthesized other procyanidin B 1 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 B 1 analogs.
The inhibitory activities of the synthetic procyanidin B 1 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. 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.

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% CO 2 : 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; Invitrogen TM (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.

Conclusions
In conclusion, we have used our synthetic methodologies to synthesize various procyanidin B 1 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.