Synthesis and antiproliferative activity of pterostilbene and -methoxy pterostilbene Mannich base derivatives against Hela cells

Fourteen novel pterostilbene (1) and 3′-methoxy pterostilbene (2) Mannich base derivatives (3–16) were synthesized via the microwave-assisted Mannich reaction of 1 or 2 with various secondary amines and formaldehyde. The regioselectivity of the reaction occurred preferentially at C-3′(and /or C-5′) position of the B-ring of stilbene. The biological testing results showed that all the target compounds exhibit antiproliferative activity against Hela cells from IC50 = 22.5–65.3μM. Compounds 1–3, 7, 11–13, and 16 displayed higher (lower IC50 values) activity than the positive control cisplatin (IC50 = 41.3μM).


Introduction
Pterostilbene (trans-3,5-dimethoxy-4 -hydroxystilbene, 1), a naturally occurring dimethylether analog of resveratrol, was first isolated from P. Santalinus (red sandalwood) and it is also found in several types of berries and grapes [1]. Pterostilbene has been shown to have a cancer chemopreventive effect similar to that of resveratrol and it is cytotoxic to a variety of tumor cell types, such as human gastric carcinoma cells [2], human prostate cancer cells [3], and lung cancer cells [4]. 3 -Methoxy pterostilbene (2), a naturally occurring homolog of pterostilbene, was isolated from Sphaerophysa salsula [5] and showed higher cytotoxic activities on some cancer cells than resveratrol [6].
Biological activity studies have shown that the development of structural analogs of antitumor agents possessing basic nitrogen atom moiety is of great value [7,8]. Anticancer studies using several classes of polyaromatic antitumour agents indicate that the introduction of a nitrogen-containing side chain increases significantly the biological activity and potency of the parent compounds. For example, it has been shown that the presence of the nitrogen moiety in flavopiridol, a synthetic nitrogen-containing flavonoid with antitumor activity against various tumor cell lines, is critical for its antitumor effect [9]. The Mannich reaction is a commonly used way to introduce nitrogenous moieties, such as aminoalkyl group to phenols. The modification of an aminoalkyl side chain in aromatic substrates permits to increase significantly the biological potency of bioactive molecules due to the greater number of molecular sites for electrophilic attack by cellular constituents, as well as due to the cascade effect of preferential chemosensitization when compared with parent compounds [10]. Furthermore, Mannich bases have been associated with increased water solubility [11]. Scheme 1 Synthesis of pterostilbene and 3 -methoxy pterostilbene Mannich base derivatives (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) Resveratrol has been the subject of numerous investigations, and its cancer chemopreventive activity has been fully demonstrated [12]. Prompted by the close structural similarity of pterostilbene and resveratrol, as well as the current high interest in the development of a pterostilbene-based therapeutic [13,14], we decided to synthesize two series of fourteen pterostilbene and 3 -methoxy pterostilbene Mannich base derivatives to be evaluated for their antiproliferative activity against human cervical carcinoma Hela cells by the standard CCK-8 assay.
Our strategy for the synthesis of the C-aminomethylated derivatives relied upon the electrophilic substitution at C-3 and/or C-5 of the stilbene B-ring. This was achieved by the Mannich reaction of 1 and 2 with formaldehyde and secondary amines in methanol, introducing a dialkylaminomethyl group in the ortho-position of the phenolic group. The general conditions of the Mannich reaction for the phenol compounds are based on the substrate, the amine, and formaldehyde ratio in alcohol with prolonged heating. It was reported that microwave irradiation can be used for the Mannich reaction because of the considerable advantages over conventional heating, such as substantial rate enhancement cleaner reaction and improvement in yield [16,17]. In our case, pterostilbene (1) or 3 -methoxypterostilbene (2), formaldehyde, and secondary amines in a 1:1.2:1.2 ratio, respectively, were stirred under microwave irradiation (600 W) for 0.5-2 h in methanol to afford the Caminomethylated derivatives 3-16. The Mannich reaction of pterostilbene (1) with methylpiperazine would lead to a dif-  The structures of all synthesized Mannich base were confirmed by 1 HNMR, 13 CNMR, and MS analysis. The 1 HNMR spectra of compounds 3-9 clearly indicate the absence of the of H-3 proton signal at δ 6.28 (and H-5 proton for compounds 8, 9) in the B-ring of pterostilbene (1). Similarly, the H-5 proton at δ 6.91 of 3 -methoxy pterostilbene (2) in the B-ring disappeared for compounds 10-16. The signal at δ 3.60-4.10 indicated the presence of the aminomethyl group at C-3 (or C-5 ) position of compounds 3-16.
The preliminary structure-activity relationship analysis revealed that pterotilbene (1) has a moderate antiproliferative activity on Hela cells. The introduction of smaller methoxy or dimethylaminomethyl side chains into the 3 -position of 1 was to increase biological activity, and the introduction of larger dialkylaminomethyl side chain into 3 -position of 1 was to decrease the activity. Based on these observations, the methoxy and dimethylaminomethyl groups were selected as suitable substituents at the 3 -position of 1. The introduction of all dialkylaminomethyl side chain into 5 -position of 3methoxy pterostilbene (2) was to decrease the activity. As the antiproliferation assay was carried out in vitro, the inhibitory effect on Hela cell variance among these compounds could be attributed to the hydrophobicity and permeability changes resulting from different substituents and position of side chains in pterostilbene (1) .

Conclusion
In Summary, 14 new pterostilbene (1) and 3 -methoxy pterostilbene (2) Mannich base derivatives 3-16 were synthesized through a microwave-assisted Mannich reaction strategy. All the compounds were tested for their antiproliferative activity against Hela cells using the standard CCK-8 assay; the results indicating that compounds 1, 2, 3, 7, 11, 12, 13 and 16 were slightly more potent than anticancer drug cisplatin (DDP). The pterostilbene skeleton can serve as a promising structural template for the development of novel antiproliferative agents deserving further investigation.

Experimental General information
Melting points were measured on an XRC-I apparatus and were uncorrected. Microwave reactions were performed using an XH-MC-1 microwave reactor (50-900 W) (Beijing Xianghu Science and Technology Development Co., China). 1 H NMR and 13 C NMR spectra were recorded on a Bruker AM-400 instrument, using tetramethylsilane as internal standard (s single; d doublet; t triplet; q quarlet; m multiplet), chemical shifts (δ) in ppm, and coupling constants (J ) in Hz. Mass spectra were recorded using a VG Autospec-3000 and a ZA-BHS spectrometer (ESI or EI ionization method). Pterostilbene (1) and 3 -methoxy pterostilbene (2) were synthesized and purified according Column chromatography was carried out on silica gel (200-300 mesh). Commercially available AR grade or chemically pure reagents were used. Anhydrous solvents were dried and redistilled prior to their use.

General procedure for the synthesis of Mannich base derivatives 3-16
Compound 1 or 2 (200 mg, 0.78 mmol) was dissolved in methanol (10 mL), then formaldehyde (0.94 mmol) and a secondary amine (0.94 mmol) were introduced to this solution. The reaction mixture was stirred under microwave irradiation (600 W) at 80 • C. The end of the reaction was determined by TLC. When the reaction was complete, the solvent was removed in vacuo. The residue was extracted with ethyl acetate (3 × 20 mL) and water (15 mL