Synthesis of Kaempferide Mannich Base Derivatives and Their Antiproliferative Activity on Three Human Cancer Cell Lines

Kaempferide (3,5,7-trihydroxy-4'-methoxyflavone, 1), a naturally occurring flavonoid with potent anticancer activity in a number of human tumour cell lines, was first semisynthesized from naringin. Based on Mannich reaction of kaempferide with various secondary amines and formaldehyde, nine novel kaempferide Mannich base derivatives 2–10 were synthesized. The aminomethylation occurred preferentially in the position at C-6 and C-8 of the A-ring of kaempferide. All the synthetic compounds were tested for antiproliferative activity against three human cancer cell lines (Hela, HCC1954, SK-OV-3) by the standard MTT method. The results showed that compounds 1, 2 and 5–10 were more potent against Hela cells with IC 50 values of 12.47–28.24 μM than the positive control cis-platin (IC 50 41.25 μM), compounds 5, 6, 8 and 10 were more potent against HCC1954 cells with IC 50 values of 8.82–14.97 μM than the positive control cis-platin (IC 50 29.68 μM), and compounds 2, 3, 5, 6 and 10 were more potent against SK-OV-3 cells with IC 50 values of 7.67-18.50 μM than the positive control cis-platin (IC 50 21.27 μM).

It has recently become apparent that the most of the important classes of drugs, especially those derived from natural products are nitrogen-containing compounds.The Mannich reaction is a versatile reaction that leads to the incorporation of amines into organic molecules.Amine moiety in drugs could enhance physicochemical properties (e.g.water solubility) and improve bioactivity and bioavailability of bioactive molecules (Joshi et al., 2013).Kaempferide can undergo regioselective Mannich reactions under certain conditions, and synthesize various Mannich base derivatives.
As part of our interest is the extension of systematic investigation of the chemistry and biological activity of flavonoids (Liu et al., 2012;Liu et al., 2014;Wu et al., 2013).Herein we report the first semisynthesis of kaempferide ( 1) from commercially low-cost naringin, and synthesis of the series of novel kaempferide Mannich base derivatives 2-10.Furthermore, all the synthetic compounds were tested for antiproliferative activity against a panel of human cancer cell lines including Hela (cervical carcinoma), HCC1954 (breast cancer) and SK-OV-3 (ovarian cancer) by the standard MTT method.
Reagents and conditions: (a) I

RESULTS AND DISCUSSION
The synthetic pathways adopted for the synthesis of kaempferide (1) and its Mannich base derivatives 2-10 are illustrated in Scheme 1,acacetin (12) could be obtained by dehydrogenation in I 2 /pyridine, regioseletive methylation, hydrolysis of glycosidic bond from the corresponding flavanone glycoside naringin, which is abundant and commercially low-cost.Benzylation of the remaining phenol hydroxyl group of 12 afforded benzyl ether 13.Oxidation of 13 to the 3-hydroxyflavone benzyl ether 14 proved to be challenging.The method was the oxidation of flavone 13 with dimethyldioxirane (DMDO) generated in situ from Oxone and acetone, flowed by acid induced rearrangement that gave the hydroxyflavone benzyl ether 14 in comparable yield.Subsequently, Pd/C catalyzed hydrogenlysis of 14 gave kaempferide (1).The sequence from naringin to kaempferide (1) was easily conducted on a large scale, and each intermediate could be purified by recrystallization.
Our strategy for the synthesis of the C-aminomethylated derivatives relied upon the electrophilic substitution at C-6 and C-8 of the A-ring of kaempferide.This was achieved by the Mannich reaction of the kaempferide (1) with formaldehyde and the secondary amines in methanol.The classical conditions of the Mannich reaction for the hydroxyl compounds are based on the substrate, the secondary amine and formaldehyde ratio in alcohol with prolonged heating (Arend et al., 1998;Sujith et al., 2009;Xu et al., 2014).In our case, kaempferide (1), formaldehyde and secondary amines in 1: 2: 2 ratio, respectively, were stirred under reflux in methanol for 3 h to afford the C-aminomethylated derivatives 2-10.The structures of the resulting Mannich bases were confirmed by 1 H NMR and mass analysis.The 1 H NMR spectra of compounds 2-10 clearly indicated the absence of the signal at δ 6.20 of H-6 (C-6) position and δ 6.46 of H-8 (C-8) position of the kaempferide in A-ring system, and a signal at δ 3.61-3.88indicated the presence of an aminomethyl group at C-6 and C-8 of kaempferide (1).
All the synthetic Mannich base derivatives were screened in vitro for antiproliferative activity against three human cancer cell lines (Hela, HCC1954 and SK-OV-3) by the standard MTT method (Hattori et al., 1952).The antiproliferative activity against these cancer cells was calculated following the formula of the inhibitory ratio and cis-platin as positive control.The inhibitory effects of kaempferide ( 1) and its Manich base derivatives 2-10 on three human cancer cells are shown in Table 1.The dose-response curve for MTT assay of compounds 1, 2 and cis-platin on Hela cells, 5, 6 and cis-platin on HCC1954 cells and 5, 6 on SK-OV-3 cells proliferation as shown in Fig. 1.Overall, most of these derivatives show a broad range of antiproliferative effect against all three cancer cell lines tested.Preliminary bioactive test demonstrated that compounds 1, 2 and 5-10 were more potent (lower IC 50 values) against Hela cells with IC 50 values of 12.47-28.24μM than the positive control cis-platin (IC 50 41.25 μM), compounds 5, 6, 8 and 10 were more potent against HCC1954 cells with IC 50 values of 8.82-14.97μM than the positive control cis-platin (IC 50 29.68μM), and compounds 2, 3, 5, 6 and 10 were more potent against SK-OV-3 cells with IC 50 values of 7.67-18.50μM than the positive control cis-platin (IC 50 21.27μM).
It is interesting to note that compound 2 to Hela cells (IC 50 12.47 μM), compound 5,6,8-10 to HCC1954 cells (IC 50 8.82-34.69μM) and compound 2,3,5-8 and 10 to SK-OV-3 cells (IC 50 7.67-24.87μM) were more an-tiproliferative activities (lower IC 50 values) than the parent compound kaempferide (1) to Hela (IC 50 15.18μM), HCC1954 (IC 50 36.27μM) and SK-OV-3 (IC 50 39.80 μM), respectively.Although the other Mannich base derivatives showed no more activity than kaempferide (1), they still exhibited some interesting inhibition on these cancer cells.Molecular recognition in the target-binding site in these cancer cells may be the reason for different behavior of these compounds.The presence of amine moiety in flavonoid molecular may increase biological potency 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.

General methods
Melting points were determined by an XRC-1 apparatus and were uncorrected.The 1 H and 13 C NMR spectra were recorded on a Bruker-AV400 spectrometer with internal standards of the different solvents.Mass spectra (MS) and high-resolution mass spectrometry (HRMS) were determined with VG Autospec-3000 or Mat 95 XP spectrometer by the EI or ESI method.The chemical shifts (δ) were measured by ppm, and coupling constant (J) was calculated in hertz (Hz).Column chromatography was carried out using 200-300 mesh silica gel (Qingdao Ocean Chemical Products of China).Commercially available AR or chemical pure reagents, and anhydrous solvent removed water and redistilled were employed.
General experimental procedure for synthesis of Mannich base derivatives.The solution of kaempferide (80 mg, 0.16 mmol) with 0.02 mL of 15% HCl(aq) in 10 mL of MeOH and formaldehyde (0.32 mmol), was stirred at room temperature, then secondary amine (0.32 mmol) was added dropwise and the reaction mixture was stirred and refluxed at 80ºC for 3 h.The solvent was removed under reduced pressure and the residue diluting with H 2 O, the solution was extracted by EtOAc (3×30 mL), the extracts were combined and the solvent was removed under reduced pressure, dried over anhydrous Na 2 SO 4 , the crude solid was recrystallized with EtOAc/ petroleum ether to afford 2-10 in 56-93% yields.
(1 -Average absorbance of treated group/Average absorbance of control group) ×100% Statistical analysis.Data represent the means of at least three separate experiments.Statistical analysis was performed using SAS statistical software.A value of p＜0.05 was considered significant.

CONCLUSION
In this paper, kaempferide (1) was first semisynthesized from naringin.Based on Mannich reaction of kaempferide with various secondary amines and formaldehyde, nine new flaovonoid Mannich base derivatives 2-10 were synthesized.The antiproliferative activities test demonstrated that compounds 1, 2 and 5-10 were more potent (lower IC 50 values) against Hela cells with IC 50 values of 12.47-28.24μM than the positive control cis-platin (IC 50 41.25 μM), compounds 5, 6, 8 and 10 were more potent against HCC1954 cells with IC 50 values of 8.82-14.97μM than the positive control cis-platin (IC 50 29.68μM), and compounds 2, 3, 5, 6 and 10 were more potent against SK-OV-3 cells with IC 50 values of 7.67-18.50μM than the positive control cis-platin (IC 50 21.27μM).The results indicated that these compounds are potential anticancer agents and are promising for further development.