Synthesis of Analogues of Gingerol and Shogaol, the Active Pungent Principles from the Rhizomes of Zingiber officinale and Evaluation of Their Anti-Platelet Aggregation Effects

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads.


Introduction
Ginger (Chinese name: Shengjiang), derived from the rhizomes of Zingiber officinale Roscoe, is a well-known spice and is most frequently prescribed as a traditional Chinese medicine for its stomachic, antiemetic, antidiarrheal, expectorant, antiasthmatic, hemostatic and cardiologic properties for the treatment of several gastrointestinal and respiratory diseases [1][2][3]. The most famous traditional medicinal application of Z. officinale is to promote blood circulation for the removal of blood stasis, a mechanism that is related to anti-platelet aggregation activity [4,5]. Numerous chemical investigations of the pungent and bioactive principles of ginger have been carried out [6][7][8][9][10][11][12][13][14][15][16][17][18][19]. The pungent principles reported from the rhizomes of Zingiber officinale include: zingerone, gingerols, gingerdiols, gingerdiones, and shogaols ( Figure 1).  In the course of our continuing research program aimed at discovering novel bioactive constituents from natural sources, thrombolytic and vasoactive activity examinations were carried out, and the ether extracts of the rhizomes of Z. officinale were found to exhibit significant anti-platelet aggregation activity and vasorelaxing effects. In our previous article [20], twenty-nine compounds were identified, and [6]-gingerol and [6]-shogaol exhibited potent anti-platelet aggregation bioactivity. These results initiated our interest in searching for more potent antiplatelet aggregation agents from the analogues of gingerol and shogaol. Therefore, in the present study eight groups of compounds ( Figure 2) were prepared and subjected to examinations of their anti-platelet aggregation activity.

Chemistry
At first, the dehydrozingerone 9 was prepared by vanillin condensation with a good yield (89%), Equation (1). Then the cross aldol condensations of α,β-unsaturated ketone 9 with different aldehydes were investigated using various bases as catalysts. The major products were the dehydrogingerols 3a-f, and the minor products dehydroshogaols 2a-f were obtained in the optimum yield (6%-15%) when lithium bis(trimethylsilyl)amide (LiHMDS) was employed. Therefore, deprotonation of 9 with LiHMDS in tetrahydrofuran at 0 °C and subsequent trapping with aldehydes Equation (2) afforded products 2a-f and 3a-f with moderate yields in a range between 50% and 66% (  Chlorination and dehydrohalogenation of alcohols 3a-f with HCl and K 2 CO 3 , respectively, produced quantitative yields of adducts 2a-f Equation (4) and reduced the occurrence of trace amounts of 10. The catalytic hydrogenation of [n]-dehydroshogaols 2a-f over palladium on charcoal afforded [n]-paradols 5 and trace amounts of secondary alcohol 11. It was surprising that [n]-dehydroparadols 6 could be obtained with the same method only reduced the amount of palladium on charcoal from 0.05 to 0.015 eq. The results are shown in Equation (5) and Table 3.   The same hydrogenation procedure was applied in [n]-dehydrogingerols 3, and a high yield of [n]-gingerols 8 was obtained (83%-86%). Dehydration of 8 with HCl/K 2 CO 3 gave approximately 85% of [n]-shogaols 4 (Equation (6), Table 4). Although there were many reagents available for the oxidation of secondary alcohols to ketone, unfortunately, most of these oxidizing agents did not show sufficient activity except in the case of Swern oxidation, which yielded a moderate amount of oxidized compound 1 (Equation (7), Table 5

Anti-Platelet Aggregation Evaluation Bioassay
Platelets circulate in the blood of mammals and are involved in hemostasis, leading to the formation of blood clots. Too many platelets form blood clots that may obstruct blood vessels and induce strokes, myocardial infarctions, and pulmonary embolisms. Sometimes this situation also results in the blockage of blood vessels to other parts of the body, including the extremities of the arms or legs [21]. The traditional medicinal use of ginger is to promote the blood circulation necessary for removing blood stasis. Therefore, synthetic derivatives were examined in the anti-platelet aggregation bioassay to test for the presence of activity. The anti-platelet aggregation results are summarized in Tables 6-13. All the tested compounds displayed significant inhibitory effects on the aggregation of washed rabbit platelets stimulated by arachidonic acid (AA). At a 10 μg/mL concentration, most of the tested compounds with the exception of 3a, 3d, and 7e caused the inhibition percentages of aggregation induced by AA (100 μM) to be higher than 90%. On the other hand, the activities of these synthetic derivatives against platelet activating factor (PAF) and thrombin (Thr) induced aggregation were insignificant.
The [n]-isodehydrogingerdiones 1a-e also showed significant inhibition of platelet aggregation induced by AA (Table 12). [7]-Isodehydrogingerdione 1c was found to be the most effective compound among this series, with an IC 50 value of 0.68 μg/mL. Moreover, an epoxide ring next to the α,β-unsaturated ketone produced derivatives 7a-f of lower potency compared with [n]-paradols 5a-f. They were only as potent as the dehydroshogaol series, with IC 50 values between 0.96 and 2.38 μg/mL. Apparently, [10]-epoxydehydroparadol 7f exhibited the most significant inhibitory effect among this series with an IC 50 value of 0.96 μg/mL (Table 13).

General
All the chemicals were purchased from Merck KGaA (Darmstadt, Germany), unless specifically indicated. Column chromatography was performed on silica gel (70-230 mesh, 230-400 mesh), and TLC monitoring was executed on Merck precoated Si gel 60 F 254 plates, using UV light to visualize the spots. The melting points of the purified compounds were determined using a Yanagimoto micromelting point measuring apparatus (Tokyo, Japan) without corrections. The UV spectra were obtained on a Hitachi UV-3210 spectrophotometer (Tokyo, Japan). The IR spectra were obtained as KBr discs on a Jasco Report-100 FT-IR spectrometer (Tokyo, Japan). 1 H and 13 C NMR spectra were recorded on a Bruker AC-200 NMR spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are shown in δ values (ppm) with tetramethylsilane as an internal standard. The EI mass and high-resolution mass spectra were measured on a VG Analytical Model 70-250S spectrometer (Micromass, Manchester, UK). Elemental analyses were performed on a Perkin-Elmer 240 analyzer (Waltham, MA, USA). (9) 10% Sodium hydroxide (7.0 g, 175 mmol) was added dropwise to a solution of vanillin (2.5 g, 16.4 mmol) in acetone (100 mL) at room temperature. The reaction mixture was stirred for 12 h, concentrated under reduced pressure, then neutralized by cold 5% HCl (aq) . The solution was extracted with EtOAc (4 × 50 mL). The organic layers were combined, washed with saturated NaCl (aq) (brine), dried over MgSO 4 , and concentrated under reduced pressure. The product was isolated on silica gel column chromatography (EtOAc/hexanes = 1/4) to afford yellow needles (2.8 g, 89% yield).            After the mixture had been stirred for 1 h, the appropriate aldehyde (31.4 mmol) was added and stirred for 3 h. The reaction was then quenched with 5% HCl (aq) at 0 °C and extracted with EtOAc (4 × 20 mL). The organic layers were combined, washed with brine, dried over Na 2 SO 4 , and concentrated under reduced pressure. Products 2 and 7 were isolated using C-18 gel column chromatography (water/methanol = 1/2).