Alkyl Phloroglucinol Derivatives from Syzygium levinei and their Differentiation-Inducing Activity

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material
• Extraction and isolation
• Acidic hydrolysis of compound 3
• Absolute configuration of glucose
• Determination of cell proliferation by MTT assay
• Morphological studies
• Determination of erythroid differentiation by benzidine test
• Supporting information
• Results and Discussion
• Acknowledgements
• References

Abstract

Eleven compounds have been identified from the whole plants of Syzygium levinei (Myrtaceae) on the basis of spectroscopic analysis and chemical methods. Among these compounds, (Z)-1-(2,6-dihydroxy-4-methoxyphenyl)-oct-5-en-1-one (1), (3E,7Z)-1-(2,6-dihydroxy-4-methoxyphenyl)-deca-3,7-dien-1-one (2), and 1-(2-O-β-D-glucopyranosyl-5,6-dihydroxy-4-methoxyphenyl)-octan-1-one (3) were characterized as new alkyl phloroglucinol derivatives, and 1-(2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one (4) was identified as a new natural product. Compounds 1, 2, and 4 were found to inhibit the proliferation of leukemia K562 and HL-60 cells and also to induce the differentiation of K562 cells as determined by the appearance of matured morphology and the increase in the intracellular hemoglobin level.

Introduction

Syzygium levinei (Myrtaceae) is an arborous species widely distributed in the south of China [1]. Previous phytochemical investigations of the genus Syzygium led to the identification of flavones [2], triterpenoids [2], tannins [3], and phenolic compounds [4], [5]. Pharmacological studies indicated that several constituents and extracts of Syzygium plants possess immunostimulant [2], antiviral [4], antimutagenic [5], antibacterial [5], and antioxidant activities [6]. To the best of our knowledge, no chemical study has been made on the species Syzygium levinei. As a continuation of our efforts to find new antitumor natural products from Chinese folk medicines, the petroleum ether extract of S. levinei was found to exhibit cytotoxicity against Ramos Burkitt’s lymphoma B cells. Further bioassay-guided isolation and systematic isolation led to the identification of three new alkyl phloroglucinol derivatives as (Z)-1-(2,6-dihydroxy-4-methoxyphenyl)-oct-5-en-1-one (1), (3E,7Z)-1-(2,6-dihydroxy-4-methoxyphenyl)-deca-3,7-dien-1-one (2), and 1-(2-O-β-D-glucopyranosyl-5,6-dihydroxy-4-methoxyphenyl)-octan-1-one (3), a new natural alkyl phloroglucinol derivative 1-(2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one (4) [7] along with seven other known compounds (5 - 11). In addition, the effect of compounds 1, 2, and 4 on cellular proliferation and differentiation in leukemia cells was investigated.

Materials and Methods

General

Optical rotations were measured with a Perkin-Elmer 241MC polarimeter or a Perkin-Elmer 341 polarimeter. UV spectra were recorded with a Beckman DU-7 spectrometer. IR spectra were recorded using a Perkin-Elmer 577 spectrometer. LR-ESI-MS were measured using a Finnigan LCQ-DECA instrument, and HR-ESI-MS data were obtained on a Mariner spectrometer. LR-EI-MS were obtained on a MAT-95 spectrometer, and HR-EI-MS on a Kratos 1H spectrometer. NMR spectra were run on a Bruker AM 400 spectrometer with TMS as internal standard. Preparative HPLC was carried out using a Varian SD-1 instrument, equipped with a Merck NW25 C₁₈ column (10 μm, 20 mm × 250 mm), and ProStar 320 UV/Vis detector. GC analysis was performed with a Shimadzu model GC-MS-QP5050A instrument equipped with a DB-1 column (0.25 mm i. d. × 30 m), and in the EI ionization mode. Column chromatographic separations were carried out using a LiChroprep RP-18 Lobar column (40 - 63 μm, Merck) and using silica gel H60 (300 - 400 mesh), zcx-II (100 - 200 mesh) (Qingdao Haiyang Chemical Group Corporation; Qingdao, P. R. China), and Sephadex LH-20 (Pharmacia Biotech AB; Uppsala, Sweden) as packing materials. HSGF254 silica gel TLC plates (Yantai Chemical Industrial Institute; Yantai, P. R. China) and RP-18 WF₂₅₄ TLC plates (Merck) were used for analytical TLC. Human erythroleukemia K562, human myelogenous leukemia HL-60 and Burkitt’s lymphoma Ramos cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The cell lines were maintained at 37 °C (5 % CO₂) in tissue culture dishes filled with growth medium (RPMI 1640 medium with 10 % heat-inactivated fetal bovine serum, 100 kU/L penicillin, and 200 kU/L streptomycin). Reagents for biological assays including benzidine dihydrochloride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and all other reagents were purchased from Sigma Co. (St. Louis, MO, USA). Compounds 1, 2 and 4 were stored at -20 °C as 100 mM solutions in ethanol. The solutions were diluted at least 1000-fold in the growth medium so that the final concentration of solvent was less than 0.1 %, which had no effect on the cell proliferation and differentiation.

Plant material

The whole plants of Syzygium levinei were collected in the suburb of Guangzhou, Guangdong province, P. R. China, in May 2003, and identified by Professor Zexian Li of the South China Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. SIMMW0306) is deposited in the Herbarium of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Extraction and isolation

Powdered air-dried whole plants of Syzygium levinei (3.0 kg) were percolated with 95 % ethanol (10 L × 3) at room temperature. The filtrate was concentrated to dryness under vacuum and then suspended in 20 % ethanol overnight. After filtration of the precipitated chlorophyll and evaporation of ethanol in the filtrate, the aqueous residue (1 L) was extracted with petroleum ether, ethyl acetate, and n-butanol (1.0 L × 3), successively, to give a petroleum ether fraction (5.5 g), an ethyl acetate fraction (23.0 g), and an n-butanol fraction (41.5g), respectively. The petroleum ether fraction (5.5 g) was subjected to chromatography over a silica gel H60 column (3.5 cm i. d. × 25 cm) with a petroleum ether-acetone gradient (10 : 1, 8 : 1, 5 : 1, 2 : 1, 1 : 1, 0 : 1, each 500 mL) as eluent to give Fr. A1 (between 0 and 600 mL, 550 mg), Fr. A2 (between 600 and 1100 mL, 1.36 g), Fr. A3 (between 1100 and 3000 mL, 2.85 g). A part of Fr. A2 (100.6 mg) was further purified over a preparative HPLC column (2.0 cm i. d. × 25 cm, RP-18), eluted with a methanol-water gradient (65 % - 80 %, 10 mL/min, 1 - 75min, 750 mL) to afford 4 (between 450 mL and 490 mL, 16.8 mg), 1 (between 520 mL and 570 mL, 21.4 mg), and 2 (between 610 mL and 660 mL, 30.4 mg). The n-butanol extract (41.5 g) was separated over a silica gel H60 column (10 cm i. d. × 25 cm) eluted with a chloroform-methanol gradient (10 : 1, 1.5 L; 5 : 1, 1.5 L; 3 : 1, 1.0 L; 2 : 1, 1.0 L; 1 : 1, 1.0 L; 0 : 1, 500 mL) to give Fr. C1 (between 0 and 1100 mL, 2.65 g), Fr. C2 (between 1100 and 1900 mL, 4.90 g), Fr. C3 (between 1900 and 3100 mL, 5.62 g), Fr. C4 (between 3100 and 4200 mL, 5.15 g), Fr. C5 (between 4200 and 5500 mL, 6.80 g), and Fr. C6 (between 5500 and 6500 mL, 11.5 g). Fr. C3 (5.62 g) was separated over a silica gel H60 column (3.5 cm i. d. × 20 cm), eluted with a chloroform-methanol gradient (10 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1, 0 : 1, each 400 mL) to give Fr. C3A (between 0 and 750 mL, 550 mg), Fr. C3B (between 750 and 1150 mL, 860 mg), Fr. C3C (between 1150 and 1700 mL, 1.82 g) and Fr. C3D (between 1700 and 2400 mL, 1.40 g). Fr. C3B (between 750 and 1150 mL, 860 mg) was separated over a LiChroprep RP-18 Lobar column eluted with methanol-water (30 %, 50 %, 70 % each 100 mL) to give 3 (between 148 mL and 175 mL, 12.5 mg).

Syzygiol A (1): amorphous white powder; UV (MeOH): λ_max = 286, 229 nm; IR (KBr): ν_max = 3303, 2953, 2852, 1635, 1589, 1520, 1429, 1375, 1317, 1234, 1203, 1078 cm^-1; LR-EI-MS: m/z (rel. int.) = 264 [M]⁺ (7), 182 (42), 167 (100); HR-EI-MS: m/z = 264.1368 [M]⁺ (calcd. for C₁₅H₂₀O₄: 264.1361); ¹H- and ¹³C-NMR data: see Table [1] and Table [2].

Syzygiol B (2): amorphous white powder; UV (MeOH): λ_max = 289, 230 nm; IR (KBr): ν_max = 3315, 3007, 2962, 1635, 1585, 1520, 1429, 1356, 1217, 1161, 1084 cm^-1; LR-EI-MS: m/z (rel. int.) = 290 [M]⁺ (10), 167 (100), 149 (18); HR-EI-MS: m/z = 290.1521 [M]⁺ (calcd. for C₁₇H₂₂O₄: 290.1508); ¹H- and ¹³C-NMR data: see Table [1] and Table [2].

Syzygioside (3): amorphous white powder; [α]_D ²¹: -90.0° (c 0.02, methanol); UV (MeOH): λ_max = 289, 229 nm; IR (KBr): ν_max = 3408, 2924, 1625, 1595, 1450, 1427, 1230, 1207, 1167, 1074, 1047cm^-1; LR-ESI-MS (positive-ion mode): m/z = 483.1 [M + Na]⁺; LR-ESI-MS (negative-ion mode): m/z = 459.0 [M - H]^-; HR-ESI-MS (negative-ion mode): m/z = 459.1828 [M - H]^- (calcd. for C₂₀H₃₁O₁₁: 459.1866); ¹H- and ¹³C-NMR data: see Table [1] and Table [2].

The structures of eight known compounds, 1-(2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one (4), 2α,3β,19α,24-tetrahydroxyurs-12-en-28-oic acid (5) [8], [9], myricitrin (6) [10], [11], quercitrin (7) [10], [11], 2,4,6-trimethoxyphenyl-1-O-β-D-(6′-O-galloyl)-glucopyranoside (8) [12], 2α,3α,19α,24-tetrahydroxyurs-12-enoic acid 28-O-β-D-glucopyranosyl ester (9) [13], 2,4,6-trimethoxyphenyl-O-β-D-glucopyranoside (10) [14], [15], and n-butyl-β-D-fructopyranoside (11) [16] were determined by comparison of the physicochemical data with those reported in literature (see Supporting Information).

Acidic hydrolysis of compound 3

Compound 3 (2 mg) dissolved in 50 % methanol (1.0 mL) containing 5 % HCl was heated in a boiling water bath for 5 hours. After cooling, the reaction mixtures were neutralized with 10 % Na₂CO₃ and glucose was identified as its sugar moiety by co-TLC of the aqueous solution with authentic glucose sample (EtOAc-MeOH-H₂O-HOAc, 13 : 3:3 : 4, Rf = 0.46).

Absolute configuration of glucose

A solution in pyridine (300 μL) of the hydrolyzed sugar from 3 (0.01 mol/L) and L-cysteine methyl ester hydrochloride (0.02 mol/L) were mixed and warmed at 60 °C for 1 h. Acetic anhydride (200 μL) was then added, and the mixture was warmed at 90 °C for another 1 h. After evaporation of pyridine and acetic anhydride under vacuum, the residue was dissolved in acetone (500 μL) and the solution (1 μL) was subjected to GLC [17]. A peak for the peracetylated thiazolidine derivatives with a retention time at 11.87 min was observed, which was identical to the derivatives of authentic D-glucose prepared in the same manner.

Determination of cell proliferation by MTT assay

Cells (5 × 10⁴ cells/mL) were incubated with different concentrations of compounds 1, 2 and 4 for 72 hours. Cell proliferation was determined with the MTT assay as described previously [18]. The assays were carried out in triplicate in at least three independent experiments.

Morphological studies

Cells (5 × 10⁴ cells/mL) were treated with each compond for 5 days. Cells were collected by centrifugation, fixed with methanol, stained with Giemsa staining solution, and observed under a microscope as described [18].

Determination of erythroid differentiation by benzidine test

Cells (5 × 10⁴ cells/mL) were treated with each compound for 5 days. The percentage of cells staining for hemoglobin was estimated by staining with benzidine/H₂O₂ essentially as previously described [19]. The blue-stained hemoglobin-positive cells were counted under a microscope. At least 120 cells were assessed for each sample.

Supporting information

Extraction and isolation of known compounds, the physicochemical data of known compounds, and ¹H-¹H COSY and main HMBC correlation signals of 1 - 3.

Results and Discussion

Compound 1 was obtained as a white amorphous powder with an elemental formula of C₁₅H₂₀O₄ determined by HR-EI-MS (m/z = 264.1368, [M]⁺). Its UV absorptions maximized at 286, 229 nm, suggesting an aromatic moiety in its structure. The IR spectrum of 1 revealed the existence of hydroxy group (3303 cm^-1), aromatic ring (1589, 1520, 1429 cm^-1) and ketone (1635 cm^-1) in its structure. In the ¹³C-NMR spectrum of 1, 15 carbon signals including two methyls, four methylenes, four sp ² methines, and five sp ² quaternary carbons were observed. In the NMR spectra of 1, a singlet at δ_H = 5.95 (2H, s), along with four carbon signals [δ_C = 104.6 (C-1), 93.9 (C-3/C-5), 163.1 (C-2/C-6), 165.2 (C-4)], suggested the existence of a symmetrical phloroglucinol subunit. Two olefinic protons were found at δ_H = 5.38 (1H, dt, J = 11.9, 5.0 Hz), 5.40 (1H, dt, J = 11.9, 5.0 Hz). Analysis of ¹H-¹H COSY and HSQC spectra revealed fragments: -C-2′-C-3′-C-4′-C-5′-C-6′-C-7′-C-8′- in its structure. The planar structure of 1 was established on the basis of its HMBC NMR spectra, in which ¹³C-¹H long-range correlation signals were observed at C-1/H-3, H-5; C-4/4-OCH₃; C-1′/H-3, H-5, H-3′; C-4′/H-2′, H-6′; C-5′/H-3′, H-7′; C-8′/H-6′. The Z configuration of the double bond was identified from the comparison of its ¹³C-NMR data with those of (Z)-3-heptene and its 3-E-isomer [20], which was further confirmed by the ¹H-¹H coupling constant of the olefinic protons (J = 11.9 Hz). The allylic carbon at δ_C = 20.2 (C-7′) and the olefinic carbons [δ_C = 132.0 (C-6′), 128.0 (C-5′)] were identical with those of the 3-Z-isomer [allylic carbon: δ_C = 20.9 (C-2); olefinic carbon: δ_C = 131.9 (C-3) and 129.2 (C-4)], but not those of the 3-E-isomer [allylic carbon: δ_C = 25.9 (C-2); olefinic carbon: δ_C = 132.4 (C-3) and 129.4 (C-4)]. Finally, the structure of 1 was determined as (Z)-1-(2,6-dihydroxy-4-methoxyphenyl)-oct-5-en-1-one, and has been assigned the trivial name syzygiol A.

Compound 2 was obtained as a white amorphous powder with an elemental formula of C₁₇H₂₂O₄ determined by HR-EI-MS (m/z = 290.1521, [M]⁺). Its ¹H- and ¹³C-NMR spectra were very similar to those of 1 except for two more sp ² methine carbons, which indicated it to be also a linear alkylene phloroglucinol with two double bonds in the side chain. In the NMR spectra of 2, a singlet at δ_H = 5.95 (2H, s), along with four carbon signals [δ_C = 104.4 (C-1), 94.0 (C-3/C-5), 163.1 (C-2/C-6), 165.6 (C-4)], suggested the existence of a symmetrical phloroglucinol subunit. Four olefinic protons were found at δ_H = 5.33 (1H, dt, J = 10.9, 6.7 Hz), 5.36 (1H, dt, J = 10.7, 6.7 Hz), 5.60 (1H, dt, J = 15.4, 6.3 Hz) and 5.71 (1H, dt, J = 15.3, 6.6 Hz), which indicated the existence of two double bonds in its structure. Analysis of its ¹H-¹H-COSY and HSQC spectra revealed fragments: -C-2′-C-3′-C-4′-C-5′-C-6′-C-7′-C-8′-C-9′-C-10′- in its structure. The planar structure of 2 was established on the basis of its HMBC spectra, in which ¹³C-¹H long-range correlation signals were observed at C-1/H-3, H-5; C-4/4-OCH₃; C-1′/H-3, H-5, H-3′; C-4′/H-2′, H-6′; C-5′/H-3′, H-7′; C-6′/H-8′; C-7′/H-9′; C-8′/H-5′, H-9′, H-10′. The Z configuration of the double bond at Δ^{7 ′} was determined using the same method as mentioned in 1 [20]. The stereochemistry of the double bond at Δ^{3 ′} was determined as E by comparison of its ¹³C-NMR data with those of E-3-octenoic acid ethyl ester and its 3-Z-isomer [21], which was further confirmed by the ¹H-¹H coupling constants of the olefinic protons (J = 15.3 Hz). Thus structure of 2 was characterized to be (3E,7Z)-1-(2,6-dihydroxy-4-methoxyphenyl)-deca-3,7-dien-1-one, and has been assigned the trivial name syzygiol B.

Compound 3 was obtained as a white amorphous powder with an elemental formula of C₂₁H₃₂O₁₁ determined by HR-ESI-MS (m/z = 459.1866, [M - H]^-). In its ¹³C-NMR spectrum, 21 carbon signals including two methyls, five methylenes, nine methines (two for sp ²), and four sp ² quaternary carbons were observed. In the ¹H-NMR spectrum of 3, an anomeric proton was found at δ_H = 5.04 (1H, d, J = 7.7 Hz), two meta-coupled protons at δ_H = 6.31 (1H, d, J = 2.0 Hz) and 6.11(1H, d, J = 2.0 Hz) revealed the existence of a 1,2,3,5-tetrasubstituted benzene ring, which was confirmed by four sp ² quaternary carbon signals [δ_C = 107.8 (C-1), 162.2 (C-2), 167.8 (C-4), 167.5 (C-6)], and two sp ² methine signals [δ_C = 95.0 (C-3), 96.8 (C-5)]. Acid hydrolysis of 3 gave the glucose as the sugar moiety by co-TLC with an authentic sample. The ¹H-¹H coupling constant (J = 7.7 Hz) of the anomeric proton indicated the glucose unit to be in the β-glycosidic form. The absolute configuration of glucose in 3 was found to belong to the D series by GC analysis of the peracetylated thiazolidine derivative [17]. Preliminary analysis of ¹H-¹H-COSY and HSQC spectra enabled the deduction of fragments -C-2′, C-3′, C-4′, C-5′, C-6′, C-7′, C-8′- in its structure. The planar structure of 3 was established on the basis of its HMBC spectra, in which ¹³C-¹H long range correlation signals were observed at C-1/H-3, H-5; C-2/H_glc-1; C-4/4-OCH₃; C-1′/H-3′; C-4′/H-2′; C-5′/H-3′, H-7′; C-6′/H-4′, H-8′. The structure of 3 was further confirmed by the NOE correlation signals between H-3 and H_glc-1; H-3 and 7-OCH₃; H-5 and 7-OCH₃; H-2′ and H-3′; H-3′ and H-4′; H-4′ and H-5′; H-5′ and H-6′; H-6′ and H-7′; H-6′ and H-8′; H-7′ and H-8′ in its NOESY spectrum. The structure of 3 was determined as 1-(2-O-β-D-glucopyranosyl-5,6-dihydroxy-4-methoxyphenyl)-octan-1-one, and has been assigned the trivial name syzygioside.

Compound 4 was identified as 1-(2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one by spectroscopic analysis and comparison of the NMR data with those reported in literature [5].

Differentiation therapy is a novel approach for the treatment of leukemia in which immature leukemia cells are induced to attain a mature phenotype when exposed to differentiation inducers, either alone or in combination with other chemotherapeutic or chemopreventive drugs. One successful example is the use of all-trans-retinoic acid in the treatment of acute myeloid leukemia, which is much safer and more effective than conventional chemotherapy [22]. Thus it is important to find new compounds with differentiation-inducing activity. Previous studies have shown that compound 4 is a synthetic DIF analogue [23] and possesses antiproliferative and differentiation-inducing effects on erythroid leukemia K562 cells [24]. Compounds 1 and 2 are analogues of 4, therefore, the three compounds were tested together for their effects on the proliferation and differentiation of HL-60 and K562 cells with 4 as a positive control. As shown in Fig. [1], compounds 1, 2 and 4 inhibited the proliferation of K562 cells (Fig. [1] A) and HL-60 cells (Fig. [1] B) in a concentration-dependent manner, with IC₅₀ values of 14.39 μM, 27.90 μM, 15.96 μM for K562 cells and 16.44 μM, 31.57 μM, 18.71 μM for HL-60 cells, respectively.

The morphology of K562 cells was changed after 5 days incubation with various concentrations of 1 or 4. After treatment with 10 μM of 1 or 4, the cells showed a prominent perinuclear vacuole or cytoplasmic inclusion, accentuation of cytoplasmic blebbing, and a decreased nuclear-to-cytoplasmic ratio. Compound 2 did not show significant morphological effects on K562 cells (Fig. [2]). To confirm K562 cell differentiation, the functional changes in K562 cells induced by 1, 2 and 4 were analyzed. As shown in Fig. [3], after 5 days of incubation with 2.5 - 20 μM of 1, 2 and 4, the percentage of benzidine-positive cells in K562 cells was significantly increased, which indicated that compounds 1 and 4 do indeed induce the differentiation of K562 cells. However, compounds 1, 2 and 4 (5 - 20 μM) did not induce significant morphological changes in HL-60 cells (data not shown).

Acknowledgements

The authors are grateful to Shanghai Committee of Science and Technology for partial financial support (No. 03DZ19228 and No. 034 319 229).

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1 These authors contribute equally to this work

Weimin Zhao

Department of Natural Products Chemistry

Shanghai Institute of Materia Medica

Shanghai Institutes for Biological Sciences

Chinese Academy of Sciences

Shanghai 201203

People’s Republic of China

Email: wmzhao@mail.shcnc.ac.cn

References

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1 These authors contribute equally to this work

Weimin Zhao

Department of Natural Products Chemistry

Shanghai Institute of Materia Medica

Shanghai Institutes for Biological Sciences

Chinese Academy of Sciences

Shanghai 201203

People’s Republic of China

Email: wmzhao@mail.shcnc.ac.cn

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