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Natural Product Communications

Polyprenylated Phloroglucinols from Hypericum maculatum

2015 Vol. 10 No. 7 1231 - 1235

Paraskev T. Nedialkova,*, Georgi Momekovb, Zlatina K. Kokanova-Nedialkovaa and Jörg Heilmannc a

Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, Dunav str. 2, 1000 Sofia, Bulgaria b Department of Pharmacology, Toxicology and Pharmacotherapy, Faculty of Pharmacy, Medical University of Sofia, Dunav str. 2, 1000 Sofia, Bulgaria c Department of Pharmaceutical Biology, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany [email protected] Received: February 18th, 2015; Accepted: March 12th, 2015

A detailed phytochemical investigation of the dichloromethane extract of the aerial parts of Hypericum maculatum Crantz. led to the isolation of four new (2-5) and six known (1a/b, 6-10) polyprenylated phloroglucinol derivatives. The new compounds were identified by means of spectral methods (MS, NMR, IR, UV) as (E)-4-(3,7-dimethylocta-2,6-dien-1-yl)-5-hydroxy-2-(3-methylbut-2-en-1-yl)-3,6-dioxocyclohexa-1,4-dien-1-yl isobutyrate (2), (E)-2-(3,7-dimethylocta-2,6dien-1-yl)-5-hydroxy-4-(3-methylbut-2-en-1-yl)-3,6-dioxocyclohexa-1,4-dien-1-yl isobutyrate (3), (E)-4-(3,7-dimethylocta-2,6-dien-1-yl)-5-hydroxy-2-(3methylbut-2-en-1-yl)-3,6-dioxocyclohexa-1,4-dien-1-yl 2-methylbutanoate (4) and (E)-2-(3,7-dimethylocta-2,6-dien-1-yl)-5-hydroxy-4-(3-methylbut-2-en-1yl)-3,6-dioxocyclohexa-1,4-dien-1-yl 2-methylbutanoate (5). The known compounds have been identified as hyperpolyphyllirin/hyperibine J (1a/b), erectquione A (6), (E)-1-(3-(3,7-dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)-2-methylpropan-1-one (7), (E)-1-(3-(3,7-dimethylocta-2,6-dien-1-yl)2,4,6-trihydroxyphenyl)-2-methylbutan-1-one (8), 1-(5,7-dihydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)chroman-8-yl)-2-methylpropan-1-one (9) and 1-(6,8dihydroxy-1,1,4a-trimethyl-2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl)-2-methylpropan-1-one (10). The stereochemistry of 1a is described for the first time. The cytotoxicity of 1-6 on SKW-3, BV-173 and K-562 tumor cell lines was determined using MTT based assays. Keywords: Hypericum maculatum, Maculatoquiones A-D, Hyperpolyphyllirin, Hyperibine J, Erectquione, Cytotoxicity.

The genus Hypericum L. (Hypericaceae) includes more than 480 species that are found in every continent of the world, except Antarctica [1]. H. maculatum Crantz. is widespread in all Bulgarian mountains and many other European countries. This species has been used in traditional medicine as either an equivalent to or substitute for the official herbal drug Hyperici herba and is given in several pharmacopoeias together with H. perforatum as a source of this drug [2]. In previous phytochemical studies of this plant, the presence of the naphthodianthrones hypericin and pseudohypericin, the acylphloroglucinol hyperforin, flavonoids, xanthones, as well as benzophenones have been established [3]. As part of our ongoing research on the phytochemistry of Hypericum species, four new (25) and six known (1a/b, 6-10) polyprenylated phloroglucinol derivatives (Figure 1) were isolated from the dichloromethane(CH2Cl2)-extract of H. maculatum. In addition, the cytotoxicity of the new compounds on SKW-3, BV-173 and K-562 tumor cell lines was established using MTT based assays. The CH2Cl2-extract of H. maculatum was subjected to VLC fractionation on silica gel. Further subsequent chromatographic procedures led to the isolation of ten polyprenylated phloroglucinol derivatives (Figure 1). The structures of these compounds were elucidated by means of NMR, MS, IR and UV spectral techniques and comparison with literature data. The signals in the 1H- and 13 C-NMR spectra of the isolated compounds were unambiguously assigned using 2D NMR techniques, i.e. COSY, HSQC and HMBC. Multiplicities were determined using 1H- and HSQC spectra. Compound 1a/b was isolated as colorless oil and degraded very quickly, especially in non-protic solvents. In order to make more stable derivatives, 1a/b was treated with CH2N2. The methylation process gave two products 1c and 1d in a ratio ca 1:3 (Figure 2) pointing to the original presence of two tautomers 1a/b.

Figure 1: Structures of isolated compounds.

Figure 2: Methylation products of 1a/b.

1232 Natural Product Communications Vol. 10 (7) 2015

The molecular formula of 1c was established as C32H48O4 on the basis of HR-ESI-MS and indicated nine degrees of unsaturation. The IR spectrum showed absorption bands at 1725 and 1648 cm-1 pointing to the presence of carbonyl groups. The 13C NMR spectrum of 1c (Table 1) exhibited signals for 32 carbons, including eleven quarternary (including three carbonyl, four olefinic, and one oxygenated sp2 carbon), five methines, five methylenes, and eleven methyls (including one oxygenated). These data indicated the characteristic signals of an acylphloroglucinol core with two nonconjugated carbonyl groups [δC 208.1 (C-9), δC 206.6 (C-10)], a 1,3-keto-enol system [δC 169.9 (C-2), δC 123.2 (C-3), δC 196.7 (C-4)], and two quaternary carbons at δC 78.3 (C-1), and δC 60.3 (C-5) [4]. Table 1: 13C NMR spectral data (in CDCl3, 298K, 150 MHz) of 1c and 1d. Carbon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 OCH3 a

1c (δC, multa) 78.3, C 169.9, C 123.2, C 196.7, C 60.3, C 42.2, CH2 44.0, CH 47.3, C 208.1, C 209.6, C 40.5, CH 21.4, CH3 21.3, CH3 23.6, CH2 122.4, CH 132.9, C 25.6, CH3 18.1, CH3 16.5, CH3 27.8, CH2 122.3, CH 133.4, C 25.9, CH3 17.9, CH3 13.7, CH3 37.6, CH2 25.2, CH2 124.7, CH 131.3, C 25.7, CH3 17.8, CH3 60.7, CH3

1d (δC, multa) 84.0, C 194.2, C 128.0, C 173.2, C 54.5, C 40.5, CH2 43.3, CH 48.7, C 207.7, C 209.4, C 42.5, CH 21.3, CH3 20.5, CH3 23.4, CH2 121.5, CH 133.0, C 25.7, CH3 17.9, CH3 16.9, CH3 27.0, CH2 122.5, CH 133.4, C 25.8, CH3 18.0, CH3 13.5, CH3 36.4, CH2 25.0, CH2 124.8, CH 131.1, C 25.7, CH3 17.7, CH3 62.3, CH3

multiplicities were determined by HSQC experiments.

The 1H NMR spectrum of 1c (Table 2) showed signals indicative of three isoprenyl groups [δH 5.01 (1H, m, H-15), δH 4.93 (1H, m, H-21), δH 5.03 (1H, m, H-28)] and an isobutyryl side chain [δH 2.38 (1H, sept, J = 6.5 Hz, H-11), δH 1.06 δH (3H, d, J = 6.5 Hz, H-12), δH 1.17 (3H, d, J = 6.5 Hz, H-13)]. Based on these data, 1c was considered as an acylphloroglucinol derivative substituted with three isoprenyl units and an isobutyryl group. In the HMBC experiment (Table 2) the signals of H2-14 [δH 3.37 (1H, dd, J = 16.4, 6.5 Hz, Ha-14), δH 3.24, dd, J = 16.4, 5.6 Hz, Hb-14)] gave cross peaks with those of C-2 (δC 169.9), C-3 (δC 123.2), and C-4 (δC 196.7) pointing to a C-3 attachment of the first isoprenyl group. The signals of H2-20 [δH 2.14 (1H, m, Ha-20), δH 1.69 (1H, m, Hb-20)] gave HMBC correlations with signals at δC 42.2 (C-6), δC 44.0 (C-7), and δC 47.3 (C-8), which is strong evidence for locating the second isoprenyl at C-7. The chemical shift difference of Hax-6 and Heq-6 (ΔδH 0.58) indicated, together with the chemical shift of C-7 (δC 44.0), that this isoprenyl group is exo orientated [5]. This was also evidenced by NOESY correlations of methyl protons Me-25 (δH 1.04, s) with H2-20 and Hax-6 (δH 1.31, dd, J = 13.8, 13.6 Hz) (Figure 3). According to the 1H-1H COSY experiment the methylene protons H2-27 [δH 2.18 (1H, m, Ha-27), δH 1.95 (1H, m, Hb-27)] gave correlations with those of H2-26 [δH 1.94 (1H, m, Ha-26), δH 1.40 (1H, m, H b -26)]. This observation, together with HMBC

Nedialkov et al.

Table 2: 1H NMR spectral data (in CDCl3, 298K, 600 MHz) and 1H,13C HMBC correlations of 1c and 1d. Position 6 7 11 12 13 14 15 17 18 19 20 21 23 24 25 26 27 28 30 31 OCH3

1c δH (J in Hz) HMBC (1H13C) 1.89 dd (13.8, 4.0, Heq), 4, 5, 7, 8, 9, 19, 20 1.31 dd (13.8, 13.6, Hax) 6 1.70 m 10, 12, 13 2.38 sept. (6.5) 10, 11, 13 1.06 d (6.5) 10, 11, 12 1.17 d (6.5) 2, 3, 4, 15, 16 3.37 dd (16.4, 6.5), 3.24 dd (16.4, 5.6) 14, 17, 18 5.01 m 15, 16, 18 1.70 s 15, 16, 17 1.69 s 4, 5, 6, 9 1.21 s 6, 7, 8, 21, 22 2.14 m, 1.69 m 7, 20, 23, 24 4.93 m 21, 22, 24 1.67 s 21, 22, 23 1.55 s 1, 7, 8, 26, 27 1.04 s 1, 7, 8, 25, 27, 28 1.94 m, 1.40 m 2.18 m, 1.95 m 5.03 m 1.67 s 1.61 s 4.03 s

26, 28, 29 26, 27, 30, 31 28, 29, 31 28, 29, 30 2

1d δH (J in Hz) HMBC (1H13C) 1.95 m (Heq), 1.38 4, 5, 7, 8, 19, 20 dd (13.6, 13.4, Hax) 1.59 m 6 1.99 sept. (6.5) 10, 12, 13 1.01 d (6.5) 10, 11, 13 1.11 d (6.5) 10, 11, 12 3.18 dd (14.9, 6.9), 2, 3, 4, 15, 16 3.13 dd (14.9, 6.4) 5.02 m 3, 14, 17, 18 1.66 s 15, 16, 18 1.69 s 15, 16, 17 1.30 s 4, 5, 6, 9 2.12 m, 1.74 m 6, 7, 21, 22 4.94 m 7 , 20, 23, 24 1.67 s 21, 22, 24 1.56 s 21, 22, 23 1.00 s 1, 7, 8, 26 1.93 m, 1.34 dd 1, 7, 8, 25, 27, 28 (15.9, 10.8) 2.11 m, 1.91 m 26, 28, 29 5.05 m 26, 27, 30, 31 1.65 s 28, 29, 31 1.59 s 28, 29, 30 3.91 s 4

Figure 3: Key NOESY correlations of 1c.

correlations between C-27 (δC 25.2) and H2-26, as well as C-26 (δC 37.6) and H2-27 suggested that the third isoprenyl was connected to C-26. The protons of Me-25 (δH 1.04, s) and H2-26 showed HMBC cross peaks with C-8, which is typical for the location of both groups in a bicyclo[3.3.1]nonane skeleton. The former signal gave a cross peak with Hax-6 in the NOESY spectrum (Figure 3), which is possible only when Me-25 is axially orientated. The three-proton singlet at δH 1.21 (Me-19) showed HMBC correlations with two carbonyls at δC 196.7 (C-4) and δC 208.1 (C-9), to a methylene at δC 42.2 (C-6), as well as to a quaternary carbon at δC 60.3 (C-5) pointing out that Me-19 is a substituent of C-5.The three-proton singlet of the artificial methoxy group at δH 4.03 gave a cross peak with the carbon signal of C-2. Finally, the remaining isobutyryl side chain has to be located at C-1. According to the evidence given above the structure of 1c was established as methoxyhyperpolyphyllirin. The second product of the methylation process (1d) showed similar spectra (UV, IR, MS and NMR) to those of 1c. The only difference between 1c and 1d was the position of the methoxy group pointing to the presence of two tautomers in the case of 1a/b. In the 1H NMR (Table 2) spectrum the signal appeared at δH 3.91 (3H, s) and gave long-range correlation with a carbon signal at δC 173.2 (C-4). In a similar manner the structure of 1d was established as methoxyhyperibine J. Of the tautomeric mixture (1a/b) 1a is identical to hyperpolyphyllirin, a polyprenylated acylphloroglucinol recently found in H. polyphyllum, while 1b is identical to hyperibine J, recently isolated from H. triquetrifolium [6]. Interestingly, for 1a neither the stereochemistry nor the presence of a tautomeric mixture has been known as it has been identified in a metabolic approach with LC/MS and NMR using 1H, HSQC and HMBC. In addition, the presence of a tautomeric mixture has not been reported for 1b, as well [6].

Polyprenylated phloroglucinols from Hypericum maculatum

Natural Product Communications Vol. 10 (7) 2015 1233

Table 3: 13C-NMR spectral data (in CDCl3, 298K, 150 MHz) of 2-5. (δC, multa)

Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1'  2'  3'  4'  5' a

2 177.5, C 149.7, C 120.8, C 185.8, C 137.7, C 145.8, C 22.3, CH2 119.4, CH 137.3, C 39.7, CH2 26.6, CH2 124.2, CH 131.4, C 25.7, CH3 17.7, CH3 16.2, CH3 23.5, CH2 118.5, CH 134.7, C 25.7, CH3 17.9, CH3 174.1, C 33.9, CH 18.9, CH3 18.9, CH3

3 177.5, C 145.7, C 137.7, C 185.8, C 120.7, C 149.7, C 23.4, CH2 118.3, CH 138.2, C 39.6, CH2 26.5, CH2 124.0, CH 131.6, C 25.7, CH3 17.7, CH3 16.3, CH3 22.4, CH2 119.6, CH 133.8, C 25.7, CH3 17.8, CH3 174.0, C 33.9, CH 18.9, CH3 18.9, CH3

4 177.5, C 149.7, C 120.7, C 185.8, C 137.7, C 145.7, C 22.3, CH2 119.4, CH 137.4, C 39.7, CH2 26.6, CH2 124.2, CH 131.4, C 25.7, CH3 17.7, CH3 16.2, CH3 23.6, CH2 118.5, CH 137.4, C 25.7, CH3 17.9, CH3 173.7, C 40.9, CH 26.6, CH2 11.5, CH3 16.6, CH3

5 177.5, C 145.7, C 137.8, C 185.8, C 120.7, C 149.7, C 23.5, CH2 118.3, CH 138.3, C 39.6, CH2 26.5, CH2 124.0, CH 131.6, C 25.7, CH3 17.7, CH3 16.3, CH3 22.4, CH2 119.6, CH 133.8, C 25.7, CH3 17.8, CH3 173.6, C 40.9, CH2 26.6, CH2 11.5, CH3 16.6, CH3

multiplicities were determined by HSQC experiments.

Table 4: 1H NMR spectral data (in CDCl3, 298K, 600 MHz) of 2-5. Position 7 8 10 11 12 14 15 16 17 18 20 21 2' 3' 4' 5' 2-OH 6-OH

2 3.16 d (7.3) 5.12 m 1.97 m 2.05 m 5.06 m 1.65 s 1.58 s 1.73 s 3.12 d (7.2) 4.99 m 1.66 s 1.69 s 2.85 sept. (7.0) 1.33 d (7.0) 1.33 d (7.0) 6.74 br. s

δH (J in Hz) 3 4 3.13 d (7.9) 3.16 d (7.3) 4.99 m 5.12 m 1.95 m 1.97 m 2.02 m 2.05 m 5.03 m 5.06 m 1.65 s 1.66 s 1.57 s 1.58 s 1.69 s 1.73 s 3.15 d (8.5) 3.12 d (7.1) 5.12 m 4.98 m 1.68 s 1.66 s 1.73 s 1.70 s 2.85 sept. (7.0) 2.66 m 1.33 d (7.0) 1.85 m, 1.62 m 1.33 d (7.0) 1.04 t (7.5) 1.31 d (7.0) 6.74 br. s 6.75 br. s

5 3.13 d (7.7) 4.99 m 1.95 m 2.03 m 5.03 m 1.65 s 1.57 s 1.70 s 3.14 d (7.5) 5.12 m 1.68 s 1.73 s 2.66 m 1.84 m, 1.61 m 1.04 t (7.4) 1.31 d (7.0) 6.75 br. s

The molecular formula of compound 2 was established as C25H34O5 from its HR-ESI-MS, which showed a protonated molecular ion peak at m/z 415.2479. The presence of a 2,5-dihexadiene-1,4-dione skeleton was suggested by the observed UV (277, 210 nm) and IR (1664, 1646, 1633, 1452 cm-1) absorptions [7]. Its IR spectrum also showed absorption bands for a hydroxyl group, as well as for ester carbonyl at 3390 and 1760 cm-1, respectively. Treatment with an alkaline solution of 2 gave compound 6, which was identified as erectquione A [7]. The 1H- and 13C-NMR spectral data (Tables 3 and 4) revealed the presence of one isoprenyl group (C-17 – C-21), one geranyl (C-7 – C-15) and one isobutyryloxy side chain (C-1' – C-4'). The 13C NMR signals of the 2,5-dihexadiene-1,4dione skeleton appeared at δC 177.5 (C-1), 149.7 (C-2), 120.8 (C-3), 185.8 (C-4), 137.7 (C-5) and 145.8 (C-6), suggesting oxygenation at C-2 and C-6. The signal of a hydroxyl proton OH-2 (δH 6.74, br. s) showed HMBC correlations (Figure 4) with signals of C-1, C-2 and C-3. The last gave long-range correlation with the signal of H2-7 (δH 3.16, d, J = 7.3 Hz), which pointing to C-3 being the attachment position of the geranyl chain. The methylene proton signals of H2-17 (δH, 3.12, d, J = 7.2 Hz) gave HMBC correlation with carbon signals of C-4, C-5 and C-6, which provided strong evidence that the prenyl side chain is connected to C-5. The remaining isobutyryl acid residue was deduced to be attached to C-6 with an ester bond. Thus, compound 2 was identified as (E)-4-(3,7-dimethylocta-2,6dien-1-yl)-5-hydroxy-2-(3-methylbut-2-en-1-yl)-3,6-dioxocyclohexa-1,4-dien-1-yl isobutyrate, and named maculatoquione A.

Figure 4: Key HMBC and NOESY correlations of 2-5.

Compound 3 shares the same molecular formula with 2. In addition, both compounds have similar UV and IR spectra. The signal of the hydroxyl proton OH-6 (δH 6.75, br. s) showed HMBC correlations (Figure 4) with signals of C-1 (δC 177.5), C-5 (δC 120.7) and C-6 (δC 149.7). The hydroxyl at C-2 was esterified with isobutyric acid and thus, the structure of 3 was established as (E)-2-(3,7dimethylocta-2,6-dien-1-yl)-5-hydroxy-4-(3-methylbut-2-en-1-yl)3,6-dioxocyclohexa-1,4-dien-1-yl isobutyrate, and named as maculatoquione B. The molecular formula of compound 4 was established as C26H36O5 by its HR-ESI-MS, which showed a protonated molecular ion peak at m/z 429.2641. Its UV and IR spectra were similar to those of compounds 2 and 3. 1H- and 13C-NMR spectra of 4 (Table 3 and 4) were almost identical to those of compound 2, but the signals of the isobutyryl moiety were replaced by signals of a 2-methylbutanoyl residue: one carbonyl (δC 173.7), one methine (δC 40.9, δH 2.66), one methylene (δC 26.6, δH 1.85 and δH 1.62) and two methyls [δC 11.5, δH 1.04 (t, J = 7.5 Hz); δC 16.6, δH 1.31 (d, J = 7.0 Hz)]. Thus, the structure of 4 was established as (E)-4-(3,7-dimethylocta-2,6dien-1-yl)-5-hydroxy-2-(3-methylbut-2-en-1-yl)-3,6-dioxocyclohexa-1,4-dien-1-yl 2-methylbutanoate, and named as maculatoquione C. Compound 5 shares the same molecular formula with 4. The signal of the hydroxyl proton OH-6 (δ H 6.75, br. s) showed HMBC correlations (Figure 4) with signals of C-1 (δ C 177.5), C-5 (δC 120.7) and C-6 (δC 149.7). In this case, the hydroxyl at C-2 was esterified with isobutyric acid. Thus, the structure of 5 was established as (E)-2-(3,7-dimethylocta-2,6-dien-1-yl)-5hydroxy-4-(3-methylbut-2-en-1-yl)-3,6-dioxocyclohexa-1,4-dien-1yl 2-methylbutanoate, and named as maculatoquione D. Maculatoquiones A-D (2-5) are new natural products and show structure similarity to erectquiones that were found in H. erectum [7]. Compounds 7-10 have been previously isolated from H. empetrifolium and were identified by comparison of their spectral data [8]. Cytotoxicity screening assay was carried out against a panel of human tumor cell lines, representative of some of the important types of neoplastic disease, namely K-562 (human chronic myeloid leukemia), SKW-3 (human T-cell leukemia, a KE-37 derivative) and BV-173 (human B-cell precursor leukemia). The tumor cells were continuously exposed to the tested compounds 1a/b, 1c, 1d, and 2-6, for 72 h and thereafter their viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-dye reduction assay. The tested compounds exerted concen-

1234 Natural Product Communications Vol. 10 (7) 2015

Table 5: Cytotoxic activity (IC50 values) of 1a/b, 1c, 1d and 2-5 against three human tumor cell lines, after 72 h exposure. Compds 1a/b 1c 1d 2 3 4 5 6

SKW-3a 5.3±1.5 4.7±1.0 17.8±8.1 18.0±3.1 12.3±1.6 16.0±1.6 16.7±2.0 22.4±2.0

IC50 (µM±SD) K-562b 21.3±1.4 23.5±3.5 68.8±2.0 77.0±5.4 30.1±4.2 35.9±2.8 28.1±4.0 36.7±2.6

BV-173c 5.7±5.7 14.0±7.8 20.6±8.7 17.9±5.3 16.2±5.8 18.1±1.5 20.5±2.7 46.3±3.6

a

human T-cell leukemia; b human chronic myeloid leukemia; c human B-cell precursor leukemia

tration-dependent cytotoxicity against the tumor cell lines, causing 50% reduction of cellular viability at low micromolar concentrations (the corresponding IC50 values are summarized in Table 5). All compounds were of similar potency; nevertheless, in all cell lines compound 1a/b and 1c exerted superior activity in comparison with 1d. Experimental General experimental procedures: Optical rotations (OR) were measured on a Schmidt+Haensch UniPol L1000. Infrared (IR) spectra were recorded on a Thermo Scientific Nicolet iS10 spectrometer in either KBr or as a film, HR-ESI-MS spectra on an Agilent Q-TOF 6540 UHD, and NMR spectra on a Bruker BioSpin (Rheinstetten, Germany) Avance III 600 spectrometer at 600 MHz (1H) and 150 MHz (13C) in CDCl3 (99.96%, Deutero, Kastellaun) at 298K. Column chromatography (CC) was carried out with either Diaion HP-20 or MCI-gel (Supelco, USA), vacuum liquid chromatography (VLC) on either silica gel (15-40 µm, Merck, Darmstadt, Germany) or Polygoprep 60-30 C-18 (20-40 µm, Macherey-Nagel, Germany), and LPLC on LiChroprep C-18 (40-63 µm, Merck, Darmstadt, Germany). Semi-preparative high performance liquid chromatography (HPLC) was performed on a Waters (Milford MA, USA) Breeze 2 high pressure binary gradient system consisting of a pump model 1525EF, manual injector 7725i and an UV detector, model 2489. Separations were achieved on a semi-preparative HPLC column Kromasil C18 (250×21.2 mm, 10 μm, flow rate 18 mL.min-1) purchased from Eka Chemicals AB (Bohus, Sweden). Thin layer chromatography (TLC) was performed on silica gel 60 F256 or RP-18 F254s plates (Merck). The chromatograms were visualized by spraying with vanillin-sulfuric acid reagent [H2SO4-AcOH-MeOH-vanillin (5:10:85:0.5, v/v/v/m)] and heating at 110°C for 2-3 min. All solvents were of HPLC grade and were purchased from either Merck or Sigma-Aldrich (Taufkirchen, Germany). All reagents were of analytical grade. Plant material: The aerial parts of Hypericum maculatum Crantz. (Hypericaceae) were collected during the flowering season from a wild habitat (Goli vrah, Vitosha Mountains) in July 2007. A voucher specimen (№ SOM-Co-1195) has been deposited at the Herbarium of the Botany Institute of Sofia (SOM). Extraction and isolation: Air-dried aerial parts (2 kg) were extracted with CH2Cl2 (4×6 L), the extracts were combined and evaporated in vacuo to give a dark-green waxy residue (154 g). This was dissolved in n-heptane and filtered through Celite. The resulting filtrate (1.5 L) was extracted with MeCN (6×600 mL). The combined MeCN layers were evaporated in vacuo. The resulting greenish oily residue (48.6 g) was divided into 2 equal portions that were subjected to VLC on silica gel 60 (d=100 mm × h= 55 mm) eluting with n-hexane, n-hexane-EtOAc mixture and EtOAc. The fraction compositions were monitored by TLC and similar fractions were combined into 8 pooled fractions (A-H). Fraction B (14.5 g) was subjected to CC on MCI-gel (4×35 cm) and eluted with water-

Nedialkov et al.

MeOH mixtures (20:80→0:100). The sub-fraction B2 was rechromatographed on MCI-gel with H2O-MeOH (20:80) and gave 1a/b (4.75 g). The sub-fraction B3 (2.98 g) was re-chromatographed on MCI-gel with H2O-MeOH (10:90). Subsequent isocratic semiprep. HPLC of mixtures of compounds 2-5 eluted with MeCNH2O (80:20, 0.1% TFA) gave 2 (58 mg), 3 (52 mg), 4 (33 mg) and 5 (55 mg). Isocratic semiprep. HPLC separation of fraction D (429 mg) using MeCN-H2O (75:25, 0.1% TFA) gave 6 (25 mg). Fraction F (8.03 g) was subjected to VLC and eluted with mixtures of n-hexane-EtOAc (80:20→60:40). Sub-fraction F3 (3.37 g) was chromatographed on MCI-gel with water-Me2CO (35:65→0:100). Further purification by semiprep. HPLC, eluting with MeCN-H2O (35:65→80:20, 0.1% TFA), gave 7 (120 mg), 8 (49 mg), 9 (49 mg) and 10 (16 mg). Methylation of 1a/b: Two hundred mg of 1a/b was treated with CH2N2 dissolved in Et2O. After evaporation of the solvent the reaction mixture was subjected to semiprep.-HPLC using MeCNH2O (84:16) as mobile phase to yield 1c (35 mg) and 1d (109 mg). Methoxyhyperpolyphyllirin (1c) Colorless oil. [α]D: +43 (c 0.10, MeOH). IR (neat): 1725, 1648, 1609, 1451 cm-1. UV/Vis λmax (MeOH) nm (log ε): 235 (3.94), 272 (4.07). 1 H NMR (600 MHz, CDCl3): Table 2. 13 C NMR (150 MHz, CDCl3): Table 1. HR-ESI-MS (+) m/z: 497.3639 [M + H]+ (calcd for C32H49O4, 497.3625). Methoxyhyperibine J (1d) Colorless oil. [α]D: +7 (c 0.10, MeOH). IR (neat): 1730, 1659, 1596, 1448 cm-1. UV/Vis λmax (MeOH) nm (log ε): 242 (3.84), 272 (3.93). 1 H NMR (600 MHz, CDCl3): Table 2. 13 C NMR (150 MHz, CDCl3): Table 1. HR-ESI-MS (+): m/z 497.3643 [M + H]+ (calcd. for C32H49O4, 497.3625). Maculatoquione A (2) Yellow oil. IR (KBr): 3390, 1760, 1664, 1646, 1633, 1452 cm–1. UV/Vis λmax (MeOH) nm (log ε): 210 (4.39) nm, 277 (4.23). 1 H NMR (600 MHz, CDCl3): Table 4. 13 C NMR (150 MHz, CDCl3): Table 3. HR-ESI-MS (+) m/z: 415.2476 [M + H]+ (calcd. for C25H35O5, 415.2479). Maculatoquione B (3) Yellow oil. IR (KBr): 3390, 1758, 1668, 1647, 1633, 1449 cm–1. UV/Vis λmax (MeOH) nm (log ε): 211 (4.21), 277 (4.13). 1 H NMR (600 MHz, CDCl3): Table 4. 13 C NMR (150 MHz, CDCl3): Table 3. HR-ESI-MS (+) m/z: 415.2478 [M + H]+ (calcd. for C25H35O5, 415.2479). Maculatoquione C (4) Yellow oil. [α]D: +68 (c 0.10, MeOH). IR (neat): 1770, 1666, 1643, 1634, 1456 cm–1. UV/Vis λmax (MeOH) nm (log ε): 212 (4.12), 277 (4.06). 1 H NMR (600 MHz, CDCl3): Table 4. 13 C NMR (150 MHz, CDCl3): Table 3.

Polyprenylated phloroglucinols from Hypericum maculatum

HR-ESI-MS (+) m/z: 429.2641 [M + H]+ (calcd. for C26H37O5, 429.2636). Maculatoquione D (5) Yellow oil. [α]D: +31 (c 0.10, MeOH). IR (neat): 3393, 1771, 1667, 1643, 1634, 1457 cm–1. UV/Vis λmax (MeOH) nm (log ε): 211 (4.20), 278 (4.08). 1 H NMR (600 MHz, CDCl3): Table 4. 13 C NMR (150 MHz, CDCl3): Table 3. HR-ESI-MS (+) m/z: 429.2634 [M + H]+ (calcd. for C26H37O5, 429.2636). Cell lines and culture conditions: The panel of cell lines used in this study consisted of K-562 (chronic myeloid leukemia), SKW-3 (KE-37 derivative, human T-cell leukemia) and BV-173 (pre-B-cell leukemia) that were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were maintained in a controlled environment – cell culture flasks at 37°C in a “Heraeus” incubator with 5% CO2 humidified atmosphere and the growth medium was 90% RPMI-1640, supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine. Cytotoxicity determination: Stock solutions of the tested compounds (1a/b, 1c, 1d and 2-5) were prepared in DMSO and were consequently diluted with RPMI-1640 medium to yield the desired final concentrations. At the final dilutions obtained, cells

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were never exposed to DMSO concentrations exceeding 0.5%. For the cytotoxicity determination cells were seeded into 96-well plates (100 μL/well at a density of 1×105 cells/mL) and exposed to various concentrations of the test compounds for 72 h. Cell survival was determined with the MTT dye-reduction assay, as previously described, with some modifications [9]. Briefly, after the incubation with the test-compound, MTT solution (10 mg/mL in PBS) was added (10 μL/well). Plates were further incubated for 4 h at 37°C and the formazan crystals obtained were dissolved by adding 100 μL/well 5% formic acid in 2-propanol. Absorption was measured on a microprocessor-controlled microplate reader (Labexim LMR1®) at 540 nm. For each concentration at least 8 wells were used. As a blank solution 100 μL RPMI 1640 medium with 10 μL MTT stock and 100 μL 5% formic acid in 2-propanol was used. Data processing and statistics: The cytotoxicity assays were carried out in 8 separate experiments. The MTT data were fitted to sigmoidal concentration–response curves and the corresponding IC50 values were calculated using non-linear regression analysis (GraphPad Prizm software package). Statistical processing exploited Student's t-test with p≤0.05 set as significance level. Acknowledgments - This study was supported by Grant 3/2013 from the Medical Science Council at the Medical University of Sofia. The authors are thankful to Dr Daniel Bücherl and Dr Petr Jirásek, University of Regensburg, Germany for measuring NMR, MS and OR.

References [1] [2] [3]

[4] [5] [6]

[7] [8]

[9]

Crockett SL, Robson NKB. (2011) Taxonomy and chemotaxonomy of the genus Hypericum. Medicinal and Aromatic Plant Science and Biotechnology, 5(SI1), 1-13. (a) Kitanov G. (2000) The dynamics and content of hypericins in Hypericum perforatum L. and Hypericum maculatum Crantz. growing in Bulgaria. Acta Pharmaceutica, 50, 65-68; (b) Mártonfi P, Repčák M, Mártonfiová L. (2006) Secondary metabolites during ontogenetic phase of reproductive structures in Hypericum maculatum. Biologia, 61, 473-478. (a) Kitanov G. (2001) Hypericin and pseudohypericin in some Hypericum species. Biochemical Systematics and Ecology, 29, 171-178; (b) Kitanov G, Blinova K. (1987) Modern state of the chemical study of species of the genus Hypericum. Chemistry of Natural Compounds, 23, 151–166; (c) Kitanov G, Nikolov N. (1991) Phenolic constituents of Hypericum maculatum. Fitoterapia, 62, 92-93; (d) Arends P. (1969) Maculatoxanthone, a new pyranoxanthone from Hypericum maculatum. Tetrahedron Letters, 55, 4893-4896; (e) Nielsen H, Arends P. (1978) Structure of the xanthone lignoid kielcorin. Phytochemistry, 17, 2040-2041; (f) Kitanov G, Nedialkov P. (1998) Mangiferin and isomangiferin in some Hypericum species. Biochemical Systematics and Ecology, 26, 647-653; (g) Zheleva-Dimitrova D, Nedialkov P, Girreser U, Kitanov G. (2012) Benzophenones and flavonoids from Hypericum maculatum and their antioxidant activities. Natural Products Research, 26, 1576-1583. Zhang J-J, Yang X-W, Ma J-Z, Liu X, Yang L-X, Yang S-C, Xu G. (2014) Hypercohones D–G, new polycyclic polyprenylated acylphloroglucinol type natural products from Hypericum cohaerens. Natural Products and Bioprospecting, 4, 73-79. (a) Grossman RB, Jacobs H. (2000) On the structures of plukenetiones B, D, and E and their relationships to other polycyclic polyprenylated acylphloroglucinols. Tetrahedron Letters, 41, 5165-5169; (b) Ciochina R, Grossman RB. (2006) Polycyclic polyprenylated acylphloroglucinols. Chemical Reviews, 106, 3963-3986. (a) Porzel A, Farag MA, Mülbradt J, Wessjohann LA. (2014) Metabolite profiling and fingerprinting of Hypericum species: a comparison of MS and NMR metabolomics. Metabolomics, 10, 574-588; (b) Mitsopoulou KP, Vidali VP, Maranti A, Couladouros EA. (2015) Isolation and structure elucidation of hyperibine J [revised structure of adhyperfirin (7-deprenyl-13-methylhyperforin)]: Synthesis of hyperibone J. European Journal of Organic Chemistry, 2015, 287–290. An T-Y, Shan M-D, Hu L-H, Liu S-J, Chen Z-L. (2002) Polyprenylated phloroglucinol derivatives from Hypericum erectum. Phytochemistry, 59, 395-398. (a) Schmidt S, Jürgenliemk G, Skaltsa H, Heilmann J. (2012) Phloroglucinol derivatives from Hypericum empetrifolium with antiproliferative activity on endothelial cells. Phytochemistry, 77, 218-225; (b) Schmidt S, Jürgenliemk G, Schmidt TJ, Skaltsa H, Heilmann J. (2012) Bi-, tri-, and polycyclic acylphloroglucinols from Hypericum empetrifolium. Journal of Natural Products, 75, 1697-1705; (c) Crockett SL, Wenzig E-M, Kunert O, Bauer R. (2008) Anti-inflammatory phloroglucinol derivatives from Hypericum empetrifolium. Phytochemistry Letters, 1, 37-43. (a) Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55-63; (b) Konstantinov SM, Eibl H, Berger MR. (1999) BCR-ABL influences the antileukaemic efficacy of alkylphosphocholines. British Journal of Haematology, 107, 365-374.