Fitoterapia 94 (2014) 70–76

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Cytotoxic activity of lignans from Justicia procumbens Hong Jin a, Hai-Long Yin b, Shi-Jun Liu a, Li Chen a, Ying Tian a, Bin Li a, Qiong Wang a,⁎, Jun-Xing Dong a,⁎ a b

Beijing Institute of Radiation Medicine, Beijing 100850, China Beijing University of Technology, Beijing 100124, China

a r t i c l e

i n f o

Article history: Received 13 December 2013 Accepted in revised form 26 January 2014 Available online 5 February 2014 Keywords: Justicia procumbens Lignans Cytotoxic activity Structure–activity relationship

a b s t r a c t Three new lignans, Pronaphthalide A (1), Procumbiene (2), and Procumbenoside J (3), along with a novel natural product Juspurpudin (4), and twelve other known lignans were isolated from Justicia procumbens. The structures of the new compounds were elucidated by extensive spectroscopic analyses and the data of 3 provided insight into the conformational equilibria existing in it. All compounds were evaluated for their in vitro cytotoxic activity against Human LoVo and BGC-823 cell lines except for compound 2, and eight of them were found to possess potent cytotoxicity. The structure–activity relationship (SAR) analysis revealed that (i) the parent structure of 2-carbonyl arylnaphthalide lactone attached with 6 and 7-OMe was the essential element; (ii) the polarity of substituents on C-4 might significantly affect the activity; (iii) a proper cyclic lipophilic group at the C-3″ and C-5″ of apiofuranose on C-4 might enhance the activity, which could optimize the application of 3 similar to VP-16. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The genus Justicia (Acanthaceae) consists of about six hundred species, and a number of lignans have been isolated from different species, many of which exhibit diverse biological activities such as cytotoxicity, antibacterial, insecticidal, antiplatelet, antiangiogenic, antiinflammatory, antiviral, central nervous system depression and stimulation properties, and among these, the significant cytotoxic activity of lignans appeals a surge of pharmacologists' interests in the development of novel compounds for cancer therapy. The whole plant of Justicia procumbens L. (Acanthaceae) mainly distributed in South China and Taiwan has long been used in folk medicine for the treatment of fever, pain, and cancer in China [1]. Several arylnaphthalide lignans have been reported as constituents of this plant [2–5]. These lignans were shown to be cytotoxic towards several cancer cell lines [6]. In a continued search for novel cytotoxic constituents, three new lignans named Pronaphthalide A (1), Procumbiene (2), and Procumbenoside J (3), along with a novel natural product, Juspurpudin (4), and twelve known lignans (5–16) (Fig. 1) identified as 5′-methoxy

⁎ Corresponding authors. Tel.: +86 10 66931314; fax: +86 10 68164257. E-mail address: [email protected] (J.-X. Dong). 0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2014.01.025

retrochinensin (5) [7], Justicidin A (6) [3], Justicidin B (7) [3], 6′-hydroxyl justicidin A (8) [8], 6′-hydroxyl justicidin B (9) [9], Tuberculatin (10) [10], Diphyllin (11) [5], Taiwanin C (12) [8], Justicidin C (13) [4], Pinoresinol (14) [11], (−)-Syringaresinol (15) [12], and Rostellulin A (16) [13] by comparison of their NMR spectral data with the published literature data were isolated from an ethanol extract of the entire plant of J. procumbens. Herein, we described the isolation, structural elucidation, cytotoxicity of these lignans, and the SAR revealed. The result of MTT assay showed that two new arylnaphthalide lignans 1 and 3 and six known compounds 6–11 exhibited potent cytotoxic activity against sensitive Human LoVo and BGC-823 cell lines in vitro (Table 3) with IC50 values in the range of 0.03– 10.0 μM, but other compounds were inactive. 2. Experimental 2.1. General experimental procedures IR spectra were recorded on the Bio-Rad FTS-65A spectrophotometer. UV spectra were recorded using the UV-2500PC spectrometer (Shimadzu, Japan). The CD spectrum was obtained on a JASCO J-815 spectrometer. Optical rotations were measured with a Perkin-Elmer 343 polarimeter. 1H and 13C NMR spectra

H. Jin et al. / Fitoterapia 94 (2014) 70–76

71

Fig. 1. Structures of compounds 1–16.

were obtained on a Bruker ECA-400 MHz and VarianUNITY INOVA 600, and the chemical shifts were given on δ (ppm) scale with TMS as an internal standard. The HR-ESI–MS spectra were measured on a 9.4 TQ-FT-MS Apex Qe (Bruker Co. Billerica, MA, US). ESI–MS spectra were measured on a Finnigan LCQDECA spectrometer. Silica gel (60–100 mesh, 200–300 mesh, Qingdao Marine Chemical Group Co., China) and Sephadex LH-20 (Pharmacia, Sweden) were employed for column chromatography. TLC was carried out using silica gel 60 (N230 mesh, Qingdao Marine Chemical Group Co.) and GF254 plates precoated with silica gel 60. Spots on TLC were visually observed under UV light and/or by spraying with anisaldehyde–H2SO4 reagent followed by heating. 2.2. Plant materials Whole plants of J. procumbens were collected in Henan province, China, in March 2011, and identified by Dr. Bin Li (Beijing Institute of Radiation Medicine). A voucher specimen (no. 2011-0702) was on deposit at Department of Medicinal Chemistry, Beijing Institute of Radiation Medicine. 2.3. Extraction and isolation The air-dried whole plant of J. procumbens (50 kg) was chipped and extracted with 60% EtOH for three times under reflux. After filtration and evaporation of the EtOH at

reduced pressure, the residues (2.1 kg) were suspended in water and then partitioned with petroleum ether, CHCl3 and n-BuOH successively. The CHCl3 extract (200 g) was subjected to silica gel column chromatography (CC) (8.5 × 120 cm, 200–300 mesh, 3.0 kg) eluted with a gradient solvent of CHCl3–MeOH (from 100:1 to 1:1) to yield 11 fractions (A–K). Fraction C was chromatographed on silica gel CC eluted with CHCl3–MeOH (from 25:1 to 10:1) to yield sixteen fractions, C1–C16. Fractions C4–C6 were purified by Sephadex LH-20 (CHCl3–MeOH, 1:1) to obtain compound 5 (16 mg). Fraction E was subjected to silica gel CC eluted with a gradient solvent of CHCl3–MeOH (from 20:1 to 2:1) to give ten fractions, E1–E10. Fractions E3–E4 were subjected to repeated silica gel CC eluted with petroleum ether–EtoAc (8:1) to obtain compounds 7 (17 mg) and 12 (8 mg). Fractions E6–E7 were chromatographed on silica gel CC repeatedly eluted with petroleum–EtoAc (5:1) to obtain compounds 6 (1.3 g) and 13 (24 mg). Fraction F was chromatographed by Sephadex LH-20 (CHCl3–MeOH, 1:1) and recrystallized to obtain compound 16 (17 mg). Fraction G was applied to silica gel CC eluted with a gradient solvent of hexane–EtOAc (10:1–2:1) to yield fifteen fractions, G1–G15. Fraction G6 was chromatographed by Sephadex LH-20 (MeOH) to obtain compound 2 (6 mg). Fraction G9 was purified by Sephadex LH-20 (CHCl3–MeOH, 1:1 and MeOH) to obtain compound 4 (60 mg). Fractions G10– G11 were applied to Sephadex LH-20 column eluted with CHCl3–MeOH (1:1) and MeOH, respectively, then followed by

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recrystallization to obtain compounds 3 (10 mg), 9 (14 mg) and 11 (16 mg). Fraction G12 was purified by Sephadex LH-20 (CHCl3–MeOH, 1:1 and MeOH) to obtain compounds 1 (9 mg), 8 (7 mg) and 15 (115 mg). Fraction G14 was chromatographed by Sephadex LH-20 (MeOH) to obtain compound 14 (16 mg). Fraction H was chromatographed by Sephadex LH-20 (CHCl3– MeOH, 1:1) to remove pigments, and the residue (120 mg) was subjected to silica gel CC eluted with CHCl3–MeOH (from 15:1 to 10:1) to obtain compound 10 (40 mg). 2.3.1. Pronaphthalide A (1) C22H20O7, white amorphous powder; [α]20 D + 27.4° (c 0.050, MeOH); UV [MeOH] λmax (log ε) 364 (3.65), 322 (3.65) nm; IR (KBr) νmax 3491 (OH), 1759, 1592 cm−1; HR-ESI–MS m/z 397.1286 [M + H]+ (calcd for C22H21O7, 397.1282); 1H and 13 C NMR (Acetone-d6) spectroscopic data, see Table 1. 2.3.2. Procumbiene (2) C20H16O7, colorless crystal; [α]20 D + 20.0° (c 0.030, MeOH); CD (MeOH) 223 (Δε + 2.19), 292 (Δε + 0.51) nm; UV [MeOH] λmax (log ε) 256 (3.88), 275 (3.68), 288 (3.74) nm; IR (KBr) νmax 3410 (OH), 1734 and 929 cm−1; HR-ESI–MS m/z 369.0974 [M + H]+ (calcd for C20H17O7, 369.0969); 1H and 13C NMR (CDCl3) spectroscopic data, see Table 1. 2.3.3. Procumbenoside J (3) C29H28O11, yellowish amorphous powder; [α]20 D − 14.7° (c 0.075, MeOH); UV [MeOH] λmax (log ε) 361 (3.42), 241 (3.17) nm; IR (KBr) νmax 3442 (OH), 1758 and 1620 cm−1; HR-ESI–MS m/z 553.1704 [M + H]+ (calcd for C29H29O11, 553.1704); 1H and 13 C NMR (CD3OD) spectroscopic data, see Table 2. 2.3.4. Juspurpudin (4) C20H16O7, yellow amorphous powder; [α]20 D − 1.5° (c 0.065, MeOH); UV [MeOH] λmax (log ε) 289 (3.95) nm; IR (KBr) νmax 3421 (OH), 1732 and 929 cm−1; HR-ESI–MS m/z 407.0516 [M + K]+ (calcd for C20H16KO7, 407.0528); 1H and 13C NMR (CD3OD), see Table 1. 2.4. Cell cultures Human LoVo cell line from American Type Culture Collection (ATCC, Rockville, MD) and BGC-823 cell line from Cancer Institute and Hospital of Chinese Academy of Medical Sciences were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal calf serum (Gibco, USA), penicillin G 100 units·mL−1 and streptomycin 100 μg·mL−1, at 37 °C under 5% CO2. 2.5. Cell viability assay Cells were plated at a density of 2 × 104 cells·mL−1 on 96-well plates and treated with different concentrations of the test compounds for 4 days. Cytotoxicity was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [14]. After addition of 10 μL MTT solution (5 mg/mL), cells were incubated at 37 °C for 4 h. After adding 150 μL DMSO, cells were shaken to mix thoroughly. The absorbance of each well was measured at 540 nm in a Multiscan photometer. The IC50 values of the effective compounds were fitted by Origin software and listed in Table 3.

Table 1 1 H and 13C NMR data for compounds 1, 2, and 4 (400 MHz for 1H, 100 MHz for 13C, TMS, δ ppm). Position 1a 1 1a 2 3 4 4a 5 6 7 8 9 10 1 2 3 4 5 6 4-OMe 6-OMe 7-OMe 4-OMe 3-OH 2b 1 2 3 4 5 6 7a 7b α C_O 1 2 3 4 5 6 7 β γa γb 3′-OCH2O-4′ 3-OCH2O-4 4c 2 3 3a 4 4a 5a 5b 1′ 2′ 3′ 4′ 5′ 6′ 7′ a b c

δH (J in Hz)

7.61, s

7.11, s 5.65, s

6.84, d (2.0)

7.06, 6.77, 4.21, 4.01, 3.71, 3.94, 7.71,

d ( 8.1) dd (8.1, 2.0) s s s s s

6.67, s

6.72, 6.64, 3.64, 3.53,

d d d d

(7.8) (7.8) (14.6) (14.6)

6.70, d (2.0)

6.77, d (7.8) 6.72, dd (7.8, 2.0) 5.67, s 4.74, 4.85, 5.98, 5.92,

d (17.6) d (17.6) d (1.4) s

5.14, s 4.08, s 4.88, br s 4.92, br s 6.62, d (1.7)

6.73, d (8.1) 6.57, dd (8.1, 1.7) 5.90, s

Spectra were recorded in Acetone-d6. Spectra were recorded in CDCl3. Spectra were recorded in CD3OD.

δC 134.9 126.6 120.1 125.6 148.6 131.2 101.5 152.7 151.4 107.1 67.1 169.6 129.1 118.3 147.0 148.1 111.8 122.5 60.0 56.2 55.7 56.1

131.4 109.2 148.2 146.3 108.7 121.6 29.4 126.8 174.7 134.0 106.7 148.5 147.9 108.4 120.0 70.6 161.0 69.7 101.6 101.1

175.8 127.8 47.1 164.7 57.7 71.7 135.2 109.7 148.9 147.5 108.5 122.5 102.0

H. Jin et al. / Fitoterapia 94 (2014) 70–76 Table 2 1 H and 13C NMR data for compound 3 in CD3OD. (400 MHz for 1H, 100 MHz for 13C, TMS, δ ppm). Position 1 2 3 4 5 6 7 8 9 10 11a 11b 12 6-OMe 7-OMe 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4a″ 4b″ 5b″ 5a″ 6″ 7″ 8″ a

δH (J in Hz)

7.54a, s

7.02a, s

5.51a, d (16.8) 5.45a, d (16.8) 3.99, s 3.70, s 6.76a, s

6.93, d (8.1) 6.74a, d (8.1) 6.05a, s 5.42a, d (2.8) 4.30, d (2.8) 4.25, d (9.5) 4.10a, d (9.5) 4.16, d (9.0) 4.29, d (9.0) 1.43, s 1.47, s

Table 3 Cytotoxic activity of compounds against LoVo and BGC-823 cell lines in vitro. Compound

δC 137.0 130.4 120.0 146.0 101.7 153.3 151.7 107.1 128.2 131.8 68.7 172.0 56.5 56.0 129.9 111.9a 149.0 149.0 109.0 124.8a 102.6 111.9 79.4 87.1 75.9 70.5 111.9 26.3 26.9

Existed in pairs at NMR room temperature (20 °C).

2.6. Statistical analysis Values were expressed as mean ± SD. Statistical analyses were performed using the Student's t-test. Differences were considered significant when associated with a probability of 5% or less (p ≤ 0.05). 3. Results and discussion Compound 1 was obtained as white amorphous powder. Its molecular formula was assigned as C22H20O7 by HR-ESI/MS (m/z 397.1286 [M + H]+, calcd. for C22H21O7, 397.1282). Its IR spectrum showed absorption bands for a hydroxy group (3491 cm−1), an aromatic γ-lactone (1759 cm−1), and an aromatic ring (1592 cm− 1). The 1H NMR spectrum of 1 (Table 1) showed characteristic arylnaphthalide signals ascribable to five aromatic protons, a phenolic proton, four methoxy groups, and a lactone methylene group. Five aromatic protons included two signals at δH 7.61 (1H, s, H-5) and 7.11 (1H, s, H-8); three others showed an ABX type substitution pattern at δH 6.84 (1H, d, J = 2.0 Hz, H-2′), 7.06 (1H, d, J = 8.1 Hz, H-5′) and 6.77 (1H, dd, J = 8.1, 2.0 Hz, H-6′); a hydroxy group at δH 7.71 (1H, s, OH-3′) could be exchangeable with D2O; and four single methoxyl groups showed signals at δH 4.21 (3H, s, OCH3-4), 4.01 (3H, s, OCH3-6), 3.71 (3H, s, OCH3-7), and 3.94 (3H, s, OCH3-4′). These signals were suggestive of the presence of a substituted

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IC50 (μM) LoVo

1 3 6 7 JR6 (8) 9 10 Diphyllin (11) 12

0.135 0.527 0.039 6.081 0.148 2.356 / 8.120 NE

BGC-823 ± ± ± ± ± ±

0.023⁎⁎ 0.047⁎⁎ 0.004⁎⁎ 6.136 0.021⁎⁎ 0.186⁎⁎

± 1.679

0.115 0.204 0.039 0.179 0.417 4.680 1.635 8.079 N20

± ± ± ± ± ± ± ±

0.035⁎ 0.055 0.009⁎⁎ 0.056 0.139 1.860 0.697 2.018

/ — not determined and NE — no effect. Compound 2 was unavailable in sufficient quantity for the cytotoxic activity assay. Other inactive compounds were not listed in the table. Values were mean ± SD. ⁎ p b 0.05 as compared with the respective positive control value (Diphyllin and JR6 were the positive controls for LoVo and BGC-823 cell lines, respectively). ⁎⁎ p b 0.01 as compared with the respective positive control value (Diphyllin and JR6 were the positive controls for LoVo and BGC-823 cell lines, respectively).

arylnaphthalene unit in compound 1, and the signal due to the γ-lactone methylene protons appearing at δH 5.65 (2H, s, H-9) indicated that it was a 1-aryl-2,3-naphthalide lignan [3]. In the 2D NOESY spectrum of 1, there were correlations between H-9 and the methoxy proton signal at δH 4.21, between H-5 and the methoxy proton signal at δH 4.01, between H-8 and the methoxy proton signal at δH 3.71, and between H-5′ and the methoxy proton signal at δH 3.94. These data clearly indicated that the four methoxy signals at δH 4.21, 4.01, 3.71 and 3.94 were assigned to C-4, C-6, C-7, and C-4′, respectively. The 13C NMR experiment displayed 22 signals, and the data (Table 1) were assigned unambiguously by performing the DEPT, HMBC and HMQC experiments. The downfield signal at δC 169.6 was due to the lactone functionality while the signal at δC 67.1 could be attributed to the methylene group adjacent to the carbonyl of the lactone functionality. Furthermore, the HMBC correlations showed cross-peaks between C-4′ (δC 148.1) and OCH3-4′, H-5′, and H-6′, which also indicated that this methoxy group was attached to C-4′. Key NOESY correlations were shown in Fig. 3 and HMBC correlations in Fig. 2. Consequently, compound 1 was characterized as 1-(3′-hydroxy-4′-methoxy)-phenyl-4,6,7trimethoxy-2,3-naphthalide, and it was named Pronaphthalide A (1). Compound 2 was obtained as colorless crystal. Its molecular formula was assigned as C20H16O7 by HR-ESI/MS (m/z 369.0974 [M + H]+, calcd. for C20H17O7, 369.0969). The IR spectrum showed absorption bands at νmax 3410 (OH), 1734 and 929 cm− 1. Total 15 protons were observed in the 1H NMR spectrum (Table 1), and it displayed 6 signals for two phenyl units with the ABX system at δH 6.67 (1H, s, H-2), 6.72 (1H, d, J = 7.8 Hz, H-5), 6.64 (1H, d, J = 7.8 Hz, H-6), 6.70 (1H, d, J = 2.0 Hz, H-2′), 6.77(1H, d, J = 7.8 Hz, H-5′) and 6.72 (1H, dd, J = 7.8, 2.0 Hz, H-6′). The signals at δH 3.53 (1H, d, J = 14.6 Hz, Ha-7) and 3.64 (1H, d, J = 14.6 Hz, Hb-7) indicated the presence of a benzylic group; besides, those at δH 4.74 (1H, d, J = 17.6 Hz, Ha-γ) and 4.85 (1H, d, J = 17.6 Hz, Hb-γ) indicated an acyloxymethylene group. The remaining signal of a methine proton at δH 5.67 (1H, s, H-7′) might be due to the

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H. Jin et al. / Fitoterapia 94 (2014) 70–76

Fig. 2. Key HMBC correlations of compounds 1–4.

attachment of the hydroxy group. The 13C NMR spectrum displayed 20 signals and showed the α-C peak at low field as in other 2-buten-4-olides [15]. Key HMBC (Fig. 2) correlations between H-7 and δC 174.7 (C_O), H-7′ and δC 69.7 (C-γ) also permitted the attachment of the hydroxy group to C-7′. Moreover, the HMBC correlations between δC 126.8 (C-α) and 161.0 (C-β) and H-7′, H-7 as well as H-γ suggested the presence of a α,β-unsaturated-γ-lactone. In the 1H NMR spectrum two methylenedioxy groups were observed: one appeared at δH 5.92 (2H, s, 3-OCH2O-4) as expected, while the other located at δH 5.98 (2H, d, J = 1.4 Hz, 3′-OCH2O-4′) with the slight doubling that might be due to atropisomerism, as the hydroxy group on the C-7′ position could hinder the rotation of the aryl along the C-7′/C-1′ bond. Its 13C NMR data were assigned unambiguously by the HMBC and HMQC correlations and compared with guayadequiene [16]. The CD spectrum of 2 showed positive absorption peaks at 223 and 292 nm, and unfortunately, a large amount of possible conformations of this compound did not allow the elucidation of its absolute configuration. Consequently, Procumbiene (2) was characterized as α-piperonyl methyl-β-(7′-hydroxy)-piperonylmethyl-α,βunsaturated-γ-butyrolactone. Compound 3 was obtained as yellowish amorphous powder. Its molecular formula was assigned as C29H28O11 by HR-ESI/MS (m/z 553.1704 [M + H]+, calcd. for C29H29O11, 553.1704), the positive-ion ESI–MS revealed a significant fragment at m/z 381.17 [M-172 + H]+, due to the cleavage of one substituent unit. In the IR spectrum, absorptions for an aromatic γ-lactone at 1758 cm−1, a hydroxyl at 3442 cm−1, an aromatic ring at 1620 cm−1 and a methylenedioxy group at 931 cm−1 were

Fig. 3. Key NOESY correlations of compounds 1 and 3.

observed. In combination of the NMR spectra and mass spectrometry, compound 3 could be logically disconnected into two fragments—Diphyllin and a sugar residue. The 1H and 13C NMR spectra of 3 (Table 2) showed an anomeric proton signal at δH 5.42⁎ (1H, d, J = 2.8 Hz, H-1″) and an anomeric carbon signal at δC 111.9 correspondingly. In addition, the 1H–1H COSY experiment gave only the connection of the anomeric proton and the vicinal proton at δH 4.30 (1H, d, J = 2.8 Hz, H-2″); the two protons not being coupled further with any other protons. The protons of one of these two methylene groups resonated at δH 4.25 (1H, d, J = 9.5 Hz, Ha-4″) and 4.10 (1H, d, J = 9.5 Hz, Hb-4″), and from the COSY spectrum these two protons were coupled to each other. The protons of one of the other two methylene groups resonated as two doublets at δH 4.29 (1H, d, J = 9.0 Hz, Ha-5″) and 4.16 (1H, d, J = 9.0 Hz, Hb-5″), and these two protons were also not coupled to any other protons, as evident from the COSY spectrum. The above data were typical for apiose furanoside. The NMR data of apiose furanoside in 3 were compatible with the presence of β-D-apiofuranose observed in Tuberculatin [10]. However, signals of H-5″ moved to lower field. Meanwhile, in a high field at δH 1.43 (3H, s, H-7″) and 1.47 (3H, s, H-8″) two methyl groups were observed. The 13C NMR experiment displayed 29 distinctive signals due to aglycon Diphyllin and a sugar residue, simultaneously, and key HMBC cross-peaks between H-5″and C-6″ (δC 111.9) and between H-7″, H-8″ and C-6″, together with the 2D NMR experiments (1H–1H COSY, NOESY, HMQC and HMBC) suggested the presence of the 3″,5″-acetonide-4-O-β-D-apiofuranosyl unit. The linkage was also established on the basis of the carbon resonances at δC 87.5 (C-3″) and 70.5 (C-5″), which were downfield by comparison with the corresponding shifts of Tuberculatin [10] at δC 79.5 (C-3″) and 64.2 (C-5″) both in CD3OD. The 1H NMR spectrum also showed characteristic signals of Diphyllin [10], while some existed as pairs of signals due to the equilibrium between two conformational isomers resulting from the slow rotation of the sugar unit at room temperature around the glucosidic linkage with the aglycone [17], which contained five aromatic protons at δH 7.54⁎ (1H, s, H-5), 7.02⁎ (1H, s, H-8), 6.93 (1H, d, J = 8.1 Hz, H-5′), 6.76⁎ (1H, s, H-2′), and 6.74⁎ (1H, d, J = 8.1 Hz, H-6′) and two methylenedioxy protons at δH 6.05⁎ (2H, s, H-7′), together with two methoxy groups at δH 3.99 (3H, s, OMe-6) and 3.70 (3H, s, OMe-7). Moreover, the γ-lactone methylene protons at δH 5.51⁎ (1H, d, J = 16.8 Hz, Ha-11) and 5.45⁎ (1H, d, J = 16.8 Hz, Hb-11) were observed to show as an AB system while they appeared as an A2 system normally, which was attributed to the influence of the

H. Jin et al. / Fitoterapia 94 (2014) 70–76

pyranosyl moiety by Al-Abed et al. [17]; however, our test bore witness to the different opinion that the acetonide apiofuranose moiety might also be the reason. The location of the acetonide apiofuranose was deduced from the HMBC (Fig. 2) experiment which showed diagnostic long-range correlations between the anomeric proton signal at δH 5.42⁎ (1H, d, J = 2.8 Hz, H-1″) and the carbon resonance at δC 146.0 (C-4). Consequently, the structure of 3 was identified as 3″,5″-acetonide-4-O-β-D-apiofuranosyldiphyllin, and it was named Procumbenoside J. We first supplemented the data of compound 4 — a novel natural product, which was an unusual secolignan. It was obtained as yellow amorphous powder and its glucoside was isolated from Justicia purpurea [18]. The molecular formula was assigned as C20H16O7 by HR-ESI/MS (m/z 407.0516 [M + K]+, calcd. for C20H16K1O7, 407.0528). The 1H NMR spectrum (Table 1) showed 15 signals in CD3OD, which contained two symmetrically substituted phenyl units with an ABX system at δH 6.73 (2H, d, J = 8.1 Hz, H-5′), 6.62 (2H, d, J = 1.7 Hz, H-2′) and 6.57 (2H, dd, J = 8.1, 1.7 Hz, H-6′), two methylene groups at δH 4.92 (1H, br s, Ha-5), 4.88 (1H, br s, Hb-5) and 4.08 (2H, s, H-4a), a benzylic methine proton at δH 5.14 (1H, s, H-3a), and two equivalent methylenedioxy groups at δH 5.90 (4H, s). The 13 C NMR spectrum showed 13 signals for 20 carbons, of which six aromatic signals and one methylenedioxy carbon were attributed to two symmetrically trisubstituted phenyl units, and the remaining six carbon signals could be derived for a carbonyl carbon, a benzylic methine, two methylenes, and two quaternary carbons. In the HMBC spectrum, the key connectivities observed from H-3a to C-2′ (δC 109.7)/C-6′ (δC 122.5) as well as to C-2 (δC 175.8) and C-4 (δC 164.7) indicated a key partial secolignan skeleton for compound 4. Full assignments of the 1H and 13C NMR signals were concluded from the detailed analysis of COSY, HMQC and HMBC spectra. Key HMBC correlations were shown in Fig. 2. Compound 4 belongs to a rare type of secolignans, of which only a few similar compounds have been previously reported [19]. It could be interesting to suggest the biosynthesis of compound 4 from a typical lignan derivative Taiwanin C (12) as shown in Scheme 1. The lignan 12 should be transformed to the intermediate which shared a structural similarity with Podophyllotoxin by the process of ring B reduction and benzylic oxidation. The following C\C bond cleavage in the intermediate and forming of α,β-unsaturated-γ-lactone would lead to the ring opened secolignan derivative 4 (Scheme 1). In this paper, we mainly reported the isolation and identification of sixteen lignans from J. procumbens, all compounds isolated except for 2 were tested for their

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potential cytotoxic activity against Human LoVo and BGC-823 cell lines according to the IC50 values, and eight of them were found to possess potent cytotoxicity. In the assay, Diphyllin (11) and 6′-hydroxyl justicidin A (8)—JR6 of this type of compounds were chosen to be positive controls since their cytotoxicity against the Human LoVo and BGC-823 cell lines, respectively, had been reported in the literature previously [10,20], then the cytotoxicity of the compounds against the LoVo and BGC-823 cell lines was compared with the respective positive control group, and finally, the result was summarized in Table 3. The IC50 values of compounds 1, 3, 6, 8, and 9 were significantly lower than that of Diphyllin on LoVo cell line and the values of compounds 1 and 6 were significantly lower than that of JR6 on BGC-823 cell line. Finally, we clearly elucidated that the activity of lignans was closely relevant to the parent structure. The SAR showed that the parent structure of 2-carbonyl arylnaphthalide lactone was a key point to the activity, and this type of compounds was more effective than 3-carbonyl-type compounds 5 and 13, secolignan compound 4, ditetrahydrofurans compounds 14 and 15, and aryltetrahydronaphthalene compound 16. Furthermore, among the 2-carbonyl-type compounds, we also observed that 6 was significantly more active than 1 (p b 0.05) against both of the cell lines; however, in turn, 1 was significantly more active than all the others in Table 3, so we supposed that the methylenedioxy of the lower pendant phenyl ring might play an important role in the cytotoxicity to a certain degree but was not decisive. On the other hand, compound 12 showed no or weaker effect when compared with compound 7 (p b 0.05), which revealed that the 6 and 7-OMe were essential, but 6′-substituent on the lower pendant phenyl ring to some extent was unnecessary. Thus, the parent structure of 2-carbonyl arylnaphthalide lactone attached with 6 and 7-OMe was the essential element. In addition, the 2-carbonyl-type compounds 3, 6, 7 and 10 were significantly more active than Diphyllin (11) against the BGC-823 cell line (p b 0.01), which suggested that the lipid– water partition coefficient resulting from the different polarity of substituents on C-4 should significantly affect their activity. The methylation, deoxidation or suitable glycosylation of C-4 might be contributed to the activity, but in general, the methylation was superior. By the way, glycosylated compounds named Majidine, Arabelline and Patavine [10] were less active than Diphyllin against the LoVo cell line, as reported by Gabbriella Innocenti, which might be due to the steric bulk of the sugar moiety. Thus, the suitable bulk of sugar was also of great importance to the cytotoxic activity, and more than two

Scheme 1. Proposed biogenesis of Juspurpudin (4).

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substituent sugar moieties could significantly lead to the activity reduction. Besides, the 3″–5″cyclic acetonide apiofuranosyl unit on compound 3 made a big difference in the activity from compound 10 against both of the cell lines (p b 0.05), which might be due to the possibility that a small size lipophilic group masked on the 3″-OH and 5″-OH on compound 3 favored the activity similar to VP-16 developed by Sandoz. The result revealed that a moderate cyclic lipophilic group of sugar was very significant; therefore, that conclusion could provide some information about glycoside modification to natural products and should be interesting considerations for antitumor compound research. Above all, the SAR revealed that the functional groups at the essential parent structure of 2-carbonyl arylnaphthalide lactone strongly affect the activity and could be ranked according to their potency as 6 and 7-OMe N 4-OMe N 3″–5″ cyclic acetonide N 4-glycosylation N 3′-OCH2O-4′. In conclusion, our research provided new data of lignans, their cytotoxicity against Human LoVo and BGC-823 cell lines, and their SAR. Those results indicated that the lignans isolated from J. procumbens might serve as lead compounds with promising therapeutic antitumor agents and we believe that they had an interesting cytotoxic activity profile that should be studied further. Conflict of interest We declare that we have no conflict of interest. Acknowledgments The authors are really grateful to Mrs. Yan Xue, Mrs. Mei-feng Xu and Mrs. Yu-mei Zhao of the National Center of Biomedical Analysis for the measurements of the HR-ESI–MS and NMR spectra. References [1] Asano J, Chiba K, Tada M, Yoshii T. Antiviral activity of lignans. and their glycosides from Justicia procumbens. Phytochemistry 1996;42:713–7.

[2] Ogiwara M, Maeda T, Kawano N. The isolation of neojusticin from Justicia procumbens LINN. Chem Pharm Bull 1970;18:862–3. [3] Okigawa M, Maeda T, Kawano N. The isolation and structure of three new lignans from Justicia procumbens Linn. var. leucantha honda. Tetrahedron 1970;26:4301–5. [4] Ohta K, Munakata K. Justicidin C and D, the 1-methoxy-2,3naphthalide lignans, isolated from Justicia procumbens L. Tetrahedron Lett 1970;11:923–5. [5] Fukamiya N, Lee KH. Antitumor agents, 81. Justicidin-A and diphyllin, two cytotoxic principles from Justicia procumbens. J Nat Prod 1986;49:348–50. [6] Day Shiow-Hwa, Lin Yi-Chen, Lin Chun-Nan. Potent cytotoxic lignans from Justicia procumbens and their effects on nitric oxide and tumor necrosis factor-α production in mouse macrophages. J Nat Prod 2002;65:379–81. [7] Cow Christopher, Charlton James L. Antiviral activity of arylnaphthalene and aryldihydronaphthalene lignans. Can J Chem 2000;78:553–61. [8] Yang Meihua, Zhou Yuan. Complete assignments of 1H and 13C NMR data for seven arylnaphthalide lignans from Justicia procumbens. Magn Reson Chem 2006;44:727–30. [9] Yang Meihua, Chen Jianmin. A new lignan from the Jian-er syrup and its content determination by RP–HPLC. J Pharm Biomed Anal 2006;41:662–6. [10] Innocenti Gabbriella, Cappelletti Elsa Mariella. Patavine, a new arylnaphthalene lignan glycoside from shoot cultures of Haplophyllum patavinum. Chem Pharm Bull 2002;50:844–6. [11] Fonseca Sebastiao F, Ruved Edmundo A. Lignans of Araucaria angustifolia and 13 C NMR analysis of some phenyltetralin lignans. Phytochemistry 1979;18:1703–8. [12] Deyama T. The constituents of Eucommia ulmoides Oliv. Chem Pharm Bull 1983;31:2993. [13] Zhang Yongli, Cheng Yongxian. Antibacterial lignans and triterpenoids from Rostellularia procumbens. Planta Med 2007;73:1596–9. [14] Wang BJ, Won SJ, Yu ZR, Su CL. Free radical scavenging and apoptotic effects of Cordyceps sinensis fractionated by supercritical carbon dioxide. Food Chem Toxicol 2005;43:543–52. [15] Sheriha GM, Amer KMA. Lignans of haplophyllum tuberculatum. Phytochemistry 1984;23:151–3. [16] Gonzales AG, Estevez-Reyes R, Mato C. Three lignans from Bupleurum salicifolium. Phytochemistry 1990;29:1981–3. [17] Al-Abed Yousef, Sabri Salim, Zarga Musa Abu. Chemical constituents of the Flora of Jordan, Part V-B. Three new arylnaphthalene lignan glucosides from Haplophyllum buxbaumii. J Nat Prod 1990;53:1152–61. [18] Kavitha Jakka, Subbaraju. Juspurpurin, an unusual secolignan glycoside from Justicia purpurea. J Nat Prod 2003;66:1113–5. [19] Jian-lin Wu, Sakai Jun-ichi, Ando Masayoshi. Bioactive secolignans from Peperomia dindygulensis. J Nat Prod 2006;69:790–4. [20] He Xiao-Li, Yang Mei-Hua, Bi Ming-Gang. JR6, a new compound isolated from Justicia procumbens, induces apoptosis in human bladder cancer EJ cells through caspase-dependent pathway. J Ethnopharmacol 2012;144:284–92.

Cytotoxic activity of lignans from Justicia procumbens.

Three new lignans, Pronaphthalide A (1), Procumbiene (2), and Procumbenoside J (3), along with a novel natural product Juspurpudin (4), and twelve oth...
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