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A new cytotoxic 1-azaanthraquinone from the stems of Goniothalamus laoticus
Fitoterapia 81 (2010) 894–896
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A new cytotoxic 1-azaanthraquinone from the stems of Goniothalamus laoticus Santi Tip-pyang a,⁎, Yawistha Limpipatwattana a, Suttira Khumkratok b, Pongpan Siripong c, Jirapast Sichaem a a b c
Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand Natural Products Research Section, National Cancer Institute, Bangkok 10400, Thailand
a r t i c l e
i n f o
Article history: Received 2 September 2009 Accepted in revised form 26 May 2010 Available online 8 June 2010 Keywords: Goniothalamus laoticus Cytotoxicity 1-azaanthraquinone Laoticuzanone A
a b s t r a c t A new 1-azaanthraquinone, named laoticuzanone A (1), and a synthetically known 3-methyl1H-1-azaanthracene-2,9,10-trione (2), together with four known compounds, Griffithazanone A (3), methyl sinapate (4), methyl p-coumarate (5), and p-hydroxyphenylethyl p-coumarate (6) were isolated from the stems of Goniothalamus laoticus. Their structures were established on the basis of spectroscopic data as well as comparisons with the previous literature data. Compound 1 showed the highest cytotoxicity against KB and HeLa cells with IC50 values of 0.68 and 0.50 μg/ml, respectively. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The genus Goniothalamus (Blume) Hook. f & Thomson belongs to the Annonaceae family and comprises 9 species in Thailand. G. laoticus (Finet & Gagnep) Bân, locally known as “Khao lam dong” is a rare ornamental plant, 4–6 m in height, distributed mainly in Northeastern of Thailand. Ethnobotanical uses of several species of the genus Goniothalamus are well known that many of these plants have provided bioactive alkaloids [1], acetogenins [2], styryl-lactones [3,4], flavonoids [5], and azaanthraquinones [6]. Several compounds isolated from the plants in this genus showed cytotoxic activity against a number of human cancer cell lines [6,7]. In addition, a water decoction derived from its stem bark is being used traditionally as a tonic and a febrifuge by the local people [8]. Our previous investigation reported the isolation of a flavonoid, styryllactone derivatives and naphthoquinone from this plant [9].
⁎ Corresponding author. Tel.: + 66 02 2187625; fax: + 66 02 2187598. E-mail address: [email protected] (S. Tip-pyang). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.05.019
We herein report the isolation and structure elucidation of a new antitumor 1-azaanthraquinone, (3R),5-dihydroxy(4S)-methyl-3,4-dihydro-2,9,10-(2H)-1-azaanthracenetrione (1), together with the antitumor activity of all isolated compounds (1–6) from the stems of G. laoticus. 2. Experimental 2.1. General HPLC: Waters® 600 controller equipped with a Waters® 2996 photodiode array detector. Melting points; Fisher-Johns Melting Point Apparatus. IR: Nicolet 6700 FT-IR spectrometer with a mercury–cadmium–telluride (MCT) detector. UV: UV2552PC UV–Vis spectrometer. Optical rotations: Jasco P-1010 polarimeter. NMR: Varian model Mercury+ 400 spectrometer operated at 400 MHz for 1H and 100 MHz for 13C nuclei. Adsorbents: Sephadex LH-20 and silica gel 60 Merck cat. No. 7729 and 7734 were used for column chromatography. Merck silica gel 60 F254 plates were used for TLC. ESIMS and HRESIMS: Mass spectrometer model VG TRIO 2000 and a Micromass LCT mass spectrometer.
S. Tip-pyang et al. / Fitoterapia 81 (2010) 894–896
2.2. Plant material The stems of G. laoticus were collected from Sakon Nakhon Province of Thailand in June, 2007 and identified by Ms. Suttira Khumkratok, a botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, where a voucher specimen (Khumkratok no. 84-08) is deposited. 2.3. Extraction and isolation Air-dried and powdered stems of G. laoticus (5.5 kg) were successively extracted in a soxhlet apparatus with CH2Cl2, EtOAc, and MeOH. The CH2Cl2 extract was concentrated under vacuum to yield 186.0 g of crude residue. This material was fractionated by vacuum liquid chromatography (VLC) over silica gel, using hexane, EtOAc and MeOH with increasing polarity, which was subjected to silica gel column eluted with a mixture of CH2Cl2/MeOH with increasing polarity to provide two fractions. Fraction 1 was further purified by Sephadex LH-20 column chromatography using 0.5:9.5 MeOH/CH2Cl2 as eluting solvent to afford 3-methyl1H-1-azaanthracene-2,9,10-trione (2, 5.2 mg) [10]. Fraction 2 was further purified by Sephadex LH-20 column chromatography, using 9.5:0.5 CH2Cl2/MeOH as eluting solvent, followed by HPLC, using ACN/H2O (8:2) as eluents to yield a new 1-azaanthraquinone, 3,5-dihydroxy-4-methyl-3,4-dihydro2,9,10-(2H)-1-azaanthracenetrione (1, 4.1 mg) and griffithazanone A (3, 3.5 mg) [11]. The methanolic extract (30 g) was similarly chromatographed on silica gel VLC using a stepwise gradient elution of MeOH and CH2Cl2, yielding three fractions. Fraction 1 was further fractionated by Sephadex LH-20 column using 100% MeOH as eluting solvent to give two fractions. Sephadex fraction 1 was further purification by preparative TLC [silica gel, CH2Cl2/MeOH (9.5:0.5)] to obtain methyl sinapate (4, 6.7 mg) [12]. Sephadex fraction 2 was further fractionated over silica gel CC, using CH2Cl2 and MeOH with increasing polarity to yield three fractions (S1–S3). Fraction S1 was further purified by Sephadex LH-20 column chromatography, using 9.5:0.5 CH2Cl2/MeOH as eluting solvent, followed by silica gel CC, using CH2Cl2/MeOH with increasing polarity to
895
afford methyl p-coumarate (5, 7.3 mg) [13]. Fraction S3 was crystallized in a mixture of CH2Cl2 and MeOH to obtain phydroxyphenylethyl p-coumarate (6, 6.2 mg) [14]. Compound 1 (Fig. 1) (3R),5-dihydroxy-(4S)-methyl-3,4dihydro-2,9,10-(2H)-1-azaanthracenetrione, brown amorphous powder; mp 208–209 °C; [α]26 + 119° (c 0.004, D MeOH); UV (MeOH) λmax (log ε): 255 (3.8), 295 (3.9) nm; positive ion ESIMS m/z: 273.59 [M + H]+; positive ion HRESIMS m/z: [M + H]+ 274.0704 (calcd for C14H11O5, 274.0715); 1H NMR (CD3COCD3, 400 MHz) and 13C NMR (CD3COCD3, 100 MHz) are shown in Table 1. 2.4. General preparation of MTPA ester [15] To a solution of 1 (1 mg) in two drops of pyridine was added (−)-MTPA chloride (50 μL), and the mixture was left at room temperature overnight. After addition of 1 ml of 1 M NaHCO3, the reaction mixture was extracted with CH2Cl2 (2 ml ×2), washed with brine, dried over anhydrous Na2SO4, and evaporated. The residue was purified on a short silica gel column (5% MeOH–CH2Cl2) to afford S-(−)-MTPA derivative (1a). The R-(+)-MTPA derivative (1b) was prepared in the same way. Selected ΔδHSR values: −0.029 (NH); +0.002 (4−CH3). 2.5. Cytotoxicity test Compounds 1–6 were subjected to in vitro cytotoxicity studies against human cervical carcinoma (HeLa) and human mouth epidermal carcinoma (KB) using the standard MTT colorimetric method [16]. 3. Result and discussion Compound 1 was obtained as brown amorphous powder with mp 208–209 °C. Its HRESIMS gave a positive molecular ion at m/z: [M + H]+ 274.0704 (calcd 274.0715), compatible with a molecular of C14H11O5. The IR spectrum confirmed this evidence by showing the quinine carbonyl and the lactam carbonyl absorptions at 1463 and 1632 cm−1, respectively. UV absorption bands (λmax = 255 and 295 nm) also confirmed the presence of the conjugated quinonoid moiety. The 1 H NMR spectrum of 1 in acetone-d6 showed three adjacent
Fig. 1. Structures of compounds 1–3.
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S. Tip-pyang et al. / Fitoterapia 81 (2010) 894–896
Table 1 1 H, 13C, HMBC and 1H–1H COSY NMR data of compound 1 in acetone-d6. Position
δC
δH (mult., J in Hz)
HMBC
COSY
2 3 4
171.2 68.7 30.5
– 4.45 (d, 6.8) 3.41 (dd, 7.2, 6.8)
– H-4 H-3
4a 5 6 7 8 8a 9 9a 10 10a 4-CH3 5-OH NH
124.0 161.0 125.1 137.6 118.8 131.0 178.2 138.2 188.2 114.2 12.0 – –
– – 7.21 (d, 8.4) 7.59 (dd, 8.4, 7.4) 7.49 (d, 7.4) – – – – – 0.98 (d, 7.2) 12.25 (s) 9.08 (br s)
– C-2, C-4, 4-CH3 C-2, C-3, C-4a, C-9a, C-10, 4-CH3 – – C-8, C-10a C-5, C-8a C-6, C-9, C-10a – – – – – C-3, C-4, C-4a, 4-CH3 C-5, C-6, C-10a –
– – H-7 H-6, H-8 H-7 – – – – – H-4 – –
aromatic proton signals at δ 7.21, 7.49, and 7.59. In addition, the spectrum showed a chelated hydroxyl proton at δ 12.25. Three other coupled signals in the 1H NMR spectrum of 1 (δ 0.98, 3H, d, J = 7.2 Hz; δ 3.41, 1H, dd, J = 7.2, 6.8 Hz; δ 4.45, 1H, d, J = 6.8 Hz) suggested the presence of a –CH(CH3)– CH(OH)– moiety. The 13C NMR spectrum of 1 permitted assignment of only some resonances due to the limited amount of material available. Three methine aromatic carbon signals at δ 125.1, 137.6 and 118.8 were assigned for C-6, C-7 and C-8. The NMR spectral data of 1 were similar to 3, except hydroxyl group at C-5. Based on an HMBC correlation, the methyl group was allocated to C-4. The methine proton at δ 3.41 correlated with two carbonyl groups at the δ 188.2 (C-10) and 171.2 (C-2), the oxymethine carbon at δ 68.7 (C-3), the methyl group at δ 12.0 (4-CH3), and two quaternary carbons at δ 124.0 (C-4a) and 138.2 (C-9a). The other HMBC correlations provided the assignments of all carbon and proton signals in 1 (Table 1 and Fig. 2). From the NOSEY and NOE difference experiments, H-3 showed no correlation to H-4 which opposed from 3. This suggested a trans-configuration for H-3 and H-4 (3R/4S or 3S/ 4R).The absolute configuration of 1 was established using Mosher's ester methodology based on the difference between the 1H NMR chemical shift of its (R) and (S)- methoxytrifluoromethylphenylacetic acid ester (MTPA) (Mosher ester). The ΔδS–ΔδR of NH and 4-CH3 of Mosher of 1 were−0.029 and +0.002, respectively. According to Mosher's assumption [17],
Fig. 2. The Key HMBC (
) and COSY (
) correlations of 1.
Table 2 In vitro cytotoxic activity against KB and HeLa cell lines of compounds 1–6. Isolated compounds
Laoticuzanone A (1) 3-Methyl-1H-1-azaanthracene-2,9,10-trione (2) Griffithazanone A (3) Methyl sinapate (4) Methyl p-coumarate (5) p-Hydroxyphenylethyl p-coumarate (6) Adriamycin (standard agent)
IC50 (μg/ml) KB cell line
HeLa cell line
0.68 5.50 5.20 79.00 57.00 69.00 0.018
0.50 4.00 3.00 52.00 37.00 80.00 0.018
only the R configuration of C-3 could have greater shielding of NH and less shielding of 4-CH3 in the (S)-MTPA derivative of 1. Thus, the structure of 1 has absolute configuration of 3R and 4S. The structure of 1 was assigned as (3R),5-dihydroxy-(4S)methyl-3,4-dihydro-2,9,10-(2H)-1-azaanthracenetrione or trivially named laoticuzanone A. Compounds 1–6 were tested for cytotoxic activity against KB and HeLa cell lines and the results were recorded in Table 2. Compound 1 showed the highest cytotoxicity against KB and HeLa cell with IC50 values of 0.68 and 0.50 μg/ml, followed by compound 3 with IC50 values of 5.20 and 3.00 μg/ ml and compound 2 with IC50 values of 5.50 and 4.00 μg/ml, respectively. In addition, compounds 4–6 were inactive to both cell lines. Acknowledgements The authors are grateful to Graduate School of Chulalongkorn University for the fellowships. The Center for Petroleum, Petrochemical, and Advanced Materials also partially supports this project. References [1] Omar S, Chee CL, Ahmad F, Ni JX, Jaber H, Huang J, et al. Phytochemistry 1992;31:4395. [2] Jiang Z, Yu D-Q. J Nat Prod 1997;60:122. [3] Hisham A, Toubi M, Shuaily W, Ajitha Bai MD, Fujimoto Y. Phytochemistry 2003;62:597. [4] Fang XP, Anderson JE, Chang CJ, Fanwick PE, McLaughlin JL. J Chem Soc Perkin Trans 1990;1:1655. [5] Seidel V, Bailleul F, Waterman PG. Phytochemistry 2000;55:439. [6] Soonthornchareonnon N, Suwanborirux K, Bavovada R, Patarapanich C, Cassady JM. J Nat Prod 1999;62:1390. [7] Lan Y-H, Chang F-R, Yu J-H, Yang Y-L, Chang Y-L, Lee S-J, et al. J Nat Prod 2003;66:487. [8] The National Identity Office. The National Identity Office, 2000. Endemic and Rare Plants of Thailand, Bangkok; 2000. p. 50–1. [9] Limpipatwattana Y, Tip-pyang S, Khumkratok S. Biochem Syst Ecol 2008;36:798. [10] Ocaña B, Espada M, Avendaño C. Tetrahedron 1995;51:1253. [11] Zhang Y-J, Kong M, Chen R-Y, Yu D-Q. J Nat Prod 1999;62:1050. [12] Noda M, Matsumoto M. Biochim Biophys Acta 1971;231:131. [13] Khong PW, Lewis KG. Aust J Chem 1976;29:1351. [14] Kaewamatawong R, Ruangrungsi N, Likhitwitayawuid K. Nat Med (Tokyo) 2007;61:349. [15] Phuwapraisirisan P, Surapinit S, Sombund S, Siripong P, Tip-pyang S. Tetrahedron Lett 2006;47:3685. [16] Kongkathip N, Kongkathip B, Siripong P, Sangma C, Luangkamin S, Niyomdecha M, et al. Bioorg Med Chem 2003;11:3179–91. [17] Ohtani I, Kusumi T, Kashman Y, Kakisawa HJ. Am Chem Soc 1991;113: 4092.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A new cytotoxic 1-azaanthraquinone from the stems of Goniothalamus laoticus Santi Tip-pyang a,⁎, Yawistha Limpipatwattana a, Suttira Khumkratok b, Pongpan Siripong c, Jirapast Sichaem a a b c
Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand Natural Products Research Section, National Cancer Institute, Bangkok 10400, Thailand
a r t i c l e
i n f o
Article history: Received 2 September 2009 Accepted in revised form 26 May 2010 Available online 8 June 2010 Keywords: Goniothalamus laoticus Cytotoxicity 1-azaanthraquinone Laoticuzanone A
a b s t r a c t A new 1-azaanthraquinone, named laoticuzanone A (1), and a synthetically known 3-methyl1H-1-azaanthracene-2,9,10-trione (2), together with four known compounds, Griffithazanone A (3), methyl sinapate (4), methyl p-coumarate (5), and p-hydroxyphenylethyl p-coumarate (6) were isolated from the stems of Goniothalamus laoticus. Their structures were established on the basis of spectroscopic data as well as comparisons with the previous literature data. Compound 1 showed the highest cytotoxicity against KB and HeLa cells with IC50 values of 0.68 and 0.50 μg/ml, respectively. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The genus Goniothalamus (Blume) Hook. f & Thomson belongs to the Annonaceae family and comprises 9 species in Thailand. G. laoticus (Finet & Gagnep) Bân, locally known as “Khao lam dong” is a rare ornamental plant, 4–6 m in height, distributed mainly in Northeastern of Thailand. Ethnobotanical uses of several species of the genus Goniothalamus are well known that many of these plants have provided bioactive alkaloids [1], acetogenins [2], styryl-lactones [3,4], flavonoids [5], and azaanthraquinones [6]. Several compounds isolated from the plants in this genus showed cytotoxic activity against a number of human cancer cell lines [6,7]. In addition, a water decoction derived from its stem bark is being used traditionally as a tonic and a febrifuge by the local people [8]. Our previous investigation reported the isolation of a flavonoid, styryllactone derivatives and naphthoquinone from this plant [9].
⁎ Corresponding author. Tel.: + 66 02 2187625; fax: + 66 02 2187598. E-mail address: [email protected] (S. Tip-pyang). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.05.019
We herein report the isolation and structure elucidation of a new antitumor 1-azaanthraquinone, (3R),5-dihydroxy(4S)-methyl-3,4-dihydro-2,9,10-(2H)-1-azaanthracenetrione (1), together with the antitumor activity of all isolated compounds (1–6) from the stems of G. laoticus. 2. Experimental 2.1. General HPLC: Waters® 600 controller equipped with a Waters® 2996 photodiode array detector. Melting points; Fisher-Johns Melting Point Apparatus. IR: Nicolet 6700 FT-IR spectrometer with a mercury–cadmium–telluride (MCT) detector. UV: UV2552PC UV–Vis spectrometer. Optical rotations: Jasco P-1010 polarimeter. NMR: Varian model Mercury+ 400 spectrometer operated at 400 MHz for 1H and 100 MHz for 13C nuclei. Adsorbents: Sephadex LH-20 and silica gel 60 Merck cat. No. 7729 and 7734 were used for column chromatography. Merck silica gel 60 F254 plates were used for TLC. ESIMS and HRESIMS: Mass spectrometer model VG TRIO 2000 and a Micromass LCT mass spectrometer.
S. Tip-pyang et al. / Fitoterapia 81 (2010) 894–896
2.2. Plant material The stems of G. laoticus were collected from Sakon Nakhon Province of Thailand in June, 2007 and identified by Ms. Suttira Khumkratok, a botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, where a voucher specimen (Khumkratok no. 84-08) is deposited. 2.3. Extraction and isolation Air-dried and powdered stems of G. laoticus (5.5 kg) were successively extracted in a soxhlet apparatus with CH2Cl2, EtOAc, and MeOH. The CH2Cl2 extract was concentrated under vacuum to yield 186.0 g of crude residue. This material was fractionated by vacuum liquid chromatography (VLC) over silica gel, using hexane, EtOAc and MeOH with increasing polarity, which was subjected to silica gel column eluted with a mixture of CH2Cl2/MeOH with increasing polarity to provide two fractions. Fraction 1 was further purified by Sephadex LH-20 column chromatography using 0.5:9.5 MeOH/CH2Cl2 as eluting solvent to afford 3-methyl1H-1-azaanthracene-2,9,10-trione (2, 5.2 mg) [10]. Fraction 2 was further purified by Sephadex LH-20 column chromatography, using 9.5:0.5 CH2Cl2/MeOH as eluting solvent, followed by HPLC, using ACN/H2O (8:2) as eluents to yield a new 1-azaanthraquinone, 3,5-dihydroxy-4-methyl-3,4-dihydro2,9,10-(2H)-1-azaanthracenetrione (1, 4.1 mg) and griffithazanone A (3, 3.5 mg) [11]. The methanolic extract (30 g) was similarly chromatographed on silica gel VLC using a stepwise gradient elution of MeOH and CH2Cl2, yielding three fractions. Fraction 1 was further fractionated by Sephadex LH-20 column using 100% MeOH as eluting solvent to give two fractions. Sephadex fraction 1 was further purification by preparative TLC [silica gel, CH2Cl2/MeOH (9.5:0.5)] to obtain methyl sinapate (4, 6.7 mg) [12]. Sephadex fraction 2 was further fractionated over silica gel CC, using CH2Cl2 and MeOH with increasing polarity to yield three fractions (S1–S3). Fraction S1 was further purified by Sephadex LH-20 column chromatography, using 9.5:0.5 CH2Cl2/MeOH as eluting solvent, followed by silica gel CC, using CH2Cl2/MeOH with increasing polarity to
895
afford methyl p-coumarate (5, 7.3 mg) [13]. Fraction S3 was crystallized in a mixture of CH2Cl2 and MeOH to obtain phydroxyphenylethyl p-coumarate (6, 6.2 mg) [14]. Compound 1 (Fig. 1) (3R),5-dihydroxy-(4S)-methyl-3,4dihydro-2,9,10-(2H)-1-azaanthracenetrione, brown amorphous powder; mp 208–209 °C; [α]26 + 119° (c 0.004, D MeOH); UV (MeOH) λmax (log ε): 255 (3.8), 295 (3.9) nm; positive ion ESIMS m/z: 273.59 [M + H]+; positive ion HRESIMS m/z: [M + H]+ 274.0704 (calcd for C14H11O5, 274.0715); 1H NMR (CD3COCD3, 400 MHz) and 13C NMR (CD3COCD3, 100 MHz) are shown in Table 1. 2.4. General preparation of MTPA ester [15] To a solution of 1 (1 mg) in two drops of pyridine was added (−)-MTPA chloride (50 μL), and the mixture was left at room temperature overnight. After addition of 1 ml of 1 M NaHCO3, the reaction mixture was extracted with CH2Cl2 (2 ml ×2), washed with brine, dried over anhydrous Na2SO4, and evaporated. The residue was purified on a short silica gel column (5% MeOH–CH2Cl2) to afford S-(−)-MTPA derivative (1a). The R-(+)-MTPA derivative (1b) was prepared in the same way. Selected ΔδHSR values: −0.029 (NH); +0.002 (4−CH3). 2.5. Cytotoxicity test Compounds 1–6 were subjected to in vitro cytotoxicity studies against human cervical carcinoma (HeLa) and human mouth epidermal carcinoma (KB) using the standard MTT colorimetric method [16]. 3. Result and discussion Compound 1 was obtained as brown amorphous powder with mp 208–209 °C. Its HRESIMS gave a positive molecular ion at m/z: [M + H]+ 274.0704 (calcd 274.0715), compatible with a molecular of C14H11O5. The IR spectrum confirmed this evidence by showing the quinine carbonyl and the lactam carbonyl absorptions at 1463 and 1632 cm−1, respectively. UV absorption bands (λmax = 255 and 295 nm) also confirmed the presence of the conjugated quinonoid moiety. The 1 H NMR spectrum of 1 in acetone-d6 showed three adjacent
Fig. 1. Structures of compounds 1–3.
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S. Tip-pyang et al. / Fitoterapia 81 (2010) 894–896
Table 1 1 H, 13C, HMBC and 1H–1H COSY NMR data of compound 1 in acetone-d6. Position
δC
δH (mult., J in Hz)
HMBC
COSY
2 3 4
171.2 68.7 30.5
– 4.45 (d, 6.8) 3.41 (dd, 7.2, 6.8)
– H-4 H-3
4a 5 6 7 8 8a 9 9a 10 10a 4-CH3 5-OH NH
124.0 161.0 125.1 137.6 118.8 131.0 178.2 138.2 188.2 114.2 12.0 – –
– – 7.21 (d, 8.4) 7.59 (dd, 8.4, 7.4) 7.49 (d, 7.4) – – – – – 0.98 (d, 7.2) 12.25 (s) 9.08 (br s)
– C-2, C-4, 4-CH3 C-2, C-3, C-4a, C-9a, C-10, 4-CH3 – – C-8, C-10a C-5, C-8a C-6, C-9, C-10a – – – – – C-3, C-4, C-4a, 4-CH3 C-5, C-6, C-10a –
– – H-7 H-6, H-8 H-7 – – – – – H-4 – –
aromatic proton signals at δ 7.21, 7.49, and 7.59. In addition, the spectrum showed a chelated hydroxyl proton at δ 12.25. Three other coupled signals in the 1H NMR spectrum of 1 (δ 0.98, 3H, d, J = 7.2 Hz; δ 3.41, 1H, dd, J = 7.2, 6.8 Hz; δ 4.45, 1H, d, J = 6.8 Hz) suggested the presence of a –CH(CH3)– CH(OH)– moiety. The 13C NMR spectrum of 1 permitted assignment of only some resonances due to the limited amount of material available. Three methine aromatic carbon signals at δ 125.1, 137.6 and 118.8 were assigned for C-6, C-7 and C-8. The NMR spectral data of 1 were similar to 3, except hydroxyl group at C-5. Based on an HMBC correlation, the methyl group was allocated to C-4. The methine proton at δ 3.41 correlated with two carbonyl groups at the δ 188.2 (C-10) and 171.2 (C-2), the oxymethine carbon at δ 68.7 (C-3), the methyl group at δ 12.0 (4-CH3), and two quaternary carbons at δ 124.0 (C-4a) and 138.2 (C-9a). The other HMBC correlations provided the assignments of all carbon and proton signals in 1 (Table 1 and Fig. 2). From the NOSEY and NOE difference experiments, H-3 showed no correlation to H-4 which opposed from 3. This suggested a trans-configuration for H-3 and H-4 (3R/4S or 3S/ 4R).The absolute configuration of 1 was established using Mosher's ester methodology based on the difference between the 1H NMR chemical shift of its (R) and (S)- methoxytrifluoromethylphenylacetic acid ester (MTPA) (Mosher ester). The ΔδS–ΔδR of NH and 4-CH3 of Mosher of 1 were−0.029 and +0.002, respectively. According to Mosher's assumption [17],
Fig. 2. The Key HMBC (
) and COSY (
) correlations of 1.
Table 2 In vitro cytotoxic activity against KB and HeLa cell lines of compounds 1–6. Isolated compounds
Laoticuzanone A (1) 3-Methyl-1H-1-azaanthracene-2,9,10-trione (2) Griffithazanone A (3) Methyl sinapate (4) Methyl p-coumarate (5) p-Hydroxyphenylethyl p-coumarate (6) Adriamycin (standard agent)
IC50 (μg/ml) KB cell line
HeLa cell line
0.68 5.50 5.20 79.00 57.00 69.00 0.018
0.50 4.00 3.00 52.00 37.00 80.00 0.018
only the R configuration of C-3 could have greater shielding of NH and less shielding of 4-CH3 in the (S)-MTPA derivative of 1. Thus, the structure of 1 has absolute configuration of 3R and 4S. The structure of 1 was assigned as (3R),5-dihydroxy-(4S)methyl-3,4-dihydro-2,9,10-(2H)-1-azaanthracenetrione or trivially named laoticuzanone A. Compounds 1–6 were tested for cytotoxic activity against KB and HeLa cell lines and the results were recorded in Table 2. Compound 1 showed the highest cytotoxicity against KB and HeLa cell with IC50 values of 0.68 and 0.50 μg/ml, followed by compound 3 with IC50 values of 5.20 and 3.00 μg/ ml and compound 2 with IC50 values of 5.50 and 4.00 μg/ml, respectively. In addition, compounds 4–6 were inactive to both cell lines. Acknowledgements The authors are grateful to Graduate School of Chulalongkorn University for the fellowships. The Center for Petroleum, Petrochemical, and Advanced Materials also partially supports this project. References [1] Omar S, Chee CL, Ahmad F, Ni JX, Jaber H, Huang J, et al. Phytochemistry 1992;31:4395. [2] Jiang Z, Yu D-Q. J Nat Prod 1997;60:122. [3] Hisham A, Toubi M, Shuaily W, Ajitha Bai MD, Fujimoto Y. Phytochemistry 2003;62:597. [4] Fang XP, Anderson JE, Chang CJ, Fanwick PE, McLaughlin JL. J Chem Soc Perkin Trans 1990;1:1655. [5] Seidel V, Bailleul F, Waterman PG. Phytochemistry 2000;55:439. [6] Soonthornchareonnon N, Suwanborirux K, Bavovada R, Patarapanich C, Cassady JM. J Nat Prod 1999;62:1390. [7] Lan Y-H, Chang F-R, Yu J-H, Yang Y-L, Chang Y-L, Lee S-J, et al. J Nat Prod 2003;66:487. [8] The National Identity Office. The National Identity Office, 2000. Endemic and Rare Plants of Thailand, Bangkok; 2000. p. 50–1. [9] Limpipatwattana Y, Tip-pyang S, Khumkratok S. Biochem Syst Ecol 2008;36:798. [10] Ocaña B, Espada M, Avendaño C. Tetrahedron 1995;51:1253. [11] Zhang Y-J, Kong M, Chen R-Y, Yu D-Q. J Nat Prod 1999;62:1050. [12] Noda M, Matsumoto M. Biochim Biophys Acta 1971;231:131. [13] Khong PW, Lewis KG. Aust J Chem 1976;29:1351. [14] Kaewamatawong R, Ruangrungsi N, Likhitwitayawuid K. Nat Med (Tokyo) 2007;61:349. [15] Phuwapraisirisan P, Surapinit S, Sombund S, Siripong P, Tip-pyang S. Tetrahedron Lett 2006;47:3685. [16] Kongkathip N, Kongkathip B, Siripong P, Sangma C, Luangkamin S, Niyomdecha M, et al. Bioorg Med Chem 2003;11:3179–91. [17] Ohtani I, Kusumi T, Kashman Y, Kakisawa HJ. Am Chem Soc 1991;113: 4092.