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A novel cytotoxic neophysalin from Physalis alkekengi var. francheti
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Chinese Chemical Letters 20 (2009) 1327–1330 www.elsevier.com/locate/cclet
A novel cytotoxic neophysalin from Physalis alkekengi var. francheti Chu Hang Zhang a,b, Zheng Tao Wang b,*, Yi Ping Yang b, Qi Shi Sun a,* a
b
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China Key Laboratory of Standardization of Chinese Medicines of Ministry of Education Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Received 11 March 2009
Abstract A new neophysalin, named 5a-hydroxy-25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A(1), along with three other known neophysalins (2–4) were isolated from the calyxes of Physalis alkekengi L. var. francheti (Mast.) Makino. The structure of 1 was determined by means of 1D and 2D NMR, UV, IR and mass spectra. Compound 1 displayed potent cytotoxicities in vitro against PC3 and LNCaP cell lines. # 2009 Zheng Tao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Physalis alkekengi var. francheti; Neophysalin; Cytotoxicity
Physalis alkekengi L. var. francheti (Mast.) Makino (Solanaceae) is widely distributed throughout tropical and subtropical regions of the world. The fruit of this herb is an edible berry and its indeciduous calyxes are used in Chinese medicine for treatment of different illnesses, such as cancer, pharyngitis, diuretic, malaria, and liver disorders. Physalins were reported to be the characteristic steroidal constituents of Physalis plants possessing a 13,14-seco16,24-cycloergostane skeleton. Since the isolation of physalin A and B in 1969, more than 20 physalins were reported from P. alkekengi var. francheti, P. angulata and P. lancifolia. Physalins are known to undergo an acid-induced benzilic acid-type rearrangement reaction yielding a newly formed skeleton named ‘‘neophysalin’’ [1]. The skeletal rearrangement involves a bond cleavage at C(15)–C(16) and a bond formation between C(14) and C(16). The first neophylin, physalin P, was isolated from P. alkekengi var. francheti in 1992 [2]. In this study, a new neophysalin, named 5a-hydroxy-25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A(1) along with three other known neophysalins, physalin P(2), 25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A(3) and 4,7didehydroneophysalin B(4) were isolated from P. alkekengi var. francheti. The structure and cytotoxic activities of compound 1 are described in this paper. The dried calyxes (10 kg) of P. alkekengi var. francheti were extracted with 80% EtOH three times under reflux. After removal of solvent in vacuo, the residue was suspended in water and extracted with petroleum ether and ethyl acetate, respectively. The ethyl acetate extracts (70 g) were chromatographed on repeated silica gel columns using
* Corresponding author. E-mail addresses: [email protected] (Z.T. Wang), [email protected] (Q.S. Sun). 1001-8417/$ – see front matter # 2009 Zheng Tao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.06.005
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Fig. 1. Structures of compounds 1–4.
gradient CH2Cl2–MeOH as eluents to afford 1–4. Compounds 2–4 were identified as physalin P, 25,27-dihydro-4,7didehydro-7-deoxyneophysalin A and 4,7-didehydroneophysalin B (Fig. 1). Compound 1, was obtained as a white amorphous solid, mp: 269–270 8C, ½a25 D þ 37 (c, 1.52, MeOH). UV (MeOH)lmax: 219.7 nm (a,b-unsaturated ketone), IR (KBr)nmax: 3438 cm1 (hydroxyl), 1790 cm1 (g-lactone), 1717 cm1 (d-lactone), 1663 cm1 (a,b-unsaturated ketone). The molecular formula C28H32O10 was established from its HRESIMS at m/z 551.1886 [M+Na]+ (calcd. for C28H32O10Na, 551.1888) with 138 of unsaturation. 1H and 13C NMR spectral data of compound 1 were shown in Table 1. The 13C NMR and HMQC spectral data revealed that 1
Table 1 1 H and 13C NMR spectral data of compound 1 (TMS, DMSO-d6, d (ppm), 1H NMR 400 MHz, Position 1 2 3 4a 4b 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15
dH 5.75 6.62 2.37 2.62
(dd, 1H, J = 2.2, 10 Hz) (ddd, 1H, J = 1.9, 5, 10 Hz) (dd, 1H, J = 5, 19.5 Hz) (br d, 1H, J = 19.5 Hz)
5.82 5.99 2.88 2.10
(br d, 1H, J = 10 Hz) (dd, 1H, J = 4.9, 10 Hz) (br t, 1H, J = 5.6 Hz) (m, 1H)
2.33 2.23 2.08 2.26
(m, (m, (m, (m,
1H) 1H) 1H) 1H)
13
C NMR 100 MHz).
dc
Position
dH
dc
202.5 127.4 142.2 37.6
16 17 18 19 20 21 22 23a 23b 24 25 26 27 28 5-OH 13-OH 14-OH
3.05 (s, 1H)
56.5 84.6 172.7 16.7 82.8 21.7 75.4 27.7
71.9 131.6 129.1 47.4 30.9 53.7 23.6 27.5 78.6 81.2 177.6
0.94 (s, 3H) 1.71 4.46 1.61 2.03
(s, 3H) (d, 1H, J = 3.8 Hz) (br d, 1H, J = 15 Hz) (dd, 1H, J = 3.8, 15 Hz)
3.43 (q, 1H, J = 7 Hz) 1.17 1.26 3.12 6.49 6.03
(d, 3H, J = 7.5 Hz) (s, 3H) (s, 1H, OH) (s, 1H, OH) (s, 1H, OH)
34.7 39.2 173.4 16.6 27.3
C.H. Zhang et al. / Chinese Chemical Letters 20 (2009) 1327–1330
1329
Fig. 2. Key HMBC and 1H-1H COSY correlations of compound 1.
contains four carbonyl groups, seven quaternary carbons including four oxygenated carbons, nine methines including four olefinics and an oxygenated carbon, four methylenes, and four methyl groups. The 1H NMR spectrum of 1 showed the presence of three tertiary methyl groups (d 0.94, 1.26, 1.71), one secondary methyl group and four olefinic protons [dH 5.75 (dd, J = 2.2, 10 Hz), 5.82 (br d, J = 10 Hz), 5.99 (dd, J = 4.9, 10 Hz), 6.62 (ddd, J = 1.9, 5, 10 Hz), indicating close structural resemblance of 1 to physalin P (2) with a 5a-hydroxyl-2,6diene-1-one system at ring A/B moiety. The 13C NMR spectrum of 1 showed the presence of a ketone group (dC 202.5), four olefinic carbons (dC 142.2, 131.6, 129.1 and 127.4) and three lactone carbonyl groups (dC 177.6, 173.4 and 172.7), but no hemiketal group characteristic for physalin derivatives was observed, all these evidences further confirmed that the structure of 1 had the neophysalin skeleton. A detailed comparison of the 1H and 13C NMR spectral data of compounds 1 and 2 indicated that they have the same substituent pattern and relative configurations in rings A–C and in their respective lactone rings [2]. This structural similarity was confirmed by correlations observed in their 2D NMR spectra. A remarkable difference between the 1H NMR spectra of 1 and 2 was the presence of a secondary methyl group [dH 1.17 (d, J = 7.5 Hz)] in 1 instead of the –OCH2CH– moiety in 2. These phenomena suggested that these two compounds vary at C-14 and C-27, with a methyl carbon at C-27 in 1 rather than the oxygenated methylene in 2.
Fig. 3. ROESY correlations of compound 1.
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HMBC correlations for the secondary methyl group [dH 1.17 (d, J = 7.5 Hz)]/C-24 (dC 34.7), C-26 (dC 173.4) inferred that the doublet methyl group was located at C-25. In the HMBC spectrum of 1, both the signals at dH 2.88(H-8), dH 2.10(H-9) and dH 3.05(H-16) correlated with the oxygenated quaternary carbon (dC 81.2, C-14). All of these phenomena indicated a hydroxyl group was attached to C-14. This assignment was supported by the molecular formula, C28H32O10, which is 2 units larger than that of 2 (Fig. 2). The presence of the HMBC correlations for 5-OH(dH 3.12)/C-4(dC 37.6), C-5(dC 71.9), C-6(dC 131.6) indicated the location of a hydroxyl group at C-5 (Fig. 2). The relative stereochemistry of 1 was established by ROESY experiments (Fig. 3). The ROE cross peaks between CH3-28 and H-23b, H-16; CH3-27 and H-23b; H-22 and H-23 suggested they are co-facial. H-16 and H-22 were already reported to be b-orientation in compound 2 [1]. Accordingly, the b-orientation was confirmed for CH3-28, CH3-27, H-23, H-22, and H-16 in 1. The observation of ROE cross peaks of 5-OH/H-4a showed 5-OH is a-oriented, and CH3-19 are b-oriented due to the observation of ROE cross peaks of CH3-19/H-4b. A detailed comparison of compounds 1 and 3 indicated that they have the same substituent pattern and relative configurations in ring C [1] so that 13-OH was confirmed to be a-oriented. Consequently, the structure of 1 was unambiguously established as 5ahydroxy-25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A. The cytotoxic activities of compound 1 were evaluated according to MTT assay against the PC-3 (human prostate cancer) and LNCaP (human prostate cancer) cell lines. The cytotoxicity data (IC50 values) are 1.38 and 9.97 mmol/L, respectively. References [1] M. Kawai, T. Ogura, Y. Butsugan, et al. Tetrahedron 47 (1991) 2103. [2] M. Kawai, A. Matsumoto, B. Makino, et al. Bull. Chem. Soc. Jpn. 66 (1993) 1299.
Chinese Chemical Letters 20 (2009) 1327–1330 www.elsevier.com/locate/cclet
A novel cytotoxic neophysalin from Physalis alkekengi var. francheti Chu Hang Zhang a,b, Zheng Tao Wang b,*, Yi Ping Yang b, Qi Shi Sun a,* a
b
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China Key Laboratory of Standardization of Chinese Medicines of Ministry of Education Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Received 11 March 2009
Abstract A new neophysalin, named 5a-hydroxy-25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A(1), along with three other known neophysalins (2–4) were isolated from the calyxes of Physalis alkekengi L. var. francheti (Mast.) Makino. The structure of 1 was determined by means of 1D and 2D NMR, UV, IR and mass spectra. Compound 1 displayed potent cytotoxicities in vitro against PC3 and LNCaP cell lines. # 2009 Zheng Tao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Physalis alkekengi var. francheti; Neophysalin; Cytotoxicity
Physalis alkekengi L. var. francheti (Mast.) Makino (Solanaceae) is widely distributed throughout tropical and subtropical regions of the world. The fruit of this herb is an edible berry and its indeciduous calyxes are used in Chinese medicine for treatment of different illnesses, such as cancer, pharyngitis, diuretic, malaria, and liver disorders. Physalins were reported to be the characteristic steroidal constituents of Physalis plants possessing a 13,14-seco16,24-cycloergostane skeleton. Since the isolation of physalin A and B in 1969, more than 20 physalins were reported from P. alkekengi var. francheti, P. angulata and P. lancifolia. Physalins are known to undergo an acid-induced benzilic acid-type rearrangement reaction yielding a newly formed skeleton named ‘‘neophysalin’’ [1]. The skeletal rearrangement involves a bond cleavage at C(15)–C(16) and a bond formation between C(14) and C(16). The first neophylin, physalin P, was isolated from P. alkekengi var. francheti in 1992 [2]. In this study, a new neophysalin, named 5a-hydroxy-25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A(1) along with three other known neophysalins, physalin P(2), 25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A(3) and 4,7didehydroneophysalin B(4) were isolated from P. alkekengi var. francheti. The structure and cytotoxic activities of compound 1 are described in this paper. The dried calyxes (10 kg) of P. alkekengi var. francheti were extracted with 80% EtOH three times under reflux. After removal of solvent in vacuo, the residue was suspended in water and extracted with petroleum ether and ethyl acetate, respectively. The ethyl acetate extracts (70 g) were chromatographed on repeated silica gel columns using
* Corresponding author. E-mail addresses: [email protected] (Z.T. Wang), [email protected] (Q.S. Sun). 1001-8417/$ – see front matter # 2009 Zheng Tao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.06.005
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C.H. Zhang et al. / Chinese Chemical Letters 20 (2009) 1327–1330
Fig. 1. Structures of compounds 1–4.
gradient CH2Cl2–MeOH as eluents to afford 1–4. Compounds 2–4 were identified as physalin P, 25,27-dihydro-4,7didehydro-7-deoxyneophysalin A and 4,7-didehydroneophysalin B (Fig. 1). Compound 1, was obtained as a white amorphous solid, mp: 269–270 8C, ½a25 D þ 37 (c, 1.52, MeOH). UV (MeOH)lmax: 219.7 nm (a,b-unsaturated ketone), IR (KBr)nmax: 3438 cm1 (hydroxyl), 1790 cm1 (g-lactone), 1717 cm1 (d-lactone), 1663 cm1 (a,b-unsaturated ketone). The molecular formula C28H32O10 was established from its HRESIMS at m/z 551.1886 [M+Na]+ (calcd. for C28H32O10Na, 551.1888) with 138 of unsaturation. 1H and 13C NMR spectral data of compound 1 were shown in Table 1. The 13C NMR and HMQC spectral data revealed that 1
Table 1 1 H and 13C NMR spectral data of compound 1 (TMS, DMSO-d6, d (ppm), 1H NMR 400 MHz, Position 1 2 3 4a 4b 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15
dH 5.75 6.62 2.37 2.62
(dd, 1H, J = 2.2, 10 Hz) (ddd, 1H, J = 1.9, 5, 10 Hz) (dd, 1H, J = 5, 19.5 Hz) (br d, 1H, J = 19.5 Hz)
5.82 5.99 2.88 2.10
(br d, 1H, J = 10 Hz) (dd, 1H, J = 4.9, 10 Hz) (br t, 1H, J = 5.6 Hz) (m, 1H)
2.33 2.23 2.08 2.26
(m, (m, (m, (m,
1H) 1H) 1H) 1H)
13
C NMR 100 MHz).
dc
Position
dH
dc
202.5 127.4 142.2 37.6
16 17 18 19 20 21 22 23a 23b 24 25 26 27 28 5-OH 13-OH 14-OH
3.05 (s, 1H)
56.5 84.6 172.7 16.7 82.8 21.7 75.4 27.7
71.9 131.6 129.1 47.4 30.9 53.7 23.6 27.5 78.6 81.2 177.6
0.94 (s, 3H) 1.71 4.46 1.61 2.03
(s, 3H) (d, 1H, J = 3.8 Hz) (br d, 1H, J = 15 Hz) (dd, 1H, J = 3.8, 15 Hz)
3.43 (q, 1H, J = 7 Hz) 1.17 1.26 3.12 6.49 6.03
(d, 3H, J = 7.5 Hz) (s, 3H) (s, 1H, OH) (s, 1H, OH) (s, 1H, OH)
34.7 39.2 173.4 16.6 27.3
C.H. Zhang et al. / Chinese Chemical Letters 20 (2009) 1327–1330
1329
Fig. 2. Key HMBC and 1H-1H COSY correlations of compound 1.
contains four carbonyl groups, seven quaternary carbons including four oxygenated carbons, nine methines including four olefinics and an oxygenated carbon, four methylenes, and four methyl groups. The 1H NMR spectrum of 1 showed the presence of three tertiary methyl groups (d 0.94, 1.26, 1.71), one secondary methyl group and four olefinic protons [dH 5.75 (dd, J = 2.2, 10 Hz), 5.82 (br d, J = 10 Hz), 5.99 (dd, J = 4.9, 10 Hz), 6.62 (ddd, J = 1.9, 5, 10 Hz), indicating close structural resemblance of 1 to physalin P (2) with a 5a-hydroxyl-2,6diene-1-one system at ring A/B moiety. The 13C NMR spectrum of 1 showed the presence of a ketone group (dC 202.5), four olefinic carbons (dC 142.2, 131.6, 129.1 and 127.4) and three lactone carbonyl groups (dC 177.6, 173.4 and 172.7), but no hemiketal group characteristic for physalin derivatives was observed, all these evidences further confirmed that the structure of 1 had the neophysalin skeleton. A detailed comparison of the 1H and 13C NMR spectral data of compounds 1 and 2 indicated that they have the same substituent pattern and relative configurations in rings A–C and in their respective lactone rings [2]. This structural similarity was confirmed by correlations observed in their 2D NMR spectra. A remarkable difference between the 1H NMR spectra of 1 and 2 was the presence of a secondary methyl group [dH 1.17 (d, J = 7.5 Hz)] in 1 instead of the –OCH2CH– moiety in 2. These phenomena suggested that these two compounds vary at C-14 and C-27, with a methyl carbon at C-27 in 1 rather than the oxygenated methylene in 2.
Fig. 3. ROESY correlations of compound 1.
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HMBC correlations for the secondary methyl group [dH 1.17 (d, J = 7.5 Hz)]/C-24 (dC 34.7), C-26 (dC 173.4) inferred that the doublet methyl group was located at C-25. In the HMBC spectrum of 1, both the signals at dH 2.88(H-8), dH 2.10(H-9) and dH 3.05(H-16) correlated with the oxygenated quaternary carbon (dC 81.2, C-14). All of these phenomena indicated a hydroxyl group was attached to C-14. This assignment was supported by the molecular formula, C28H32O10, which is 2 units larger than that of 2 (Fig. 2). The presence of the HMBC correlations for 5-OH(dH 3.12)/C-4(dC 37.6), C-5(dC 71.9), C-6(dC 131.6) indicated the location of a hydroxyl group at C-5 (Fig. 2). The relative stereochemistry of 1 was established by ROESY experiments (Fig. 3). The ROE cross peaks between CH3-28 and H-23b, H-16; CH3-27 and H-23b; H-22 and H-23 suggested they are co-facial. H-16 and H-22 were already reported to be b-orientation in compound 2 [1]. Accordingly, the b-orientation was confirmed for CH3-28, CH3-27, H-23, H-22, and H-16 in 1. The observation of ROE cross peaks of 5-OH/H-4a showed 5-OH is a-oriented, and CH3-19 are b-oriented due to the observation of ROE cross peaks of CH3-19/H-4b. A detailed comparison of compounds 1 and 3 indicated that they have the same substituent pattern and relative configurations in ring C [1] so that 13-OH was confirmed to be a-oriented. Consequently, the structure of 1 was unambiguously established as 5ahydroxy-25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A. The cytotoxic activities of compound 1 were evaluated according to MTT assay against the PC-3 (human prostate cancer) and LNCaP (human prostate cancer) cell lines. The cytotoxicity data (IC50 values) are 1.38 and 9.97 mmol/L, respectively. References [1] M. Kawai, T. Ogura, Y. Butsugan, et al. Tetrahedron 47 (1991) 2103. [2] M. Kawai, A. Matsumoto, B. Makino, et al. Bull. Chem. Soc. Jpn. 66 (1993) 1299.