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Bioassay-guided fractionation of an ethanol extract of a Madagascar collection of the bark of Scutia myrtina led to the isolation of three new anthrone-anthraquinones, scutianthraquinones A, B and C (1-3), one new bisanthrone-anthraquinone, scutianthraquinone D (4), and the known anthraquinone, aloesaponarin I (5). The structures of all compounds were determined using a combination of 1D and 2D NMR experiments, including COSY, HSQC, HMBC, and ROESY sequences, and mass spectrometry. All the isolated compounds were tested against the A2780 human ovarian cancer cell line for antiproliferative activities, and against the chloroquine-resistant Plasmodium falciparum strains Dd2 and FCM29 for antiplasmodial activities. Compounds 1, 2 and 4 showed weak antiproliferative activities against the A2780 ovarian cancer cell line, while compounds 1 – 4 exhibited moderate antiplasmodial activities against P. falciparum Dd2 and compounds 1, 2, and 4 exhibited moderate antiplasmodial activities against P. falciparum FCM29
In our continuing search for biologically active natural products from tropical rainforests as part of an International Cooperative Biodiversity Groups (ICBG) program, we obtained an extract from the bark of Scutia myrtina collected in Madagascar. The roots of Scutia myrtina have been investigated previously, and afforded two perylenequinones with in vitro anthelmintic activity but no in vivo activity.2 In addition, several cyclopeptide alkaloids have been isolated from other Scutia species,3-6 some of which showed moderate antimicrobial activity.3 In our research, the extract was selected for bioassay-guided fractionation on the basis of its antiproliferative activity against the A2780 human ovarian cancer cell line, with an IC50 value of 6 μg/mL. The crude extract afforded three new anthrone-anthraquinones (1-3), a new bisanthrone-anthraquinone (4) and the known anthraquinone aloesaponarin I (5)7 after solvent partition and reversed-phase C18 HPLC. Herein we report the structural elucidation of the new compounds and their bioactivities against the A2780 human ovarian cancer cell line and two strains of the malaria parasite Plasmodium falciparum.
Scutianthraquinone A (1, Figure 1) was obtained as a light-brown amorphous solid. Its molecular formula was established as C39H32O13 on the basis of its molecular ion peak in its HRFAB mass spectrum. Its UV-VIS spectra showed characteristic absorptions of anthraquinones at 248, 272, 310 and 475 nm.8 The 1H NMR spectrum of 1 (Table 1) in CD3OD showed signals for an AB system at δH 8.64 (d, J = 7.8 Hz, H-3) and δH 7.86 (d, J = 7.8 Hz, H-4), an ABC system at δH 6.86 (br, d, J = 7.7 Hz, H-2′), δH 7.33 (t, J = 7.7 Hz, H-3′) and δH 6.71 (br, d, J = 7.7 Hz, H-4′), two aromatic proton singlets at δH 7.52 (s, H-5) and δH 6.66 (s, H-5′), two methoxyl groups at δH 3.89 (s, 7-COOCH3) and δH 3.88 (s, 7′-COOCH3), and two methyl groups at δH 2.49 (s, C-8-CH3) and δH 2.77 (s, C-8′-CH3). The 1H NMR spectrum in DMSO-d6 also showed resonances for four hydroxyl protons at δH 13.68, 13.24, 11.80 and 10.99, indicating that each was intramolecularly hydrogen bonded. In the 13C NMR spectrum six carbonyl carbons, 24 aromatic carbons, one oxygenated quaternary carbon, two methoxyl carbons, one methine carbon, one methylene carbon and four methyl carbons were identified.
On the basis of its UV, 1H NMR, and 13C NMR spectra and its molecular formula, the structure of 1 was assigned as an anthrone-substituted anthraquinone.
The complete 1H NMR and 13C NMR assignments and connectivities of 1 were determined from a combination of COSY, HSQC, HMBC and ROESY data. The COSY spectrum showed correlations that confirmed the connectivity of H-3 and H-4, and of H-2′, H-3′ and H-4′. In the HMBC spectrum, the correlations of H-4 and H-5 to C-10 (δC 183.6) and H-4′ and H-5′ to C-10′ (δC 78.2) indicated the positions of these protons. The position of CH3-8 was established by three-bond HMBC correlations from the protons of CH3-8 and H-5 to C-7 and C-8a (δC 131.8, 123.5) along with a two-bond correlation from the protons of CH3-8 to C-8 (δC 143.5). The position of CH3-8′ was established by three-bond HMBC correlations from protons of CH3-8′ and H-5′ to C-7′ and C-8′a (δC 126.6, 124.1) along with two-bond correlations from protons of CH3-8′ to C-8′ (δC 141.9). The presence of two carbomethoxy groups (COOCH3-7, COOCH3-7′) was indicated by HMBC correlations from their methyl protons to the corresponding carbonyl carbons (COOCH3-7, 7′, δC 170.6, 169.9), and their positions were established by ROESY correlations (Fig. 2) and by comparison of 13C NMR data for C-5/6/7/8/8a/10a and C-5/6′/7′/8′/8′a/10′a of 1 and the corresponding carbons of the known anthraquinone aloesaponarin I (5).7 A ROESY correlation from the protons of 7′-COOCH3 to those of CH3-8′ unambiguously located the carbomethoxy group at C-7′ instead of C-6′. A corresponding ROESY correlation from the protons of COOCH3-7 to CH3-8 was not observed, but the almost identical chemical shifts between C-5/6/7/8/8a/10a and C-5′/6′/7′/8′/8′a/10′a in the 13C NMR of 1 indicated that COOCH3-7 must reside on C-7 instead of C-6. Finally, the chemical shifts of C-5/6/7/8/8a/10a showed the same pattern as those of C-5/6/7/8/8a/10a of 5, offering further confirmation of the assigned positions of the carbomethoxy groups.
The anthrone and anthraquinone moieties were connected through the bond between C-2 and C-10′, as evidenced by the key HMBC correlation of H-3 to C-10′ (δC 78.2) (Figure 2). The presence of a 2-methylbutanoyl group was established by COSY correlations between a methine proton (δH 2.53, m) and methyl (δH 1.15/1.12, d, J = 7.2 Hz) and methylene protons (δH 1.69, m; δH 1.53, m). The methylene protons were part of an ethyl group, as shown by the COSY correlations to methyl protons at δH 0.85 (t, J = 7.4 Hz) (Figure 1).
Supporting evidence for the 2-methylbutanoyl group was provided by HMBC correlations from its methylene protons (δH 1.69, m; δH 1.53, m) and methyl protons (δH 1.15/1.12, d, J = 7.2 Hz) to a carbonyl group (δC 174.9) (Figure 2). The group was assigned to the C-10′ position by ROESY correlations from CH3-2″ (δH 1.15/1.12, d, J = 7.2 Hz) to H-3 and H-4′ and from H3-4″ (δH 0.85, t, J = 7.4 Hz) to H-4 and H-5′ (Figure 3). The negative ion ESI-MS/MS of 1 gave a series of fragment ions which were entirely consistent with the assigned structure. The structure of 1, except for the absolute configurations of C-10′ and the 2-methylbutanoyl group, was thus established.
Analysis of the 1H and 13C NMR spectra of 1 indicated the existence of two atropisomers. The signals for H-3′ in its 1H NMR spectrum in CD3OD appeared as two overlapping triplets, while the signal for H-5′ appeared as two singlets; each signal integrated for only one proton. In addition, the signal of CH3-2″ of the 2-methylbutanoyl group was split into two overlapping doublets, and that of H3-4″ appeared as two overlapping triplets. The splittings of the signals for C-8, C-4′, C-5, and the carbons of the 2-methylbutanoyl group were also observed in the 13C NMR spectrum. A series of 1H NMR spectra of 1 were obtained in DMSO-d6 at a series of elevated temperatures, and the split signals of the above protons either merged completely (H-3′ and H-5′) or partially (the protons of the two methyl groups in the 2-methylbutanoyl group) as the temperature increased. An example is shown in Table 2. The atropisomerism of 1 presumably arises because the rotation of the C-2–C-10′ bond is restricted by steric effects.
Scutianthraquinone B (2, Figure 1) was obtained as a light-brown amorphous solid. Its molecular formula was established as C38H30O13, differing from that of 1 by a CH2 group, on the basis of its [M-H] peak in its negative ion HRESI mass spectrum. The UV-VIS spectra showed characteristic features of anthraquinones at 248, 272, 310 and 475 nm. The 1H NMR spectrum of 2 was almost identical to that of 1, except that the signals from the C-10′ substituent lacked signals for one methylene group compared with 1 (Table 1). This observation indicated that an isobutanoyl group in 2 had replaced the 2-methylbutanoyl group in 1. HMBC correlations of the H-3a″ and H-3b″ methyl protons (δH 1.17/1.15, d, J = 7.2 Hz) to C-1″ (δC 174.0) and C-2″ (δC 35.0) and TOCSY correlations between the H3-3a″/H3-3b″ methyl protons and H-2″ (δH 2.68, m) were in agreement with the above assignment. The other HMBC and TOCSY correlations of 2 showed the same patterns as those of 1, and confirmed the similarity between 2 and 1. An attempt to determine if the isobutanoyl group was located at C-10′ by ROESY correlations by the same strategy applied to 1 was not successful, due to the small quantity of 2 and a corresponding weak signal. The substituent was thus assigned to C-10′ based on the close similarity of the 1H NMR chemical shifts of 1 and 2. If the isobutanoyl group were located at any other position significant differences in these shifts would be expected, as observed for the monomeric anthraquinone aloesaponarin I and its acetylated form.9 In addition four intramolecularly hydrogen bonded hydroxyl protons were observed at δH 14.89, 13.25, 11.92 and 10.79 in the 1H NMR spectrum of 2 in DMSO-d6. The LC-ESI-MS/MS profile of 2 was essentially identical to that of 1, after allowing for the molecular weight differences, and thus also supported the proposed structure of 2. The proposed fragmentation pattern is shown in Figure S1. Based on the arguments above, the structure of 2 was assigned as shown in Figure 1.
Scutianthraquinone C (3, Figure 1) was obtained as a light-brown amorphous solid. Its molecular formula was established as C34H24O12 on the basis of its molecular ion peak in its HRFAB mass spectrum. The UV-VIS spectra showed characteristic features of anthraquinones at 248, 272, 310 and 475 nm. The 1H NMR spectrum of 3 was almost identical to that of 1 except for the absence of signals for the 2-methylbutanoyl group in 1, indicating the presence of a free hydroxyl group at C-10′. The HMBC and COSY spectra and LC-ESI-MS/MS profile (see proposed fragmentation pattern in Figure S1) of 3 supported the above deduction. Thus, the structure of 3 was established as shown in Figure 1.
Scutianthraquinone D (4, Figure 4) was obtained as a light-brown amorphous solid. Its molecular formula was established as C61H52O20 on the basis of its [M-H] peak in its negative ion HRESI mass spectrum. The UV-VIS spectra showed characteristic features of anthraquinones at 248, 272, 310, 353 and 475 nm. In the 1H NMR spectrum of 4 in CD3OD, two AB systems (δH 8.67, d, J = 7.8 Hz, H-3 and δH 7.95, d, J = 7.8 Hz, H-4, and δH 8.32, d, J = 8.5 Hz, H-3′ and δH 6.92, d, J = 8.5 Hz, H-4′) and one ABX system (δH 6.85, br, d, J = 8.1 Hz, H-2″, δH 7.30, t, J = 8.1 Hz, H-3″ and δH 6.62, br, d, J = 8.1 Hz, H-4″) were observed, and three aromatic proton singlets (δH 7.53, H-5 and δH 6.60/6.59, 6.58/6.57, H-5′, H-5″) also appeared. Singlets for three methoxyl protons (δH 3.90, 3.89, 3.84) and three methyl protons (δH 2.53, 2.60, 2.78) were also observed. In the 1H NMR spectrum of 4 in DMSO-d6, signals for six intramolecularly hydrogen bonded hydroxyl protons (δH 14.92, 13.66, 13.23, 11.91, 10.77 and 9.71) were also observed. The COSY spectrum of 4 confirmed the aforementioned AB and ABC systems, and COSY correlations of the protons from two 2-methylbutanoyl groups also were observed. Careful comparison of 1H NMR and COSY data of 4 to those of 1 indicated the presence of an additional monomeric anthrone unit in 4, indicating a trimeric structure. The existence of six intramolecularly hydrogen bonded hydroxyl protons and comparison of the chemical shifts and coupling patterns in the 1H NMR of 4 to those of 1 indicated that the two 2-methylbutanoyl groups must be located at C-10 and C-10′, and established the structure of 4 as the bisanthrone-anthraquinone shown in Figure 1. The LC-ESI-MS/MS spectrum of 4 fully supported the proposed structure, and the proposed fragmentation pathways of 4 are shown in Figure S2.
Only a few anthrone-anthraquinones with the two moieties connected by a bond between the C-2 position of the anthraquinone unit and C-10′ of the anthrone unit have been reported,10-16 and compounds 1-3 are novel examples of this relatively rare structural type. Compound 4 is the first reported bisanthrone-anthraquinone isolated from Nature.
The isolated compounds were tested against the A2780 human ovarian cancer cell line and were also tested against chloroquine-resistant strains of Plasmodium falciparum Dd2 and FCM29. The results are shown in Table 3. Compounds 1-5 exhibited weak antiproliferative activities against the A2780 human ovarian cancer cell line, with the trimer 4 being slightly more active than dimers 1, 2 and 3, and the monomer 5 showed the weakest activity. Compounds 1-5 showed moderate antiplasmodial activities against the chloroquine-resistant P. falciparum Dd2, with IC50 values in the range of 1-6 μM; the dimers 1-3 showed slightly better activities than the trimer 4, and the monomer 5 displayed the least activity. The dimers 1 and 2 with C-10′ substituents exhibited slightly better activities than the dimer 3 with a free hydroxyl group at C-10′. With respect to the activities against P. falciparum FCM29, compounds 1, 2, and 4 showed moderate activities with IC50 values from 1.2 to 5.6 μM compared to chloroquine (IC50 = 0.41 μM). The selectivities of compounds 1 – 5 for antiplasmodial as opposed to antiproliferative activities, as determined by the quotient of antiproliferative activity and antiplasmodial activity, vary between 0.8 and 6 (Table 3).
These selectivities are lower than would be desirable for future drug development, and this fact, coupled with the chemical complexity of the compounds, makes them challenging compounds to develop. Some related anthrone-anthraquinones had selectivities of up to 400 between the chloroquine-sensitive 3D7 strain of P. falciparum and the KB cell line,17 so it is possible that compounds of this class might ultimately be developable as antimalarial agents. This conclusion is supported by the fact that some phenylanthraquinones, including monomeric18,19 and dimeric examples,20 have promising antimalarial bioactivities.
Optical rotations were recorded on a JASCO P-2000 polarimeter. UV spectra were obtained on a Shimadzu UV-1201 spectrophotometer. NMR spectra were obtained on Bruker Avance 600, JEOL Eclipse 500, Varian Inova 400, and Varian Unity 400 spectrometers. HRFAB mass spectra were obtained on a JEOL-JMS-HX-110 instrument. HRESI mass spectra were obtained on an Agilent 6220 TOF LC/MS. LC-ESIMS was performed on Agilent 1100 and Thermo TSQ Quantum instruments. Chemical shifts are given in δ (ppm), and coupling constants (J) are reported in Hz. HPLC was performed using Shimadzu LC-10A pumps coupled with a Varian Dynamax semipreparative column (250 × 10 mm). The HPLC instrument employed a Shimadzu SPD-M10A diode array detector.
The A2780 ovarian cancer cell line assay was performed at Virginia Polytechnic Institute and State University as previously reported.21 The A2780 cell line is a drug sensitive ovarian cancer cell line.22
Antiplasmodial assays with the chloroquine-resistant strain Plasmodium falciparum FCM29 were performed at the Centre National d'Application des Recherches Pharmaceutiques. Assays with the chloroquine-resistant Plasmodium falciparum Dd2 were performed at Georgetown University. Both assays used the previously reported Cybr green method.23
Bark of the climbing shrub Scutia myrtina (Burm. f.) Kurz (Rhamnaceae) was collected in the Montagne des Français region, a dry forest on limestone, Antsiranana, Madagascar, at an elevation of 280 m, at 12.24.41 S, 49.22.17 E, on February 14, 2005. Its assigned collection number is Randrianasolo.S (SSR) et al. 517. The collection was made from a woody liana, with one pair of spines in each node, green fruit becoming black when mature, brown seeds, growing on slope. Its vernacular name is Roiavotra. Scutia myrtina has a large distribution from Africa to south east Asia. This species is somewhat variable but the variation is chaotic and nearly coextensive throughout the vast range, so that the recognition of infraspecific taxa is not practicable. Voucher specimens have been deposited at herbaria of the Centre National d'Application des Recherches Pharmaceutiques, Madagascar (CNARP); the Parc Botanique et Zoologique de Tsimbazaza, Madagascar (TAN); the Missouri Botanical Garden, St. Louis, Missouri (MO); and the Muséum National d'Histoires Naturelles, Paris, France (P).
The dried and powdered bark of Scutia myrtina (254 g) was extracted at room temperature with EtOH (1.2 L) for 24 h. After filtration, the solvent was evaporated to dryness under reduced pressure, affording a crude ethanolic extract (7.86 g).
A total of 2.5 g of extract was supplied to Virginia Polytechnic Institute and State University, and this had an IC50 value of 12 μg/mL against A2780 cells. This extract (2.0 g) was suspended in aqueous MeOH (90%MeOH-H2O, 60 mL) and extracted with hexanes (3 × 60 mL). The aqueous layer was then diluted to 60% MeOH (v/v) with H2O and extracted with CH2Cl2 (3 × 90 mL). The CH2Cl2 extract (82.2 mg) was found to be the most active against A2780 cells (IC50 = 6 μg/mL) and was separated via semipreparative HPLC over a C18 column using MeOH-H2O (75:25) to afford 9 fractions (I-IX). Fractions II, III, VI, VII and VIII afforded aloesaponarin I (5, 1.2 mg, tR 19.6 min), scutianthraquinone C (3, 1.6 mg, tR 21.4 min), scutianthraquinone B (2, 1.0 mg, tR 31.6 min), scutianthraquinone D (4, 2.0 mg, tR 34.6 min) and scutianthraquinone A (1, 2.9 mg, tR 38.5 min), respectively.
Light-brown amorphous solid; [α]D25 +60.8 (c 0.06, CHCl3); UV (MeOH)λmax nm (log ε): 203 (4.35), 248 (4.09), 272 (4.01), 310 (4.03), 351 sh (3.83) and 475 sh (3.36); HRFABMS m/z: 708.1790 [M]- (Calcd for C39H32O13: 708.1843); LC-ESIMS m/z (rel. int.): [M-H]- 707 (1), 675 (21), 643 (1), 605 (4), 577 (100), 573 (14), 545 (40), 541 (2), 517 (3); 1H NMR: see Table 1; 13C NMR (CD3OD): 192.2 (C-9/C-9′), 190.9 (C-9/C-9′), 183.6 (C-10), 174.9 (C-10′-OCOCH(CH3)CH2CH3), 170.6 (C-7-COOCH3), 169.9 (C-7′-COOCH3), 163.2 (C-1′), 159.8 (C-1), 149.1 (C-6), 149.1 (C-6′), 143.6 (C-4′a), 143.5 (C-8), 141.9 (C-8′), 140.6 (C-2), 138.6 (C-10a/c-10′a), 136.0 (C-10a/C-10′a), 135.9 (C-3′), 133.9 (C-4a), 131.8 (C-7/8a), 131.7 (C-3), 126.6 (C-7′/8a′), 124.1 (C-7′/8a′), 123.5 (C-7/8a), 119.2 (C-4), 119.1 (C-9′a), 118.6 (C-9a), 118.4 (C-2′), 118.1/118.0 (C-4′), 113.9 (C-5), 112.7/112.6 (C-5′), 78.2 (C-10′), 53.0 (C-7-COOCH3), 53.0 (C-7′-COOCH3), 43.0 (C-10′-OCOCH(CH3)CH2CH3), 28.0/27.9 (C-10′-OCOCH(CH3)CH2CH3), 21.2 (8′-CH3), 20.6 (8-CH3), 17.0/16.9 (C-10′-OCOCH(CH3)CH2CH3), 12.0 (C-10′-OCOCH(CH3)CH2CH3).
Light-brown amorphous solid; [α]D25 +134.8 (c 0.04, CHCl3); UV (MeOH)λmax nm (log ε): 203 (4.13), 248 (3.85), 272 (3.78), 310 (3.74), 353 sh (3.54) and 475 (3.08); HRESIMS m/z: 693.1614 [M-H]- (Calcd for C38H30O13: 693.1608); LC-ESIMS m/z (rel. int.): 693 (1) [M-H]-, 661 (48), 629 (4), 605 (12), 577 (100), 573 (22), 545 (80), 541 (2), 517 (8); 1H NMR: see Table 1.
Light-brown amorphous solid; [α]D25 +122.2 (c 0.04, MeOH); UV (MeOH)λmax nm (log ε): 203 (4.24), 248 (3.98), 272 (3.92), 310 (3.90), and 475 sh (3.29); HRFABMS m/z 624.1296 [M]- (Calcd for C34H24O12: 624.1268); LCESIMS m/z (rel. int.): 623 (12) [M-H]-, 605 (4), 591 (100), 577 (88), 573 (28), 545 (84), 541 (64), 517 (12), 513 (10), 487 (8), 459 (4), 311 (5); 1H NMR: see Table 1.
Light-brown amorphous solid; [α]D25 -34.7 (c 0.04, CHCl3); UV (MeOH)λmax nm (log ε): 203 (4.64), 248 (4.40), 272 (4.34), 310 (4.25), 353 (4.12) and 475 sh (3.51); HRESIMS m/z: 1103.2977 [M-H]- (Calcd for C61H52O20: 1103.2979); LC-ESIMS m/z (rel. int.): 1103 (20) [M-H]-, 1071 (1), 1001 (6), 899 (40), 871 (100), 867 (14), 839 (4), 811(1); 1H NMR: see Table 1.
Light-brown amorphous solid; UV (MeOH) λmax nm (log ε): 215 (4.00), 270 (3.87), 280 (3.86), 307 sh (3.53), 408 (3.27), 430 (3.26) and 475 sh (2.98); HRFABMS m/z: 312.0609 [M]- (Calcd for C17H12O6: 312.0634); 1H NMR (400 MHz, CDCl3) δ 7.80 (1H, s, H-5), 7.78 (1H, br, d, J = 8.0 Hz, H-4), 7.63 (1H, t, J = 8.0 Hz, H-3), 7.32 (1H, br, d, J = 8.0 Hz, H-2), 4.07 (3H, s, 7-COOCH3), 2.98 (3H, s, 8-CH3); 1H NMR (500 MHz, CD3OD) δ 7.70 (1H, br, d, J = 7.8 Hz, H-4), 7.65 (1H, t, J = 7.8 Hz, H-3), 7.61 (1H, s, H-5), 7.28 (1H, br, d, J = 7.8 Hz, H-2), 3.94 (3H, s, 7-COOCH3), 2.69 (3H, s, 8-CH3); 13C NMR (500 MHz, CD3OD) δ 191.1 (C-9), 184.0 (C-10), 169.9 (7-COOCH3), 163.7 (C-1), 161.7 (C-6), 143.3 (C-8), 138.8 (C-10a), 137.0 (C-3), 134.3 (C-4a), 131.7 (C-8a/7), 125.7 (C-2), 124.0 (C-7/8a), 119.7 (C-4), 118.4 (C-9a), 113.7 (C-5), 53.2 (7-COOCH3), 20.6 (8-CH3).
This project was supported by the Fogarty International Center, the National Cancer Institute, the National Science Foundation, the National Heart Lung and Blood Institute, the National Institute of Mental Health, the Office of Dietary Supplements, and the Office of the Director of NIH, under Cooperative Agreement U01 TW00313 with the International Cooperative Biodiversity Groups. This support is gratefully acknowledged. We also thank Dr. Mehdi Ashraf-Khorassani, Dr. Hugo Azurmendi and Mr. William Bebout for assistance in obtaining LC-ESIMS, NMR and HRFAB mass spectra, respectively. Field work essential for this project was conducted under a collaborative agreement between the Missouri Botanical Garden and the Parc Botanique et Zoologique de Tsimbazaza and a multilateral agreement between the ICBG partners, including the Centre National d'Applications des Recherches Pharmaceutiques. We gratefully acknowledge courtesies extended by the Government of Madagascar (Ministère des Eaux et Forêts).
Supplementary data: Proposed mass spectrometric fragmentation schemes for compounds 1 – 4. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmc.2009. xx.yyy.
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