This Issue is Dedicated to the Memory of Professor Ivano Morelli

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1076 Natural Product Communications Vol. 1 (12) 2006 Piacente et al. prior to or at the time of virus infection. Further experiments confirmed that it inhibited the binding of gp120 to sCD4 in a dose dependent manner. At a concentration corresponding to its EC 50 , compound 4 inhibited the interaction between gp120 and CD4 by 55%. The activity exerted by betulin esters (9-11) is very much lower than that reported for betulinic acid and its derivatives by Hashimoto et al. [8]. Also orthosphenic acid (13) showed an anti-HIV activity lower than that reported for salaspermic acid [10], from which it differs only by the occurrence of a further α-OH group at C-2. Experimental General procedures: Optical rotations were measured on a Jasco DIP 1000 polarimeter. UV spectra were obtained on a Beckman DU 670 spectrometer. IR measurements were obtained on a Bruker IFS-48 spectrometer. Melting points were determined using a Bausch & Lomb apparatus. Accurate molecular weights were measured by a Voyager DE mass spectrometer. Samples were analyzed by matrix assisted laser desorption ionization (MALDI) mass spectrometry. A mixture of analyte solution and α-cyano-4-hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass calibration was performed with the ions from ACTH (fragment 18-39) at 2465.1989 Da and Angiotensin III at 931.5154 Da as internal standards. ESI-MS analyses were performed using a ThermoFinnigan LCQ Deca XP Max ion trap mass spectrometer equipped with Xcalibur software. NMR experiments were performed on a Bruker DRX-600 spectrometer at 300 K. All the 2D-NMR spectra were acquired in CD 3 OD. Standard pulse sequence and phase cycling were used for DQF-COSY, HSQC, HMBC and ROESY spectra. TLC was performed on silica gel F254 (Merck) plates, and reagent grade chemicals (Carlo Erba) were used throughout. Plant material: Maytenus macrocarpa was collected in the Ucayali Region (Pucallpa), Peru. A voucher specimen is deposited at the Department of Pharmaceutical Sciences, Salerno, Italy. Extraction and isolation: The dried and powdered bark of M. macrocarpa (310 g) was defatted with light petroleum and then extracted by maceration at room temperature with CHCl 3 until exhaustion. The CHCl 3 extract was concentrated under reduced pressure to a syrupy consistency (2.5 g). The crude extract was chromatographed on a silica gel column using CHCl 3 and increasing amounts of MeOH (up to 20%). After monitoring by TLC [Si gel plates, CHCl 3 -MeOH (9:1)], the fractions were combined to give A (350 mg), B (150 mg), C (200 mg) and D (280 mg). Fractions A-D were submitted to HPLC on a μ-Bondapack C-18 column (30 cm x 7.8 mm i.d., flow rate 2.0 mL/min) using MeOH: H 2 O in the ratio 85:15 for A-C and 3:1 for D (isocratic conditions). Pure 11 (12 mg, Rt = 12 min), 9 (8 mg, Rt = 15 min) and 10 (6 mg, Rt = 19 min) were obtained from A; 12 (9 mg, Rt = 24 min) from B; 7 (5 mg, Rt = 20 min), 8 (8 mg, Rt = 19 min) from C; 1 (7 mg, Rt = 8 min), 2 (5 mg, Rt = 12 min), 3 (12 mg, Rt = 10 min), 4 (10 mg, Rt = 15 min), 5 (7 mg, Rt = 11 min), 6 (9 mg, Rt = 16.5 min), and 13 (18 mg, Rt = 7 min) from D. Antiviral assays: The anti-HIV activity and toxicity of compounds 1-13 were assessed in C8166 cells infected with HIV-1 MN . Cells were cultured in RPMI 1640 with 10% fetal calf serum. Forty-thousand cells per microtiter plate well were mixed with 5-fold dilutions of compounds prior to addition of 10 CCID 50 units of virus and incubated for 5-6 days. Formation of syncytia was examined from 2 days post-infection. The inhibition of HIV-infection was determined by examining syncytia, by estimating antigen gp120 by ELISA, and by measuring cell viability for virus-infected cells and uninfected cell controls using the XTT-formazan method. Virus infectivity assay: The total progeny virus was titrated in microtiter plates using double dilutions of freshly collected supernatants and C8166 cells. The end point was determined by examining syncytia formation and by the XTT-formazan method. Virus titer (CCID 50 ) is expressed as the reciprocal of the dilution that gave a 50% end point. To measure the effects of compounds on virus infectivity, HIV-1 MN (10 4 -10 5 CCID 50 ) was incubated with test compound at 37°C for 1h, the virus was serially diluted, and the infectivity end-point determined. Gp120-sCD4 interaction assay: Gp120-sCD4 interaction was measured by ELISA; sCD4 was bound to microtiter plate wells at a concentration of 0.05 μg/well. Various dilutions of compounds were mixed with equal volumes of recombinant gp120 (0.04 μg/mL) and added to CD4 coated wells. After incubation at 37°C for 3-5 h, the binding of gp120 was detected using human anti-HIV serum and antihuman Ig conjugated to horseradish peroxidase.
Triterpenes from Maytenus macrocarpa Natural Product Communications Vol. 1 (12) 2006 1077 Using WIACALC (Pharmacia LKB), the percent inhibition was calculated from linear logarithmic plots using three concentrations of gp120 alone as standard. Macrocarpoic acid A (1) MP: 272-274ºC. [α] D : +48.2º (c 0.1, CHCl 3 ). Rf : 0.6 (CHCl 3 -MeOH, 9:1). IR (KBr): 3480, 2970-2880, 1690, 1450, 1360, 1230, 1070 cm -1 . 1 H NMR (600 MHz, MeOH): 0.82 (3H, s, Me-23), 0.94 (3H, s, Me-28), 1.00 (3H, s, Me-25), 1.03 (3H, s, Me-24), 1.05 (3H, s, Me-26), 1.26 (3H, s, Me-29), 1.33 (3H, s, Me-27), 3.19 (1H, dd, J = 3.5 and 11.5 Hz), 3.62 (1H, dd, J = 3.0 and 11.0 Hz), 5.24 (1H, t, J = 3.5). 13 C NMR (150 MHz MeOH): 15.7 (CH 3 , C-25), 16.1 (CH 3 , C-24), 17.3 (CH 3 , C-26), 19.0 (CH 2 , C-6), 24.4 (CH 2 , C-11), 25.8 (CH 3 , C-28), 26.3 (CH 3 , C-27), 26.5 (CH 2 , C-15), 28.2 (CH 2 , C-2), 28.3 (CH 3 , C-23), 29.0 (CH 2 , C-16), 33.4 (CH 2 , C-7), 34.1 (CH 3 , C-29), 37.8 (C, C-10), 38.9 (CH 2 , C-1), 39.5 (C, C-17), 40.1 (C, C-4), 40.7 (CH 2 , C-21), 41.2 (C, C-8), 43.5 (C, C-14), 44.3 (CH 2 , C-19), 46.2 (C, C-20), 48.9 (CH, C-9), 49.9 (CH, C-18), 56.5 (CH, C-5), 78.8 (CH, C-22), 79.6 (CH, C-3), 123.5 (CH, C-12), 144.5 (C, C-13), 181.1 (C, C-30). ESI-MS: 473 [M + H + ] , 495 [M + Na + ] . HRMS-MALDI: m/z [M + Na + ] calcd for C 30 H 48 NaO 4 495.3450, found 495.3458. Macrocarpoic acid B (2) MP: 264-266ºC. [α] D : +76.2º (c 0.1, CHCl 3 ). Rf : 0.7 (CHCl 3 -MeOH, 9:1). IR (KBr): 3450, 2980-2840, 1730, 1710, 1450, 1380, 1230, 1120 cm -1 . 1 H NMR (600 MHz, MeOH): 0.95 (3H, s, Me-28), 1.09 (3H, s, Me-24), 1.11 (6H, s, Me-23, Me-26), 1.13 (3H, s, Me-25), 1.26 (3H, s, Me-29)1.33 (3H, s, Me-27), 3.63 (1H, dd, J = 3.0 and 11.0 Hz), 5.25 (1H, t, J = 3.5). 13 C NMR (150 MHz MeOH): 15.5 (CH 3 , C-25), 17.2 (CH 3 , C-26), 20.3 (CH 2 , C-6), 21.7 (CH 3 , C-24), 24.0 (CH 2 , C-11), 25.8 (CH 3 , C-28), 26.2 (CH 3 , C-27), 26.3 (CH 2 , C-15), 26.6 (CH 3 , C-23), 26.7 (CH 2 , C-7), 29.0 (CH 2 , C-16), 34.3 (CH 3 , C-29), 35.0 (CH 2 , C-2), 37.8 (C, C-10), 39.5 (C, C-17), 40.1 (CH 2 , C-1), 40.6 (CH 2 , C-21), 40.5 (C, C-8), 43.4 (C, C-14), 44.2 (CH 2 , C-19), 46.0 (C, C-20), 48.0 (C, C-4), 47.5 (CH, C-9), 49.8 (CH, C-18), 56.0 (CH, C-5), 78.7 (CH, C-22), 123.4 (CH, C-12), 143.5 (C, C-13), 181.0 (C, C-30), 219.0 (C, C-3). ESI-MS: 471 [M + H + ] , 493 [M + Na + ] . HRMS-MALDI: m/z [M + Na + ] calcd for C 30 H 46 NaO 4 493.3294, found 493.3301. Macrocarpol A (7) MP: 310-312ºC. [α] D : +42.0º (c 0.1, CHCl 3 ). Rf : 0.7 (CHCl 3 -MeOH, 9:1). IR (KBr): 3440, 2930, 1680, 1600, 1188 cm -1 . 1 H NMR (600 MHz, MeOH): 0.88 (3H, s, Me-29), 0.96 (3H, s, Me-23), 0.97 (3H, s, Me-30), 1.01 (3H, s, Me-24), 1.08 (3H, s, Me-25), 1.09 (3H, s, Me-26), 1.17 (3H, s, Me-27), 3.08 (1H, d, J = 11.0 Hz, H-28a), 3.59 (1H, d, J = 11.0 Hz, H-28b), 4.58 (1H, dd, J = 3.5, 12.0 Hz), 5.21 (1H, t, J = 3.5, H-12), 6.32 (1H, d, J = 15.9), 6.83 (1H, d, J = 8.0, H-3’, H-5’), 7.50 (1H, d, J = 8.0, H-2’, H-6’), 7.64 (1H, d, J = 15.9), 13 C NMR (150 MHz MeOH): 16.3 (CH 3 , C-25), 17.3 (CH 3 , C-26), 17.5 (CH 3 , C-24), 17.8 (CH 3 , C-29), 18.8 (CH 2 , C-6), 21.6 (C, C-30), 23.9 (CH 3 , C-27), 24.1 (CH 2 , C-16), 24.2 (CH 2 , C-11), 25.9 (CH 2 , C-2), 26.7 (CH 2 , C-15), 28.3 (CH 3 , C-23), 32.2 (CH 2 , C-21), 34.3 (CH 2 , C-7), 36.5 (CH, C-22), 37.7 (C, C-17), 38.0 (C, C-10), 38.7 (C, C-4), 39.8 (CH 2 , C-1), 40.7 (CH, C-20), 41.3 (CH, C-8), 41.4 (CH, C-19), 43.3 (C, C-14), 49.4 (CH, C-9), 55.3 (CH, C-18), 56.7 (CH, C-5), 70.5 (CH 3 , C-28), 115.8 (CH, C-3’, C-5’), 116.4 (CH, C-8’), 125.1 (CH, C-12), 127.3 (C, C-1’), 129.5 (CH, C-2’, C-6’), 140.8 (C, C-13), 143.8 (CH, C-7’), 157.4 (C, C-4’) 167.2 (C, C-9’). ESI-MS: 589 [M + H + ] , 611 [M + Na + ] . HRMS-MALDI: m/z [M + Na + ] calcd for C 39 H 56 NaO 4 611.4076, found 611.4082. Acknowledgments - The authors thanks Dr Juan de Dioz Zuniga Quiroz of Agro Selva Zuniga, Parque Caceres Dorregaray 86-C Pueblo Libre, Lima 21, Peru, for providing the plant material. References [1] Piacente S, De Tommasi N, Pizza C. (1999) Laevisines A and B: two new sesquiterpene-pyridine alkaloids from Maytenus laevis. Journal of Natural Products, 62, 161-163.
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