Triterpenes from Maytems macrocarpo Natural Product Communications Vol.1(12)2006 1075 allowed us to clarify the difference between the two acyl moiety identified as(E)-p-coumaroyl.In the compounds hile.in the case of 3.the ROES HMBC spectrum,the proton at 84.58 (H-3) 1(Me-30) 0 correlated with the carbon resonance at 8 167.2 H-22 28 ted I hu tha 1 s126(Me-29 3-(E)-coumaroyluvac d latio d to the The known compounds maytenfolic that 1 3B.22a-dihvdr 2-cn-30-oic carpoic acid A. maytenfolic acid (5)[16].22-epi-trip totrit acid A (6)[16].3-(E)-caf 阁 Compound 2 showed,in the ESI-MS in positive ion mode,the quasi-molecular ion [M+H]at m/471, 6ncolceular coumais)-caffeoylbetulin (11) spec secopets .11x2 with those 1.33,onc one methine to an oxygen The co-occurrence in M.macrocarpa of triterpenes aring carbor belonging to different classes is an unusual finding 30 For maytenfolic acid (3).previously isolated from M at6123.4.1435.181.0 and 2190 inc 2 aierst antileukemic xoolean-12-eno acid 13 ity has been lin esters nounds 7.8 are esters of uvaol.fo hich antiproliferative and antileukemic activity at 83.19 in the 'H NMR spectrum and 79.7 in the have been reported [20]. C NMR spectrum,observed for 1.It was confirmed Table:Anti-HIVactivity of omounds 1-13 by the long range correlations observed in the HMBC the signals at 6 1.09 Compounds proton (C-3) Thus 2 was assigned 00 a 00 22a-hydroxy-olean -0X0-30-01c acid.and named mac ula 8 und which showed the molecular ion peak [M+H 00002050m 00006500 at m/589.The 'H NMR spectrum showed seven methyl groups at 0.88(d,J=6.5 Hz),0.96(s),0.97 (d,J=6.5Hz.1.01(s.1.08(s,1.09(s,and1.17 EC =CO (s).two signals ascribable to a primary 3.08 and 3.59 (each IH,d, 1.0Hz a me ne proton linked to On the basis of the anti-HIV activity reported for the derivative bet linic acid [8-91. nd to closely related to orthosphenic acid [10],the anti 683 HIV activity of compounds 1-13 in C8166 cells HZ) 02 80 infected with HIV-IMN was tested.The most active typical of a 1.4-disub ing The NMR spectros above (Tabl h the me anism the chemical shift of C-3 and the occurrence of anTriterpenes from Maytenus macrocarpa Natural Product Communications Vol. 1 (12) 2006 1075 allowed us to clarify the difference between the two compounds. While, in the case of 3, the ROESY spectrum showed correlations between the signal at δ 1.22 (Me-30) and the signals at δ 2.36 (H-18), 3.52 (H-22), and 1.01 (Me-28), these effects were absent in the ROESY spectrum of 1, where the signal at δ 1.26 (Me-29) showed a correlation with that at δ 1.33 (Me-27). This evidence led to the conclusion that 1 was 3β,22α-dihydroxy-olean-12-en-30-oic acid, which was named macrocarpoic acid A. Compound 2 showed, in the ESI-MS in positive ion mode, the quasi-molecular ion [M+H]+ at m/z 471, corresponding to the molecular formula C30H46O4. The compound exhibited, in its 1 H NMR spectrum, seven singlet methyl groups (δ 0.95, 1.09, 1.11 x 2, 1.13, 1.26, 1.33), one olefinic proton (δ 5.25), and one methine proton (δ 3.63) linked to an oxygen bearing carbon. In the 13C NMR spectrum the occurrence of signals for 30 carbons, including peaks at δ 123.4, 143.5, 181.0 and 219.0, suggested that 2 was an oxoolean-12-enoic acid [13]. By comparison of NMR data of 2 with those of 1 it was speculated that the position of the carbonyl group (δ 219.0) was at C-3 on the basis of the absence in 2 of the signals at δ 3.19 in the 1 H NMR spectrum and 79.7 in the 13C NMR spectrum, observed for 1. It was confirmed by the long range correlations observed in the HMBC spectrum between the proton signals at δ 1.09 (Me-23) and 1.11 (Me-24) and the carbon resonance at δ 219.0 (C-3). Thus 2 was assigned as 22α-hydroxy-olean-12-en-3-oxo-30-oic acid, and named macrocarpoic acid B. The molecular formula C39H56O4 was assigned to compound 7 on the basis of the ESI-MS spectrum, which showed the quasi-molecular ion peak [M+H]+ at m/z 589. The 1 H NMR spectrum showed seven methyl groups at δ 0.88 (d, J = 6.5 Hz), 0.96 (s), 0.97 (d, J = 6.5 Hz), 1.01 (s), 1.08 (s), 1.09 (s), and 1.17 (s), two signals ascribable to a primary alcoholic function at δ 3.08 and 3.59 (each 1H, d, J = 11.0 Hz), and a signal for a methine proton linked to an oxygen-bearing carbon at δ 4.58 (dd, J = 3.5 and 11.0 Hz). Further features were signals due to three olefinic protons at δ 5.21 (t, J = 3.5), 6.32 (d, J = 15.9 Hz ), and 7.64 (d, J = 15.9 Hz ), and signals at δ 6.83 (2H, d, J = 8.0 Hz) and 7.50 (2H, d, J = 8.0 Hz), typical of a 1,4-disubstituted aromatic ring. The 13C NMR spectroscopic data of 7 were similar to those of urs-12-en-3β,28-diol, known as uvaol [14], except for the chemical shift of C-3 and the occurrence of an acyl moiety identified as (E)-p-coumaroyl. In the HMBC spectrum, the proton at δ 4.58 (H-3) correlated with the carbon resonance at δ 167.2 (C=O), indicating that the (E)-coumaroyl group was located at C-3. Thus compound 7 was identified as 3-(E)-coumaroyluvaol, and named macrocarpol A. The known compounds maytenfolic acid (3) [11], triptotriterpenonic acid A (4) [15], 22-epi￾maytenfolic acid (5) [16], 22-epi-triptotriterpenonic acid A (6) [16], 3-(E)-caffeoyluvaol (8) [14], 3-(E)-p￾coumaroylbetulin (9) [17], 3-(Z)-p-coumaroylbetulin (10) [18], 3-(E)-caffeoylbetulin (11) [18], nepeticin (12) [12], and orthosphenic acid (13) [12] were identified by comparison of their spectroscopic data with those reported in the literature. The co-occurrence in M. macrocarpa of triterpenes belonging to different classes is an unusual finding. For maytenfolic acid (3), previously isolated from M. diversifolia [11], antileukemic activity has been reported [11], while compounds 9-11, betulin esters, have been reported to exert antitumor promoting activity [19]. Compounds 7-8 are esters of uvaol, for which antiproliferative and antileukemic activity have been reported [20]. Table 1: Anti-HIV activity of compounds 1-13. Compounds EC50a TC50b 1 10 50 2 10 50 3 10 80 4 1 35 5 100 200 6 50 100 7 10 50 8 10 100 9 12 62 10 10 25 11 5 50 12 10 50 13 10 80 EC50a = concentration (μg/mL) that reduced by 50% the production of gp120 in infected C8166 cells. TC50b = concentration (μg/mL) that caused 50% cytotoxicity to uninfected C8166 cells. On the basis of the anti-HIV activity reported for the lupane derivative betulinic acid [8-9], and, to a minor extent, for salaspermic acid, a friedelane derivative closely related to orthosphenic acid [10], the anti￾HIV activity of compounds 1-13 in C8166 cells infected with HIV-1MN was tested. The most active compound was 4, with an EC50 value of 1μg/mL and a selectivity index above 30 (Table 1). The investigation of the mechanism of action of 4 revealed that it was more effective when added either