Isolation, Structural Modification, and HIV Inhibition of Pentacyclic

Article pubs.acs.org/jnp

Isolation, Structural Modification, and HIV Inhibition of Pentacyclic Lupane-Type Triterpenoids from Cassine xylocarpa and Maytenus cuzcoina Oliver Callies,† Luis M. Bedoya,‡,§ Manuela Beltrán,‡ Alejandro Muñoz,‡ Patricia Obregón Calderón,‡ Alex A. Osorio,† Ignacio A. Jiménez,† José Alcamí,‡ and Isabel L. Bazzocchi*,† †

Instituto Universitario de Bio-Orgánica “Antonio González”, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain ‡ Unidad de Inmunopatología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Pozuelo Km.2, 28220-Majadahonda, Madrid, Spain § Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid, Ramón y Cajal s/n, 28040, Madrid, Spain S Supporting Information *

ABSTRACT: As a part of our investigation into new anti-HIV agents, we report herein the isolation, structure elucidation, and biological activity of six new (1−6) and 20 known (7−26) pentacyclic lupane-type triterpenoids from the stem of Cassine xylocarpa and root bark of Maytenus cuzcoina. Their stereostructures were elucidated on the basis of spectroscopic and spectrometric methods, including 1D and 2D NMR techniques. To gain a more complete understanding of the structural requirements for anti-HIV activity, derivatives 27−48 were prepared by chemical modification of the main secondary metabolites. Sixteen compounds from this series displayed inhibitory effects of human immunodeficiency virus type 1 replication with IC50 values in the micromolar range, highlighting compounds 12, 38, and 42 (IC50 4.08, 4.18, and 1.70 μM, respectively) as the most promising anti-HIV agents. dimethylsuccinyl)betulinic acid], a first-in-class HIV-1 maturation inhibitor.7,8 In our previous search for anti-HIV agents from plants, pentacyclic triterpenoids from the oleanane series isolated from Celastraceae species have demonstrated potent anti-HIV activity.9 In continuing research toward the discovery of naturally occurring antiretroviral agents, the current study reports the isolation, structural modification, and anti-HIV evaluation of a series of pentacyclic lupane-type triterpenoids with unusual substitution pattern on the triterpene scaffold. This series included 26 compounds (1−26), isolated from Cassine xylocarpa and Maytenus cuzcoina, and 22 analogues (27−48), 13 of which are reported for the first time. Their structures have been elucidated on the basis of spectrocopic analysis, including 1D and 2D NMR techniques (COSY, HSQC, HMBC, and ROESY), and spectrometric methods. The compounds have been tested for their effects on HIV type 1 replication, and the most effective inhibitors, those with a percentage of HIV-1 inhibition higher than 50%, were selected for further IC50 calculations to compare potencies. The structure−activity relationship revealed that the regiosubstitu-

T

hree decades into the acquired immunodeficiency syndrome (AIDS) epidemic, 35 million people are living with this disease worldwide and, up to now, 39 million people have died of AIDS-related illness. Currently, antiretroviral therapy has dramatically decreased morbidity and mortality and is capable of suppressing viral loads to undetectable levels, making AIDS a chronic disease. However, despite these advances, toxicity problems, patient adherence, adverse effects, and, particularly, the emergence of drug-resistant strains of human immunodeficiency virus (HIV), along with restricted accessibility in resource-poor areas, emphasize the urgent need for new anti-HIV drugs with simpler treatment regimens, acceptable toxicity, and novel mechanisms of action.1 Natural compounds play an important role in overcoming the current urgency in anti-HIV/AIDS therapies. In fact, various natural compounds are currently in preclinical or clinical studies for the treatment of HIV infections.2 In particular, lupane-type pentacyclic triterpenoids have been extensively studied as anti-HIV agents, providing a versatile structural platform for the discovery of new biologically active compounds.3 Over the last two decades, many derivatives of the well-known lupane-type triterpenoid betulinic acid (3βhydroxylup-20(29)-en-28-oic acid) have been reported to inhibit HIV replication,4−6 including bevirimat [3-O-(3′,3′© XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 18, 2014

A

DOI: 10.1021/np501025r J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Article

RESULTS AND DISCUSSION Natural Lupane Triterpenoids. Repeated chromatography on Sephadex LH-20 and silica gel of the CH2Cl2 fraction of the stems of C. xylocarpa and n-hexane−Et2O extract of the root barks of M. cuzcoina yielded six new triterpenoids (1−6) along with 20 known lupane metabolites (7−26). Compound 1 gave a molecular formula of C30H48O3, as established on the basis of the 13C NMR and HREIMS spectra, requiring seven indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3462 cm−1) and carbonyl (1701 cm−1) groups. The 1H NMR spectrum (Table 1) showed six methyl groups [δH 0.80, 0.91, 1.14, 1.41, 1.42, and 1.43 (each 3H, s)], one methylene group [δH 2.23 (ddd, J = 2.8, 4.8, 14.8 Hz), 2.79 (td, J = 6.4, 14.8 Hz), H2-2)], one hydroxymethylene group at δH 4.12 (2H, br s, H-30), one oxymethine proton [δH 4.48 (1H, br s, H-6)], and two deshielded exocyclic methylene protons at δH 4.91 and 4.94 (2H, br s, H-29). The 13C NMR spectrum (Table 2) showed 30 carbon resonances, which were further classified by DEPT experiments as six methyls, 11 methylenes, six methines, a ketocarbonyl, an olefinic quaternary carbon, and five quaternary carbons, in agreement with the molecular formula. The carbonyl carbon resonance at δC 216.7 indicated the presence of a ketocarbonyl moiety, the two sp2 carbons at δC 154.6 (C) and 106.9 (CH2) were consistent with a Δ20(29) double bond

tion and oxidation degree are key to modulating the potency of HIV inhibition.

Table 1. 1H NMR Dataa (δ, CDCl3, J are Given in Parentheses in Hertz) for Compounds 1−6 position

1b

2c

3d

1

1.25, 1.97 ddd (3.0, 6.5, 13.1) 2.23 ddd (2.8, 4.8, 14.8) 2.79 td (6.4, 14.8)

1.20, 1.93 ddd (2.9, 6.4, 13.1) 2.21 ddd (2.9, 4.8, 15.0) 2.79 td (6.4, 15.0)

1.38, 1.91 ddd (4.4, 7.5, 13.3) 2.46 ddd (4.4, 7.6, 15.6) 2.51 ddd (7.4, 9.9, 15.7)

1.12 4.47 1.61 1.68 1.33 1.43 1.25

1.36 1.50 1.46

9 11 12

1.15 4.48 br s 1.63 dd (2.5, 15.7) 1.68 dd (3.7, 15.7) 1.40 1.41, 1.52 1.12, 1.49

13 15

1.77 1.02, 1.76

1.78 dt (3.7, 12.4) 0.99, 1.75

1.73 1.05, 1.70

16

1.40, 1.52

18 19 21 22 23 24 25 26 27 28

1.49 2.30 dt (5.3, 11.0) 1.34, 2.08 1.28, 1.42 1.14 s 1.41 s 1.43 s 1.42 s 0.91 s 0.80 s

1.42, 1.53 ddd (2.6, 4.5, 13.2) 1.36 2.77 1.24 1.37, 1.44 1.14 s 1.40 s 1.41 s 1.40 s 0.88 s 0.83 s

1.45, 1.54 ddd (2.6, 4.5, 13.1) 1.66 2.74 dt (4.3, 11.2) 1.40, 2.20 1.39 1.09 s 1.05 s 0.95 s 1.09 s 0.97 s 0.85 s

29

4.91 br s 4.94 br s 4.12 br s

5.92 s 6.29 s 9.51 s

5.71 s 6.25 s

2 3 5 6 7

30

d (2.1) br s dd (2.8, 15.0) dd (3.7, 14.9)

4c

1.38 1.30, 1.44 1.03, 1.28

5c

6b

0.90, 1.68

3.78 dd (4.9, 11.1)

1.64

1.75 ddd (3.6, 4.8, 13.8)

1.61, 2.68 ddd (5.7, 8.6, 14.5) 2.42, 2.46 ddd (5.5, 8.8, 15.0)

3.14 dd (5.6, 8.7) 0.69 d (1.8) 4.53 dd (2.9, 5.7) 1.62 dd (2.8, 14.4) 1.68 1.30 1.40, 1.55 1.31, 1.89

3.49 t (3.0) 1.22 dd (2.0, 11.8) 1.43, 1.50 1.37, 1.43

1.82 1.02 ddd (2.3, 4.6, 13.9) 1.81 1.35, 1.50 ddd (2.2, 4.8, 12.8) 1.34 1.81 1.24, 1.88 1.10, 1.30 1.06 s 1.15 s 1.21 s 1.38 s 0.93 s 0.82 s 1.12 s 1.23 s

1.48 1.48

1.64 dd (3.6, 13.2) 2.14 br d (12.9) 1.11 dd (4.7, 13.7) 1.63 1.65 1.12, 1.68

1.48 3.91 td (5.3, 10.7) 1.12, 1.92 1.83 1.17, 1.62

1.22, 1.92

1.98

1.56 t (11.9) 2.38 dt (6.0, 11.2) 1.41, 1.96 1.04, 1.84 0.91 s 0.83 s 0.90 s 1.03 s 1.00 s 3.34 d (10.7) 3.80 dd (1.2, 10.7) 4.56 dd (1.3, 2.2) 4.67 d (2.1) 1.67 s

1.70 2.42 1.99 1.86 1.12 1.08 1.09 1.07 1.04 3.37 3.78 4.64 4.74 1.72

s s s s s d (10.8) d (10.8) br s br s s

a

Signals without multiplicity are overlapped and deduced by HSQC experiments. bRecorded at 400 MHz. cRecorded at 600 MHz. dRecorded at 500 MHz. B



Article

Table 2. 13C NMR Dataa (δ, CDCl3) for Compounds 1−6 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a

1b 42.1, 34.4, 216.7, 48.9, 56.5, 69.6, 42.2, 40.0, 50.6, 36.7, 21.3, 26.7, 37.1, 42.9, 27.4, 35.3, 43.1, 48.9, 43.7, 154.6, 31.7, 39.8, 25.0, 23.7, 17.0, 17.1, 14.8, 17.7, 106.9, 65.0,

CH2 CH2 C Cd CH CH CH2 C CH C CH2 CH2 CH C CH2 CH2 C CHd CH C CH2 CH2 CH3 CH3 CH3 CH3 CH3 CH3 CH2 CH2

2c 42.1, 34.5, 216.8, 49.0, 56.6, 69.7, 42.2, 40.0, 50.5, 36.7, 21.3, 29.7, 36.8, 43.2, 27.4, 35.3, 43.0, 50.6, 42.1, 157.2, 32.7, 39.8, 25.0, 23.7, 16.9, 17.1, 14.7, 17.8, 133.2, 195.1,

3b CH2 CH2 C C CH CH CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH3 CH3 CH3 CH3 CH3 CH3 CH2 CH

39.6, 34.1, 218.2, 47.3, 54.9, 19.6, 33.6, 40.7, 49.6, 36.8, 21.5, 29.7, 37.8, 42.8, 27.3, 35.4, 43.1, 50.9, 40.7, 146.3, 32.9, 39.7, 26.6, 21.0, 15.9, 15.8, 14.4, 17.9, 124.9, 171.2,

4c

CH2 CH2 C C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH3 CH3 CH3 CH3 CH3 CH3 CH2 C

40.7, 27.6, 79.1, 39.6, 55.5, 69.1, 42.4, 40.4, 50.9, 36.6, 21.6, 28.8, 36.6, 43.8, 27.5, 35.5, 44.6, 48.4, 49.9, 73.5, 29.2, 40.2, 27.7, 16.9, 17.8, 17.2, 15.2, 19.2, 24.8, 31.6,

CH2 CH2 CH C CH CH CH2 C CH Cd CH2 CH2 CHd C CH2 CH2 C CH CH C CH2 CH2 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

5b 75.9, 36.4, 76.6, 37.4, 47.8, 18.4, 34.0, 42.9, 51.2, 37.4, 23.7, 25.2, 36.9, 41.7, 27.1, 29.2, 43.6, 48.8, 47.8, 150.3, 29.7, 34.0, 27.7, 21.9, 11.7, 16.2, 14.8, 60.6, 109.8, 19.0,

CH CH2 CH Cd CHd CH2 CH2d C CH Cd CH2 CH2 CH C CH2 CH2 C CH CHd C CH2 CH2d CH3 CH3 CH3 CH3 CH3 CH2 CH2 CH3

6b 41.8, 34.0, 218.3, 47.5, 54.6, 19.4, 33.9, 42.2, 54.5, 38.0, 70.1, 37.2, 36.1, 42.2, 26.8, 33.6, 47.3, 47.8, 47.4, 149.5, 29.4, 28.8, 27.2, 20.5, 16.5, 16.6, 14.4, 60.3, 110.1, 18.9,

CH2 CH2 C C CH CH2 CH2 Cd CH C CH CH2 CH Cd CH2 CH2 C CH CH C CH2 CH2 CH3 CH3 CH3 CH3 CH3 CH2 CH2 CH3

Data are based on DEPT, HSQC, and HMBC experiments. bRecorded at 100 MHz. cRecorded at150 MHz. dOverlapping signals.

showed NOE correlations of the proton at δH 4.48 with the Me-23 and H-5 (δH 1.15), which confirmed a β-axial orientation of the hydroxy group. All of these data and comparison with reported data for betulone10 established the structure of 1 as 6β,30-dihydroxylup-20(29)-en-3-one. Compound 2 exhibited an [M+] ion peak at m/z 454 in the EIMS, and its molecular formula C30H46O3 (HREIMS) indicated two fewer hydrogen atoms than 1. Its 1H and 13C NMR data (Tables 1 and 2) were similar to those of 1. The main difference was that the resonances assigned to the hydroxymethylene group in 1 were replaced by those for a formyl group in 2, together with the shift of the signals corresponding to C-30 from δH 4.12, δC 65.0 in 1 to δH 9.51, δC 195.1 in 2, suggesting that 2 was the formyl derivative of 1. This suggestion was supported by 2D NMR data analysis, which verified the 1D NMR data, relative configuration, and regiochemical assignments. Accordingly, the structure of 2 was defined as 6β-hydroxy-3-oxolup-20(29)-en-30-al. Compound 3 gave an ion peak at m/z 454 in the EIMS, and its molecular formula was C30H46O3 by HREIMS and 13C NMR data. It showed spectroscopic data similar to compounds 1 and 2. The NMR data of 3 showed that the resonances for the C-19 propenal group in compound 2 were replaced by those assignable to a propenoic moiety in 3 [δH 2.74 dt (1H, J = 4.3, 11.2 Hz, H-19) and 5.71, 6.25 (2H, s, H-29), δC 40.7 (CH-19), 146.3 (C-20), 124.9 (CH2-29), and 171.2 (C-30)]. A complete set of 2D NMR spectra (COSY, HSQC, and HMBC) was acquired for 3 in order to gain the complete and

characteristic of a lupane skeleton, and resonances at δC 69.6 (CH) and 65.0 (CH2 ) were due to oxymethine and oxymethylene carbons, respectively. These data accounted for two indices of hydrogen deficiency, and the remaining five were ascribed to a pentacyclic ring system in 1. All these data, along with the co-occurrence of 20 known lupane derivatives, suggest that compound 1 is a lupane triterpenoid with one carbonyl and one primary and one secondary hydroxy group. The regiochemistry of the functional groups was determined by an HMBC experiment, showing 3JCH correlations of Me-23 and -24 (δH 1.14 and 1.41, respectively) with a quaternary carbon at δC 48.9 (C-4) and a carbonyl carbon at δC 216.7, which located the carbonyl group at C-3. The position of the oxymethine group at C-6 (δH 4.48) was determined by 3JCH correlations with two quaternary carbons at δC 40.0 (C-8) and δC 36.7 (C-10). The hydroxymethylene group was sited at C-30 since the signal at δH 4.12 showed 2JCH correlation to vinylic C20 (δC 154.6) and 3JCH correlation to vinylic CH2−29 (δC 106.9). The orientation of the C-6 oxymethine proton was established on the basis of theoretical calculations of the coupling constants, using an MM3 force field (PCModel V 9.2) and comparison with experimental data and confirmed by a ROESY experiment. Furthermore, the J6,5 and J6,7 values when calculated for the minimum energy conformer of compound 1 showed smaller coupling constants [J = 1.2 Hz (H6eq-H5ax), 2.7 Hz (H6eq-H7ax), 3.5 Hz (H6eq-H7eq)] compared to its 6-epimer (Figure S17, Supporting Information), which was in agreement with the broad singlet assigned to H-6. The ROESY experiment C



Article

Scheme 1. Synthesis of Compounds 27−38a

a

Reagents: (a) RCOX, pyridine, CH2Cl2. (b) p-TsCl, DMAP, CH2Cl2. (c) TBDMSCl, DMAP, CH2Cl2. (d) PCC, CH2Cl2.

as α on the basis of the small coupling constant (δH 3.49, t, J = 3.0 Hz) and the correlations observed in a 1D-NOESY experiment between H-3β and H-2α, H-2β, Me-23, and Me-24 (δH 0.91 and 0.83). The orientation of the C-1 hydroxy group was assigned as β by the coupling constants (δH 3.78, J = 4.9, 11.1 Hz) and the correlations (H-1α/H-2α, H-5, and H-9) observed in a 1D-NOESY experiment. All these data and comparison with reported data for glochidiol11 established the structure of 5 as 1β,3α,28-trihydroxylup-20(29)-ene. The structure of compound 6, with the molecular formula C30H48O3, was determined by analysis of its 1H and 13C NMR data (Tables 1 and 2) and 2D NMR experiments. The most significant 1H NMR signals are those corresponding to a ketocarbonyl group (δC 218.3), an oxymethine group [δH 3.91 (1H, td, J = 5.3, 10.7 Hz), δC 70.1], and an oxymethylene group [δH 3.37 (1H, d, J = 10.8 Hz), δH 3.78 (1H, d, J = 10.8 Hz), δC 60.3], along with the characteristic signals of the Δ20(29)-ene functionality of a lupane skeleton. The position of the C-11 hydroxy group was defined by an HMBC experiment in which the methine proton at δH 1.48 (H-9) showed 2J correlation with the signal at δC 70.1 (C-11). The primary hydroxy group was located at C-28 by the 3J correlations between the oxymethylene protons and the carbon resonances at δC 33.6 (C16) and δC 28.8 (C-22). The orientation of the C-11 hydroxy group was assigned on the basis of the 1H NMR coupling constants and confirmed by a NOESY experiment, showing correlation between the oxymethine proton at δH 3.91 and Me26 (δH 1.07). These data and comparison with literature data for betulone11 established the structure of 6 as 11α,28dihydroxylup-20(29)-en-3-one. The known compounds were identified by comparison of their experimental and reported spectroscopic data for 3oxolup-20(29)-en-30-al (7),12 3-oxo-30-hydroxylupane (8),13 lupenone (9),11 6β,20-dihydroxylupan-3-one (10),10 glochidiol (11),11 betulone (12),11 rigidenol (13),11 glochidone (14),11 11α-hydroxyglochidone (15),14 lupeol (16),11 25-hydroxylupeol (17),15 3β,30-dihydroxylupane (18),16 lupan-3β-caffeate (19),11 betulin-3β-caffeate (20),11 3-epi-nepeticin (21),14 nepeticin (22),11 3-epi-betulin (23),11 betulin (24),11 3-epiglochidiol (25),11 and ochraceolide A (26).17 Lupane Triterpenoid Analogues. As a part of our investigation into new anti-HIV agents, we focused on structural modifications of the main secondary metabolites 8,

unambiguous assignment of the 1H and 13C NMR resonances as listed in Tables 1 and 2, respectively. Thus, the structure of 3 was established as 3-oxolup-20(29)-en-30-oic acid. Compound 4 was assigned the molecular formula C30H52O3 on the basis of the HRESIMS at m/z 483.3814 [M + Na]+ (calcd 483.3814) and supported by 13C NMR data (Table 2). The 1H NMR spectrum (Table 1) showed eight methyl singlets (δH 0.84−1.41) and two oxymethine protons at δH 3.14 (dd, J = 5.6, 8.7 Hz, H-3) and 4.53 (dd, J = 2.9, 5.7 Hz, H-6). These data suggested that compound 4 was a trisubstituted lupane triterpenoid. The multiplicity and 1H NMR shift of the oxymethine proton at δH 3.14 indicate the presence of a C-3 hydroxy group and were confirmed by an HMBC experiment in which the oxymethine proton showed 2JCH correlation with C-4 (δC 39.6) and 3JCH correlations with Me-23 (δC 27.7), Me-24 (δC 16.9), and C-5 (δC 55.5). The hydroxy group at C-6 was deduced by the 3JCH correlations of the signal at δH 4.53 with the C-8 and C-10 quaternary carbons. Furthermore, the position of a tertiary hydroxy group in the molecule was determined by the 2JCH correlations of Me-29 (δH 1.12) and Me-30 (δH 1.23) with an oxygen-bearing tertiary carbon at δC 73.5 (C-20). The orientation of the secondary hydroxy groups at C-3 and C-6 was established on the basis of coupling constants and confirmed by a ROESY experiment. Thus, the observed NOEs of H-3 and H-6 with Me-23 (δH 1.06) and H-5 (δH 0.69) indicated a β-orientation for both hydroxy groups. All these data and comparison with literature data10 established the structure of 4 as 3β,6β,20-trihydroxylupane. Compound 5 was assigned the molecular formula C30H50O3 by 13C NMR and HREIMS data. The 1H and 13C NMR spectra (Tables 1 and 2) showed characteristic signals of a lupane triterpenoid with two hydroxymethine groups [δH 3.49 (t, J = 3.0 Hz), δC 76.6 and 3.78 (dd, J = 4.9, 11.1 Hz), δC 75.9] and one hydroxymethylene group [δH 3.34 (d, J = 10.7 Hz), 3.80 (dd, J = 1.2, 10.7 Hz), δC 60.6]. COSY, HSQC, and HMBC experiments permitted the complete and unambiguous assignment of the chemical shifts of 5. Correlations observed in an HMBC experiment between Me-23 and Me-24 with the signal at δC 76.6 located one hydroxymethine group at C-3, correlation of Me-25 with C-1 (δC 75.9) placed the other hydroxymethine group at C-1, while correlations of CH2-28 with C-16 and C-22 sited the primary alcohol functionality at C-28. The orientation of the C-3 hydroxy group was assigned D



Article


9, 13, 18, and 24 from the species under study. Thus, 22 derivatives (27−48), seven of which (30−32, 35, 36, 40, and 48) are reported for the first time, were synthesized. The series of betulin derivatives 27−38 with C-3, C-28, or both substitutions were obtained according to Scheme 1. Compounds 27−36 were prepared by reaction of betulin (24) with different acyl chlorides, anhydrides, or tosyl chloride. Silylation of 24 with tert-butyldimethylsilyl chloride (TBDMSCl) provided the 28-silyl ether analogue 37, and oxidation of 24 with pyridinium chlorochromate (PCC) yielded the dicarbonyl compound 38. Since functionalization of the C-19 isopropenyl moiety of the lupane framework is rare,18,19 28-O-acetylbetulin (27)20 was subjected to transformations by its hydroxylation into 40, epoxidation into 41, and allylic oxidation into 42 (Scheme 2).

a Reagents: (a) Ac2O, Et3N, DMAP, CH2Cl2. (b) (i) PhSeCl, EtOAc. (ii) MCPBA, pyridine.

while the known derivatives were identified by comparison of their observed and reported spectroscopic data (Tables 3 and 4). We include in this study a series of lupane-type triterpenoid analogues modified at C-1, C-2, C-3, C-6, C-11, C-25, C-28, C29, or C-30, which showed unusual substitution patterns on the triterpenoid skeleton. Biological Evaluation. The isolated lupane triterpenoids 1−26 and derivatives 27−48 were tested for their inhibitory effect of human immunodeficiency virus type 1 replication. The anti-HIV properties of the known natural compounds 921 and 244,22 and derivatives 27,23 34,4 and 3821 have been previously reported. Nevertheless, we include these compounds in our studies in order to broaden the structure−activity studies, and moreover, the antiviral procedures were different from the ones used herein. Anti-HIV screening was performed infecting a lymphoblastoid cell line (MT-2) with an X4 tropic recombinant virus (NL4.3-Ren) in the presence of triterpenoids at a concentration of 10 μM (Table S29, Supporting Information). Compounds 1−3, 7, 10−12, 15, 17, 22, 23, 26, 38, 39, 42, and 43, with a percentage of HIV-1 inhibition higher than 50%, were subjected to IC50 calculations to compare potencies (Table 3). These assays were performed infecting again MT-2 cells with an X4 tropic HIV (NL4.3-Ren).24 Viability was also evaluated under the same conditions but adding RPMI medium instead of viral supernatants. Fivefold serial dilution concentrations were tested (from 100 to 0.032 μM). Compounds 7 and 42 exhibited higher activity, with an IC50 value around 1.5 μM, followed by 38 and 12, with an IC50 value around 4 μM. Moreover, compounds 10 and 17 also showed IC50 values below 7 μM. The selectivity index (SI: ratio CC50/IC50) (Table 3) was high for compounds 12 and 10 and moderate for 42. Among the most potent compounds, 38 showed an SI between 4.78 and 23.9, and compound 7, although displaying a potent effect on HIV (IC50 1.35 μM), possessed a high cell toxicity (SI 4.2). Therefore, compounds 12, 38, and 42 are the most promising for further study as antiretroviral agents (Figure 1). A preliminary structure−activity relationship study of the reported lupane-type triterpenoids as HIV replication inhibitors (Table 5) indicated that compounds with two oxygenated positions exhibited improvement of activity compared to the corresponding monooxygenated analogues (17, 22, 24, and 25 vs 16, 15 vs 14 or 1 vs 8). The replacement of a hydroxy by a carbonyl group (ketocarbonyl or formyl) at C-3 and/or C-28 improved the activity (27 vs 39 or 24 vs 38), suggesting that this functionality would act as a H-bond acceptor with the receptor. Moreover, the effect of acyl, tosyl (33), and silyl (37) moieties indicated that the attachment of those groups does not positively affect their inhibitory activity. The regiochemistry seems to be an important element for activity, as contributions of substituents at C-11 (22) and C-25 (17) are greater than those at C-1 (25) and C-28 (24). Studies for optimizing the


a

Reagents: (a) PCC, CH2Cl2. (b) NMO, OsO4, THF/H2O. (c) MCPBA, NaHCO3, CH2Cl2. (d) SeO2, EtOH.

To investigate the anti-HIV effect of substitution patterns of the A-ring, compound 39 was obtained by oxidation of 27 with PCC (Scheme 2), while treatment of 9 with phenyltrimethylammonium tribromide (PTAB) in THF afforded compound 45 (Scheme 3). Scheme 3. Synthesis of Compounds 43−45a

a

Reagents: (a) Ac2O, pyridine. (b) PTAB, THF.

As shown in Scheme 3, treatment of compounds 8 and 18 with Ac2O in CH2Cl2 in the presence of DMAP and Et3N afforded esters 43 and 44, respectively. Chlorination of the isopropenyl group of 11α-O-acetylrigidenol (46) with phenylselenyl chloride (PhSeCl) in EtOAc and sequential addition of pyridine and m-chloroperoxybenzoic acid (MCPBA) yielded compounds 47 and 48 (Scheme 4). The structures of the new derivatives were determined via 1D and 2D NMR techniques, E



Article

Table 3. 1H NMR Dataa (δ, CDCl3, J are Given in Parentheses in Hertz) for Compounds 30−32, 35, 36, 40, and 48 30b

position 1 2 3 11 19 23 24 25 26 27 28

32b

35b

36b

40b

48b 7.80 d (10.4) 5.75 d (10.4)

3.21 dd (5.1, 11.2)

3.19 dd (5.0, 11.3)

3.21 dd (6.0, 11.0)

3.20 dd (6.8, 11.6)

4.85 dd (4.9, 11.6)

2.54 0.99 0.78 0.85 1.02 1.08 4.12 4.54 4.63 4.74 1.72

2.56 0.97 0.77 0.85 1.02 1.10 4.20 4.61 4.63 4.74 1.72

2.53 0.99 0.78 0.85 1.02 1.08 4.10 4.51 4.63 4.73 1.72

2.47 0.99 0.78 0.84 1.00 1.05 3.86 4.28 4.61 4.71 1.70

2.58 1.01 0.94 1.00 1.05 1.13 4.20 4.65 4.65 4.75 1.74

3.21 dd (4.5, 11.1) 5.18 dt (4.8, 11.2)

29 30 a

31b

dt (5.7, 11.2) s s s s s d (10.8) d (10.8) s s s

dt (5.7, 10.8) s s s s s d (12.4) d (12.4) s s s

dt (6.0, 11.0) s s s s s d (11.3) d (11.3) s s s

dt (6.8, 11.2) s s s s s d (10.6) d (10.6) s s s

dt (5.8, 11.0) s s s s s d (11.0)

s s

0.99 0.78 0.85 0.99 1.08 3.85 4.34 3.44 3.66 1.21

s s s s s d d d d s

(11.1) (11.1) (10.7) (10.7)

1.13 1.11 1.18 1.09 1.02 0.81

s s s s s s

5.04 br s 5.10 br s 4.03 s

Signals without multiplicity are overlapped and deduced by HSQC experiments. bRecorded at 400 MHz.

Table 4. 13C NMR Dataa (δ, CDCl3) for Compounds 30−32, 35, 36, 40, and 48 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a

30b 38.8, 27.4, 79.0, 38.9, 55.4, 18.3, 34.8, 40.9, 50.4, 37.2, 20.8, 25.2, 37.7, 42.8, 27.2, 29.7, 47.8, 49.0, 46.7, 150.1, 30.0, 34.2, 28.0, 15.4, 16.2, 16.1, 14.8, 63.3, 109.9, 19.2,

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH3 CH3 CH3 CH3 CH3 CH2 CH2 CH3

31b

32b

38.7, CH2 27.4, CH2 79.0, CH 38.9, C 55.3, CH 18.3, CH2 34.8, CH2 40.9, C 50.4, CH 37.2, C 20.8, CH2 25.3, CH2 37.7, CH 42.8, C 27.2, CH2 29.7, CH2 47.8, C 49.0, CH 46.7, CH 150.2, C 30.1, CH2 34.2, CH2 28.0, CH3 15.4, CH3 16.1,c CH3 16.1,c CH3 14.8, CH3 63.3, CH2 109.9, CH2 19.2, CH3

38.7, CH2 27.4, CH2 79.0, CH 38.9, C 55.3, CH 18.3, CH2 34.7, CH2 40.9, C 50.4, CH 37.7,c C 20.8, CH2 25.2, CH2 37.7,c CH 42.8, C 27.1, CH2 29.7, CH2 47.8, C 48.9, CH 46.7, CH 150.0, C 30.0, CH2 34.2, CH2 28.0, CH3 15.3, CH3 16.1, CH3 16.0, CH3 14.8, CH3 63.6, CH2 110.0, CH2 19.2, CH3

35b 38.4, 28.9, 80.6, 37.8, 55.4, 18.2, 34.6, 40.9, 50.3, 37.1, 20.8, 25.2, 37.6, 42.7, 27.1, 29.1, 47.8, 48.9, 46.4, 150.2, 29.7, 34.5, 28.0, 16.5, 16.1, 16.0, 14.7, 62.6, 109.8, 19.1,


36b 38.5, 23.9, 81.7, 38.2, 55.6, 18.3, 34.8, 41.0, 50.4, 37.2, 20.9, 25.2, 37.7, 42.8, 27.2, 29.7, 47.8, 49.0, 46.8, 150.4, 30.1, 34.2, 28.2, 17.0, 16.2, 16.1, 14.8, 63.4, 110.0, 19.2,


40b

48b

38.6, CH2 26.9, CH2 78.8, CH 38.5, C 55.0, CH 18.0, CH2 34.6, CH2 41.2, C 50.1, CH 36.8, C 21.1, CH2 26.8, CH2 36.5, CH 43.2, C 27.1, CH2 28.8, CH2 47.6, C 48.2, CH 46.9, CH 74.8, C 30.1, CH2 34.2, CH2 27.7, CH3 15.9,c CH3 15.9,c CH3 15.1, CH3 14.8, CH3 62.3, CH2 66.9, CH2 24.9, CH3

163.2, CH 124.5, CH 205.2, C 45.2, C 52.7, CH 18.9, CH2 34.4, CH2 42.7, C 43.1,c CH 39.8, C 73.4, CH 22.7, CH2 36.9, CH 43.3, C 27.1, CH2 35.2, CH2 43.1,c C 49.1, CH 46.6, CH 150.4, C 29.4, CH2 39.6, CH2 28.8, CH3 21.4, CH3 20.2, CH3 17.4, CH3 14.3, CH3 17.9, CH3 112.9, CH2 48.2, CH2

Data are based on DEPT, HSQC, and HMBC experiments. bRecorded at 100 MHz. cOverlapping signals.

their precursor 27. Moreover, the impact of the formyl group was underlined by the presence of such functional group at C30 in the most active compounds of this series, 2 (5% RLUs), 7 (0% RLUs), and 42 (0% RLUs). Our results are in agreement with previous data that reported the importance of an α,βunsaturated formyl group for the anti-HIV activity. 28 Furthermore, these results show that other positions in the skeleton are an interesting alternative to broaden the structural

lupane triterpenoid scaffold have mainly targeted modifications at C-3 and C-28,4,25−27 while the isopropenyl group has been less investigated.18,19 The importance of the C-19 moiety was evident, as changes of this domain rendered analogues more potent (3, 7, and 8 vs 9), suggesting that compounds with C-30 oxygenation displayed significant improvements in potency as compared to those having an unsubstituted C-30. Following these results, analogues 40 and 41 are slightly more potent than F



Article

Figure 1. Anti-HIV activity and toxicity of the most potent lupanes. MT-2 cells were infected with HIV (NL4.3-Ren) in the presence of different concentrations of lupanes. Cell viability was measured in parallel but in uninfected cells. Bevirimat was used as control.

displayed strong inhibition of HIV-1 replication with IC50 values lower than 5 μM. The relevant structural pharmacophores were the formyl group and oxidation level of the molecule. Further studies will be conducted to elucidate the mechanism implicated in the anti-HIV activity of these promising compounds.

Table 5. Evaluation of Anti-HIV Activity of Selected Active Lupanesa compoundb 1 2 3 7 10 11 12 15 17 22 23 26 38 39 42 43 bevirimat

IC50 μM ± SD 9.5 7.9 ∼100 1.4 7.0 13.9 4.1 8.7 6.9 10.4 8.8 39.0 4.2 13.4 1.7 13.1 0.01

± 2.6 ± 2.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 3.3 3.2 1.8 1.9 1.9 5.0 0.5 2.8 0.00 2.0 0.2 2.9 0.00

CC50 ± SD

SI

>20 < 100 10.4 >100 5.7 ± 3.9 >100 >20 < 100 >100 60.4 ± 54.8 >20 < 100 >20 < 100 >20 < 100 >100 >20 < 100 >20 < 100 14.2 ± 2.4 36.2 ± 3.4 >10

>2.10 < 10.48 1.3 4.2 >14.3 >1.4 < >24.5 6.9 >2.9 < >1.9 < >2.3 < >2.6 >4.8 < >1.5 < 8.2 2.8 >1000

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 241 automatic polarimeter in CHCl3 at 20 °C. UV spectra were obtained on a JASCO V-560 spectrophotometer in absolute EtOH. IR (film) spectra were measured on a Bruker IFS 55 spectrophotometer. 1H (400, 500, or 600 MHz) and 13 C (100, 125, or 150 MHz) NMR spectra were recorded on Bruker Avance 400, 500, and 600 spectrometers; chemical shifts were referred to the residual solvent signal (CDCl3: δH 7.26, δC 77.0); DEPT, COSY, ROESY (spin lock field 2500 Hz), NOESY (mixing time 500 ms), HSQC, and HMBC (optimized for J = 7.7 Hz) experiments were carried out with the pulse sequences given by Bruker. EIMS and HREIMS were obtained on a Micromass Autospec spectrometer, and HRESIMS (positive mode) was measured on a LCT Premier XE Micromass Electrospray spectrometer. Silica gel 60 (particle size 15− 40 and 63−200 μm, Machery-Nagel) and Sephadex LH-20 (Pharmacia Biotech) were used for column chromatography, while silica gel 60 F254 (Machery-Nagel) was used for analytical and preparative TLC. Centrifugal planar chromatography was performed by a Chromatotron instrument (model 7924T, Harrison Research Inc., Palo Alto, CA, USA) on manually coated silica gel 60 GF254 (Merck) using a 1, 2, or 4 mm plates. The spots were visualized by UV light and heating silica gel plates sprayed with H2O−H2SO4−HOAc (1:4:20). All solvents used were analytical grade (Panreac). The reagents were purchased from Aldrich and used without further purification. Plant Material. Cassine xylocarpa (Vent.) was collected in August 2004 at the Parque Nacional El Imposible (Province of Ahuachapán,

7.2

14.4 9.6 11.4 23.9 7.5

a

CI50 (inhibitory concentration 50%) and CC50 (cytotoxic concentration 50%) were calculated using GrapPad Prism software. SI: selectivity index (CC50/IC50). SD: standard deviation. bCompounds not included in the table were inactive (

Isolation, Structural Modification, and HIV Inhibition of Pentacyclic

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