Isolation, structural modification, and HIV inhibition of pentacyclic lupane-type triterpenoids from cassine xylocarpa and maytenus cuzcoina

Article abstract of DOI:10.1021/np501025r

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.

Full text of DOI:10.1021/np501025r

Article
pubs.acs.org/jnp
Isolation, Structural Modification, and HIV Inhibition of Pentacyclic
Lupane-Type Triterpenoids from Cassine xylocarpa and Maytenus
cuzcoina
†
‡,§
‡
‡
́ ́
, Alejandro Munoz, Patricia Obregon Calderon
,‡
Oliver Callies, Luis M. Bedoya, Manuela Beltran
́
̃
†
†
‡
,†
Alex A. Osorio, Ignacio A. Jimen
́
ez, Jose
́
Alcamí, and Isabel L. Bazzocchi*
†
Instituto Universitario de Bio-Organ
8206 La Laguna, Tenerife, Spain
́ ́ ́
ica “Antonio Gonzalez”, Universidad de La Laguna, Avenida Astrofísico Francisco Sanchez 2,
3
‡
Unidad de Inmunopatología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Pozuelo Km.2,
8220-Majadahonda, Madrid, Spain
2
§
́
Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid, Ramon 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 stereo-
structures 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 IC values in the micromolar range, highlighting compounds 12, 38, and 42 (IC 4.08, 4.18, and 1.70 μM, respectively) as
50
50
the most promising anti-HIV agents.
hree decades into the acquired immunodeficiency
dimethylsuccinyl)betulinic acid], a first-in-class HIV-1 matura-
tion inhibitor.
7
,8
T
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,
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
9
activity. 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-
1
acceptable toxicity, and novel mechanisms of action.
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
2
clinical studies for the treatment of HIV infections. 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
3
compounds. Over the last two decades, many derivatives of the
well-known lupane-type triterpenoid betulinic acid (3β-
hydroxylup-20(29)-en-28-oic acid) have been reported to
Received: December 18, 2014
4
−6
inhibit HIV replication,
including bevirimat [3-O-(3′,3′-
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np501025r
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Journal of Natural Products
Article
tion and oxidation degree are key to modulating the potency of
HIV inhibition.
RESULTS AND DISCUSSION
■
Natural Lupane Triterpenoids. Repeated chromatogra-
phy on Sephadex LH-20 and silica gel of the CH Cl fraction of
2
2
the stems of C. xylocarpa and n-hexane−Et O extract of the
2
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 C H O , as
3
0
48
3
13
established on the basis of the C NMR and HREIMS spectra,
requiring seven indices of hydrogen deficiency. The IR
−1
spectrum indicated the presence of hydroxy (3462 cm ) and
−1
1
carbonyl (1701 cm ) groups. The H NMR spectrum (Table
) showed six methyl groups [δ 0.80, 0.91, 1.14, 1.41, 1.42,
1
H
and 1.43 (each 3H, s)], one methylene group [δ 2.23 (ddd, J
H
=
2.8, 4.8, 14.8 Hz), 2.79 (td, J = 6.4, 14.8 Hz), H -2)], one
2
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 δ 4.91 and 4.94
H
(
3
2H, br s, H-29). The 13C NMR spectrum (Table 2) showed
0 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 δ 216.7 indicated the presence
C
2
of a ketocarbonyl moiety, the two sp carbons at δ 154.6 (C)
C
20(29)
and 106.9 (CH ) were consistent with a Δ
double bond
2
1
a
Table 1. H NMR Data (δ, CDCl , J are Given in Parentheses in Hertz) for Compounds 1−6
3
b
c
d
c
c
b
position
1
1
2
3
4
5
6
1.25, 1.97 ddd (3.0, 6.5, 1.20, 1.93 ddd (2.9, 6.4, 1.38, 1.91 ddd (4.4, 7.5, 0.90, 1.68
3.1) 13.1) 13.3)
3.78 dd (4.9, 11.1) 1.61, 2.68 ddd (5.7, 8.6,
14.5)
1
2
2.23 ddd (2.8, 4.8, 14.8) 2.21 ddd (2.9, 4.8, 15.0) 2.46 ddd (4.4, 7.6, 15.6) 1.64
1.75 ddd (3.6, 4.8, 2.42, 2.46 ddd (5.5, 8.8,
1
3.8)
15.0)
2.79 td (6.4, 14.8)
2.79 td (6.4, 15.0)
2.51 ddd (7.4, 9.9, 15.7)
3
5
6
7
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
3.49 t (3.0)
1.15
1.12 d (2.1)
4.47 br s
1.36
1.50
1.46
1.22 dd (2.0, 11.8) 1.48
1.43, 1.50
4.48 br s
1.63 dd (2.5, 15.7)
1.61 dd (2.8, 15.0)
1.68 dd (3.7, 14.9)
1.33
1.37, 1.43
1.48
1.68 dd (3.7, 15.7)
9
1.40
1.38
1.30
1.64 dd (3.6, 13.2) 1.48
1
1
1
2
1.41, 1.52
1.12, 1.49
1.43
1.30, 1.44
1.03, 1.28
1.40, 1.55
2.14 br d (12.9)
3.91 td (5.3, 10.7)
1.25
1.31, 1.89
1.11 dd (4.7, 13.7) 1.12, 1.92
1
.63
1
1
3
5
1.77
1.78 dt (3.7, 12.4)
0.99, 1.75
1.73
1.82
1.65
1.83
1.02, 1.76
1.05, 1.70
1.02 ddd (2.3, 4.6, 13.9) 1.12, 1.68
.81
1.42, 1.53 ddd (2.6, 4.5, 1.45, 1.54 ddd (2.6, 4.5, 1.35, 1.50 ddd (2.2, 4.8, 1.22, 1.92
3.2) 13.1) 12.8)
1.66 1.34
1.17, 1.62
1
1
6
1.40, 1.52
1.98
1
1
1
2
2
2
2
2
2
2
2
8
9
1
2
3
4
5
6
7
8
1.49
1.36
1.56 t (11.9)
2.38 dt (6.0, 11.2)
1.41, 1.96
1.04, 1.84
0.91 s
1.70
2.30 dt (5.3, 11.0)
1.34, 2.08
1.28, 1.42
1.14 s
2.77
2.74 dt (4.3, 11.2)
1.40, 2.20
1.39
1.81
2.42
1.24
1.24, 1.88
1.10, 1.30
1.06 s
1.99
1.37, 1.44
1.14 s
1.40 s
1.41 s
1.40 s
0.88 s
0.83 s
1.86
1.09 s
1.12 s
1.08 s
1.09 s
1.07 s
1.04 s
3.37 d (10.8)
1.41 s
1.05 s
1.15 s
0.83 s
1.43 s
0.95 s
1.21 s
0.90 s
1.42 s
1.09 s
1.38 s
1.03 s
0.91 s
0.97 s
0.93 s
1.00 s
0.80 s
0.85 s
0.82 s
3.34 d (10.7)
3
.80 dd (1.2, 10.7) 3.78 d (10.8)
2
3
9
0
4.91 br s
5.92 s
6.29 s
9.51 s
5.71 s
6.25 s
1.12 s
1.23 s
4.56 dd (1.3, 2.2)
4.67 d (2.1)
1.67 s
4.64 br s
4.74 br s
1.72 s
4.94 br s
4.12 br s
a
b
c
d
Signals without multiplicity are overlapped and deduced by HSQC experiments. Recorded at 400 MHz. Recorded at 600 MHz. Recorded at 500
MHz.
B
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Table 2. 13C NMR Data (δ, CDCl ) for Compounds 1−6
a
3
b
c
b
c
b
b
position
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
42.1, CH2
34.4, CH2
216.7, C
42.1, CH2
34.5, CH2
216.8, C
39.6, CH2
34.1, CH2
218.2, C
40.7, CH2
27.6, CH2
79.1, CH
39.6, C
75.9, CH
36.4, CH2
76.6, CH
41.8, CH2
34.0, CH2
218.3, C
d
d
48.9, C
49.0, C
47.3, C
37.4, C
47.5, C
d
56.5, CH
69.6, CH
42.2, CH2
40.0, C
56.6, CH
69.7, CH
42.2, CH2
40.0, C
54.9, CH
19.6, CH2
33.6, CH2
40.7, C
55.5, CH
69.1, CH
42.4, CH2
40.4, C
47.8, CH
18.4, CH2
34.0, CH2
42.9, C
54.6, CH
19.4, CH2
d
33.9, CH
2
d
42.2, C
50.6, CH
36.7, C
50.5, CH
36.7, C
49.6, CH
36.8, C
50.9, CH
51.2, CH
54.5, CH
38.0, C
d
d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
36.6, C
37.4, C
21.3, CH2
26.7, CH2
37.1, CH
42.9, C
21.3, CH2
29.7, CH2
36.8, CH
43.2, C
21.5, CH2
29.7, CH2
37.8, CH
42.8, C
21.6, CH2
28.8, CH2
23.7, CH2
25.2, CH2
36.9, CH
41.7, C
70.1, CH
37.2, CH2
36.1, CH
d
36.6, CH
43.8, C
d
42.2, C
27.4, CH2
35.3, CH2
43.1, C
27.4, CH2
35.3, CH2
43.0, C
27.3, CH2
35.4, CH2
43.1, C
27.5, CH2
35.5, CH2
44.6, C
27.1, CH2
29.2, CH2
43.6, C
26.8, CH2
33.6, CH2
47.3, C
d
48.9, CH
43.7, CH
154.6, C
50.6, CH
42.1, CH
157.2, C
50.9, CH
40.7, CH
146.3, C
48.4, CH
49.9, CH
73.5, C
48.8, CH
47.8, CH
47.4, CH
149.5, C
d
47.8, CH
150.3, C
31.7, CH2
39.8, CH2
25.0, CH3
23.7, CH3
17.0, CH3
17.1, CH3
14.8, CH3
17.7, CH3
106.9, CH2
65.0, CH2
32.7, CH2
39.8, CH2
25.0, CH3
23.7, CH3
16.9, CH3
17.1, CH3
14.7, CH3
17.8, CH3
133.2, CH2
195.1, CH
32.9, CH2
39.7, CH2
26.6, CH3
21.0, CH3
15.9, CH3
15.8, CH3
14.4, CH3
17.9, CH3
124.9, CH2
171.2, C
29.2, CH2
40.2, CH2
27.7, CH3
16.9, CH3
17.8, CH3
17.2, CH3
15.2, CH3
19.2, CH3
24.8, CH3
29.7, CH2
34.0, CH2
27.7, CH3
21.9, CH3
11.7, CH3
16.2, CH3
14.8, CH3
60.6, CH2
109.8, CH2
29.4, CH2
28.8, CH2
27.2, CH3
20.5, CH3
16.5, CH3
16.6, CH3
14.4, CH3
60.3, CH2
110.1, CH2
18.9, CH3
d
31.6, CH3
19.0, CH3
a
b
c
d
Data are based on DEPT, HSQC, and HMBC experiments. Recorded at 100 MHz. Recorded at150 MHz. Overlapping signals.
characteristic of a lupane skeleton, and resonances at δ 69.6
showed NOE correlations of the proton at δ 4.48 with the
C
H
(
CH) and 65.0 (CH ) were due to oxymethine and
Me-23 and H-5 (δH 1.15), which confirmed a β-axial
2
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.
orientation of the hydroxy group. All of these data and
comparison with reported data for betulone established the
10
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 C H O (HREIMS)
3
0
46
3
1
13
indicated two fewer hydrogen atoms than 1. Its H and
C
The regiochemistry of the functional groups was determined
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 δ 4.12, δ 65.0 in 1 to δ 9.51, δ
C
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.
3
by an HMBC experiment, showing J correlations of Me-23
CH
and -24 (δH 1.14 and 1.41, respectively) with a quaternary
carbon at δ 48.9 (C-4) and a carbonyl carbon at δ 216.7,
C
C
which located the carbonyl group at C-3. The position of the
H
C
H
3
oxymethine group at C-6 (δ 4.48) was determined by J
H
CH
correlations with two quaternary carbons at δ 40.0 (C-8) and
C
δ 36.7 (C-10). The hydroxymethylene group was sited at C-30
C
2
since the signal at δ 4.12 showed J correlation to vinylic C-
0 (δC 154.6) and J correlation to vinylic CH −29 (δ
06.9). The orientation of the C-6 oxymethine proton was
H
CH
3
2
1
CH
2
C
Compound 3 gave an ion peak at m/z 454 in the EIMS, and
1
3
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 (H -H ), 2.7
its molecular formula was C H O by HREIMS and
C
30
46
3
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 [δ 2.74 dt (1H, J
H
= 4.3, 11.2 Hz, H-19) and 5.71, 6.25 (2H, s, H-29), δ 40.7
6
eq
5ax
C
Hz (H -H ), 3.5 Hz (H -H )] compared to its 6-epimer
(CH-19), 146.3 (C-20), 124.9 (CH -29), and 171.2 (C-30)]. A
6
eq
7ax
6eq
7eq
2
(
Figure S17, Supporting Information), which was in agreement
complete set of 2D NMR spectra (COSY, HSQC, and HMBC)
with the broad singlet assigned to H-6. The ROESY experiment
was acquired for 3 in order to gain the complete and
C
DOI: 10.1021/np501025r
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Journal of Natural Products
Article
a
Scheme 1. Synthesis of Compounds 27−38
a
Reagents: (a) RCOX, pyridine, CH Cl . (b) p-TsCl, DMAP, CH Cl . (c) TBDMSCl, DMAP, CH Cl . (d) PCC, CH Cl .
2
2
2
2
2
2
2
2
unambiguous assignment of the H 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.
1
as α on the basis of the small coupling constant (δ 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
H
Compound 4 was assigned the molecular formula C H O
(δ 0.91 and 0.83). The orientation of the C-1 hydroxy group
30
52
3
H
+
on the basis of the HRESIMS at m/z 483.3814 [M + Na]
was assigned as β by the coupling constants (δ 3.78, J = 4.9,
H
1
3
(
calcd 483.3814) and supported by C NMR data (Table 2).
11.1 Hz) and the correlations (H-1α/H-2α, H-5, and H-9)
1
The H NMR spectrum (Table 1) showed eight methyl singlets
observed in a 1D-NOESY experiment. All these data and
11
(
5
δ 0.84−1.41) and two oxymethine protons at δ 3.14 (dd, J =
comparison with reported data for glochidiol established the
H
H
.6, 8.7 Hz, H-3) and 4.53 (dd, J = 2.9, 5.7 Hz, H-6). These
structure of 5 as 1β,3α,28-trihydroxylup-20(29)-ene.
data suggested that compound 4 was a trisubstituted lupane
triterpenoid. The multiplicity and H NMR shift of the
The structure of compound 6, with the molecular formula
C H O , was determined by analysis of its H and C NMR
30 48 3
1
1
13
oxymethine proton at δ 3.14 indicate the presence of a C-3
data (Tables 1 and 2) and 2D NMR experiments. The most
significant H NMR signals are those corresponding to a
H
1
hydroxy group and were confirmed by an HMBC experiment in
2
which the oxymethine proton showed J correlation with C-4
ketocarbonyl group (δ 218.3), an oxymethine group [δ 3.91
CH
C
H
3
(
δ 39.6) and J correlations with Me-23 (δ 27.7), Me-24
(1H, td, J = 5.3, 10.7 Hz), δ 70.1], and an oxymethylene group
C
CH
C
C
(
δ 16.9), and C-5 (δ 55.5). The hydroxy group at C-6 was
[δ 3.37 (1H, d, J = 10.8 Hz), δ 3.78 (1H, d, J = 10.8 Hz), δ
C
C
H
H
C
3
20(29)
deduced by the J correlations of the signal at δ 4.53 with
the C-8 and C-10 quaternary carbons. Furthermore, the
position of a tertiary hydroxy group in the molecule was
60.3], along with the characteristic signals of the Δ
-ene
CH
H
functionality of a lupane skeleton. The position of the C-11
hydroxy group was defined by an HMBC experiment in which
2
2
determined by the J correlations of Me-29 (δ 1.12) and
the methine proton at δ 1.48 (H-9) showed J correlation with
CH
H
H
Me-30 (δ 1.23) with an oxygen-bearing tertiary carbon at δC
the signal at δ 70.1 (C-11). The primary hydroxy group was
located at C-28 by the J correlations between the oxy-
H
C
3
7
3.5 (C-20). The orientation of the secondary hydroxy groups
at C-3 and C-6 was established on the basis of coupling
methylene protons and the carbon resonances at δ 33.6 (C-
C
constants and confirmed by a ROESY experiment. Thus, the
16) and δ 28.8 (C-22). The orientation of the C-11 hydroxy
group was assigned on the basis of the H NMR coupling
C
1
observed NOEs of H-3 and H-6 with Me-23 (δ 1.06) and H-5
H
(
δ 0.69) indicated a β-orientation for both hydroxy groups. All
constants and confirmed by a NOESY experiment, showing
H
10
these data and comparison with literature data established the
correlation between the oxymethine proton at δ 3.91 and Me-
H
structure of 4 as 3β,6β,20-trihydroxylupane.
26 (δ 1.07). These data and comparison with literature data
for betulone established the structure of 6 as 11α,28-
dihydroxylup-20(29)-en-3-one.
The known compounds were identified by comparison of
their experimental and reported spectroscopic data for 3-
H
11
Compound 5 was assigned the molecular formula C H O
3
30
50
13
1
13
by C NMR and HREIMS data. The H and C NMR spectra
Tables 1 and 2) showed characteristic signals of a lupane
triterpenoid with two hydroxymethine groups [δ 3.49 (t, J =
(
H
1
2
13
3
.0 Hz), δ 76.6 and 3.78 (dd, J = 4.9, 11.1 Hz), δ 75.9] and
oxolup-20(29)-en-30-al (7), 3-oxo-30-hydroxylupane (8),
C
C
11
10
one hydroxymethylene group [δ 3.34 (d, J = 10.7 Hz), 3.80
lupenone (9), 6β,20-dihydroxylupan-3-one (10), glochidiol
(11), betulone (12), rigidenol (13), glochidone (14),
11α-hydroxyglochidone (15), lupeol (16), 25-hydroxylu-
peol (17), 3β,30-dihydroxylupane (18), lupan-3β-caffeate
(19), betulin-3β-caffeate (20), 3-epi-nepeticin (21),
nepeticin (22), 3-epi-betulin (23), betulin (24), 3-epi-
glochidiol (25), and ochraceolide A (26).
H
11
11
11
11
(
dd, J = 1.2, 10.7 Hz), δ 60.6]. COSY, HSQC, and HMBC
C
1
4
11
experiments permitted the complete and unambiguous assign-
ment 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
15
16
1
1
11
14
11
11
11
11
17
hydroxymethine group at C-1, while correlations of CH -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
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,
2
D
DOI: 10.1021/np501025r
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Journal of Natural Products
, 13, 18, and 24 from the species under study. Thus, 22
Article
a
9
Scheme 4. Synthesis of Compounds 46−48
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. Com-
pounds 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.
a
Reagents: (a) Ac O, Et N, DMAP, CH Cl . (b) (i) PhSeCl, EtOAc.
2
3
2
2
(ii) MCPBA, pyridine.
Since functionalization of the C-19 isopropenyl moiety of the
1
8,19
20
lupane framework is rare,
28-O-acetylbetulin (27) was
while the known derivatives were identified by comparison of
their observed and reported spectroscopic data (Tables 3 and
subjected to transformations by its hydroxylation into 40,
epoxidation into 41, and allylic oxidation into 42 (Scheme 2).
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, C-
9, or C-30, which showed unusual substitution patterns on the
a
Scheme 2. Synthesis of Compounds 39−42
2
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
21
anti-HIV properties of the known natural compounds 9 and
4
,22
23
4
21
24
and derivatives 27, 34, and 38 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
4
3, 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
(
24
cells with an X4 tropic HIV (NL4.3-Ren). Viability was also
evaluated under the same conditions but adding RPMI medium
instead of viral supernatants. Fivefold serial dilution concen-
trations 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 CC /IC ) (Table
a
Reagents: (a) PCC, CH Cl . (b) NMO, OsO , THF/H O. (c)
2
2
4
2
MCPBA, NaHCO , CH Cl . (d) SeO , EtOH.
3
2
2
2
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 phenyl-
trimethylammonium tribromide (PTAB) in THF afforded
compound 45 (Scheme 3).
50
50
3
) was high for compounds 12 and 10 and moderate for 42.
Among the most potent compounds, 38 showed an SI between
.78 and 23.9, and compound 7, although displaying a potent
effect on HIV (IC 1.35 μM), possessed a high cell toxicity (SI
a
Scheme 3. Synthesis of Compounds 43−45
4
50
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) Ac O, pyridine. (b) PTAB, THF.
2
As shown in Scheme 3, treatment of compounds 8 and 18
with Ac O in CH Cl in the presence of DMAP and Et N
2
2
2
3
afforded esters 43 and 44, respectively. Chlorination of the
isopropenyl group of 11α-O-acetylrigidenol (46) with phenyl-
selenyl 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
DOI: 10.1021/np501025r
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Journal of Natural Products
Article
1
a
Table 3. H NMR Data (δ, CDCl , J are Given in Parentheses in Hertz) for Compounds 30−32, 35, 36, 40, and 48
3
b
b
b
b
b
b
b
position
30
31
32
35
36
40
48
1
2
3
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) 3.21 dd (4.5, 11.1)
2.54 dt (5.7, 11.2) 2.56 dt (5.7, 10.8) 2.53 dt (6.0, 11.0) 2.47 dt (6.8, 11.2) 2.58 dt (5.8, 11.0)
1
1
2
2
2
2
2
2
1
9
3
4
5
6
7
8
5.18 dt (4.8, 11.2)
0.99 s
0.97 s
0.99 s
0.99 s
1.01 s
0.94 s
1.00 s
1.05 s
1.13 s
4.20 d (11.0)
4.65
0.99 s
1.13 s
1.11 s
1.18 s
1.09 s
1.02 s
0.81 s
0.78 s
0.77 s
0.78 s
0.78 s
0.78 s
0.85 s
0.85 s
0.85 s
0.84 s
0.85 s
1.02 s
1.02 s
1.02 s
1.00 s
0.99 s
1.08 s
1.10 s
1.08 s
1.05 s
1.08 s
4.12 d (10.8)
4.20 d (12.4)
4.61 d (12.4)
4.63 s
4.10 d (11.3)
4.51 d (11.3)
4.63 s
3.86 d (10.6)
4.28 d (10.6)
4.61 s
3.85 d (11.1)
4.34 d (11.1)
3.44 d (10.7)
3.66 d (10.7)
1.21 s
4
.54 d (10.8)
4.63 s
.74 s
1.72 s
29
4.65
5.04 br s
5.10 br s
4.03 s
4
4.74 s
4.73 s
4.71 s
4.75 s
1.74 s
30
1.72 s
1.72 s
1.70 s
a
b
Signals without multiplicity are overlapped and deduced by HSQC experiments. Recorded at 400 MHz.
Table 4. 13C NMR Data (δ, CDCl ) for Compounds 30−32, 35, 36, 40, and 48
a
3
b
b
b
b
b
b
b
position
30
31
32
35
36
40
48
1
2
3
4
5
6
7
8
9
38.8, CH2
27.4, CH2
79.0, CH
38.9, C
38.7, CH2
27.4, CH2
79.0, CH
38.9, C
38.7, CH2
27.4, CH2
79.0, CH
38.9, C
38.4, CH2
28.9, CH2
80.6, CH
37.8, C
38.5, CH2
23.9, CH2
81.7, CH
38.2, C
38.6, CH2
26.9, CH2
78.8, CH
38.5, C
163.2, CH
124.5, CH
205.2, C
45.2, C
55.4, CH
18.3, CH2
34.8, CH2
40.9, C
55.3, CH
18.3, CH2
34.8, CH2
40.9, C
55.3, CH
18.3, CH2
34.7, CH2
40.9, C
55.4, CH
18.2, CH2
34.6, CH2
40.9, C
55.6, CH
18.3, CH2
34.8, CH2
41.0, C
55.0, CH
18.0, CH2
34.6, CH2
41.2, C
52.7, CH
18.9, CH2
34.4, CH2
42.7, C
c
50.4, CH
37.2, C
50.4, CH
37.2, C
50.4, CH
50.3, CH
37.1, C
50.4, CH
37.2, C
50.1, CH
36.8, C
43.1, CH
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
37.7, C
39.8, C
20.8, CH2
25.2, CH2
37.7, CH
42.8, C
20.8, CH2
25.3, CH2
37.7, CH
42.8, C
20.8, CH2
25.2, CH2
20.8, CH2
25.2, CH2
37.6, CH
42.7, C
20.9, CH2
25.2, CH2
37.7, CH
42.8, C
21.1, CH2
26.8, CH2
36.5, CH
43.2, C
73.4, CH
22.7, CH2
36.9, CH
43.3, C
c
37.7, CH
42.8, C
27.2, CH2
29.7, CH2
47.8, C
27.2, CH2
29.7, CH2
47.8, C
27.1, CH2
29.7, CH2
47.8, C
27.1, CH2
29.1, CH2
47.8, C
27.2, CH2
29.7, CH2
47.8, C
27.1, CH2
28.8, CH2
47.6, C
27.1, CH2
35.2, CH
2
c
43.1, C
49.0, CH
46.7, CH
150.1, C
49.0, CH
46.7, CH
150.2, C
30.1, CH2
34.2, CH2
28.0, CH3
48.9, CH
46.7, CH
150.0, C
48.9, CH
46.4, CH
150.2, C
49.0, CH
46.8, CH
150.4, C
48.2, CH
46.9, CH
74.8, C
49.1, CH
46.6, CH
150.4, C
30.0, CH2
34.2, CH2
28.0, CH3
15.4, CH3
16.2, CH3
16.1, CH3
14.8, CH3
63.3, CH2
109.9, CH2
19.2, CH3
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
29.7, CH2
34.5, CH2
28.0, CH3
16.5, CH3
16.1, CH3
16.0, CH3
14.7, CH3
62.6, CH2
109.8, CH2
19.1, CH3
30.1, CH2
34.2, CH2
28.2, CH3
17.0, CH3
16.2, CH3
16.1, CH3
14.8, CH3
63.4, CH2
110.0, CH2
19.2, CH3
30.1, CH2
34.2, CH2
27.7, CH3
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
c
15.4, CH
15.9, CH
3
3
c
c
16.1, CH
15.9, CH3
15.1, CH3
14.8, CH3
62.3, CH2
66.9, CH2
24.9, CH3
3
3
c
16.1, CH
14.8, CH3
63.3, CH2
109.9, CH2
19.2, CH3
19.2, CH3
a
b
c
Data are based on DEPT, HSQC, and HMBC experiments. Recorded at 100 MHz. Overlapping signals.
lupane triterpenoid scaffold have mainly targeted modifications
their precursor 27. Moreover, the impact of the formyl group
was underlined by the presence of such functional group at C-
30 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,β-
unsaturated formyl group for the anti-HIV activity.
Furthermore, these results show that other positions in the
skeleton are an interesting alternative to broaden the structural
4
,25−27
at C-3 and C-28,
less investigated.
while the isopropenyl group has been
The importance of the C-19 moiety was
1
8,19
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
2
8
F
DOI: 10.1021/np501025r
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Journal of Natural Products
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.
Table 5. Evaluation of Anti-HIV Activity of Selected Active
Lupanes
displayed strong inhibition of HIV-1 replication with IC50
values lower than 5 μM. The relevant structural pharmaco-
phores 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.
a
b
compound
IC50 μM ± SD
CC50 ± SD
SI
1
2
3
7
9.5 ± 2.6
7.9 ± 2.6
∼100
>20 < 100
10.4
>2.10 < 10.48
1.3
>100
1.4 ± 0.2
7.0 ± 3.3
13.9 ± 3.2
4.1 ± 1.8
8.7 ± 1.9
6.9 ± 1.9
10.4 ± 5.0
8.8 ± 0.5
39.0 ± 2.8
4.2 ± 0.00
13.4 ± 2.0
1.7 ± 0.2
13.1 ± 2.9
0.01 ± 0.00
5.7 ± 3.9
>100
4.2
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 241 automatic polarimeter in CHCl at
■
10
11
12
15
17
22
23
26
38
39
42
43
>14.3
>20 < 100
>100
>1.4 < 7.2
>24.5
3
2
0 °C. UV spectra were obtained on a JASCO V-560 spectropho-
60.4 ± 54.8
>20 < 100
>20 < 100
>20 < 100
>100
6.9
tometer in absolute EtOH. IR (film) spectra were measured on a
1
>2.9 < 14.4
>1.9 < 9.6
>2.3 < 11.4
>2.6
Bruker IFS 55 spectrophotometer. H (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 (CDCl : δH 7.26, δC 77.0); DEPT,
3
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 H O−H SO −HOAc (1:4:20). All solvents used
>20 < 100
>20 < 100
14.2 ± 2.4
36.2 ± 3.4
>10
>4.8 < 23.9
>1.5 < 7.5
8.2
2.8
bevirimat
>1000
a
CI50 (inhibitory concentration 50%) and CC50 (cytotoxic concen-
tration 50%) were calculated using GrapPad Prism software. SI:
b
selectivity index (CC /IC ). SD: standard deviation. Compounds
50
50
not included in the table were inactive (<50% replication inhibition at
0 μM). All values are the mean of at least three independent
1
experiments.
2
2
4
diversity in the search for new natural product-based anti-HIV
agents.
were analytical grade (Panreac). The reagents were purchased from
Aldrich and used without further purification.
In summary, a set of 48 lupane-type triterpenoids were tested
for anti-HIV activity. Three compounds from this series
Plant Material. Cassine xylocarpa (Vent.) was collected in August
2004 at the Parque Nacional El Imposible (Province of Ahuachapan,
́
G
DOI: 10.1021/np501025r
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Journal of Natural Products
Article
5
El Salvador) and identified by J. Monterrosa. A voucher specimen
3β,6β,20-Trihydroxylupane (4): colorless, amorphous solid; [α]2
D
(
Max Sandoval ISB874) was deposited in the Herbarium of Jardin
Botanico La Laguna, El Salvador. Maytenus cuzcoina Loesener was
collected at Huayllabamba-Urquillos, Province of Urubamba, Cusco
Peru), in December 1998, and was identified by Prof. Alfredo
́
−4 (c 0.5, CHCl ); IR (film) ν 3388, 2927, 2856, 1721, 1460, 1380,
1166, 1020, 758, 471 cm ; for H (400 MHz, CDCl ) and C NMR
) data, see Tables 1 and 2; EIMS m/z (%) 442 [M
O] , 424 (6), 409 (5), 400 (0.8), 351 (0.8), 218 (21), 187 (42),
149 (58), 123 (100), 95 (55), 81 (51); HRESIMS m/z 483.3814
3
max
−1 1 13
́
3
(100 MHz, CDCl
3
+
(
− H
2
Tupayachi. A voucher specimen (cuz 02765 A.T. 1004 MO) was
deposited in the herbarium of Vargas, Department of Botany, in the
National University of San Antonio Abad de Cusco.
+
(calcd for C30
H O Na [M + Na] 483.3814).
52 3
1β,3α,28-Trihydroxylup-20(29)-ene (5): colorless, amorphous
+4 (c 0.3, CHCl ); IR (film) νmax 3305, 2920, 2858,
1450, 1374, 1248, 1040, 1008, 983, 880, 756 cm ; for H (400 MHz,
) data, see Tables 1 and 2;
EIMS m/z (%): 458 (M , 14), 440 (100), 427 (39), 422 (32), 409
26), 391 (16), 273 (12), 245 (28), 219 (31), 201 (48), 189 (79), 175
89), 159 (30), 135 (47), 119 (53), 95 (62); HREIMS m/z 458.3748
solid; [α]2
5
Extraction and Isolation. The dried stems (1.2 kg) of C.
xylocarpa were extracted with EtOH in a Soxhlet apparatus.
Evaporation of the solvent under reduced pressure provided 89.5 g
of crude extract, which was partitioned into a CH Cl −H O (1:1, v/v)
D 3
−1
1
CDCl
) and 13C NMR (100 MHz, CDCl
3
3
+
2
2
2
(
(
[
solution. Removal of the CH Cl from the organic-soluble extract
2
2
under reduced pressure yielded 27.3 g of residue, which was
chromatographed on silica gel (1 kg) eluting with mixtures of
hexanes−EtOAc of increasing polarity (0−100%) to obtain 48
fractions, which were combined into six fractions (A−F) on the
basis of their TLC profile. These fractions were repeatedly subjected to
+
M ] (calcd for C H O , 458.3760).
30 50
3
1
1α,28-Dihydroxy-3-oxolup-20(29)-ene (6): colorless, amorphous
solid; [α]D +30 (c 0.2, CHCl ); IR (film) ν 3438, 3071, 2928, 2868,
698, 1643, 1462, 1383, 1071, 1030, 757 cm ; for H (400 MHz,
CDCl ) and C NMR (100 MHz, CDCl ) data, see Tables 1 and 2;
EIMS m/z (%) 456 (M , 32), 438 (71), 425 (50), 407 (61), 233 (26),
19 (37), 201 (89), 189 (91), 175 (77), 161 (41), 121 (82), 107 (91),
25
3
max
−1
1
1
13
column chromatographies over Sephadex LH-20 (hexanes−CHCl −
3
3
3
+
MeOH, 2:1:1) and silica gel (hexanes−EtOAc of increasing polarity
2
9
4
5
−50%), centrifugal planar chromatography, and preparative TLC
+
5 (100); HREIMS m/z 456.3604 [M ] (calcd for C H O ,
using mixtures of hexanes−EtOAc (8:2), hexanes−Et O (3:7), or
30 48
3
2
56.3603).
General Procedure for Esterification of Betulin (24).
Compounds 27−36. A solution of betulin (24), dry pyridine
0.5−1.5 mL), and an excess of the corresponding acid derivative in
dry CH Cl (2−4 mL) was stirred at 0 °C under an Ar atmosphere
and different sets of conditions, which are specified below for each
procedure. The progress was monitored by TLC using hexanes−
EtOAc (7:3). After the mixture was concentrated to dryness under
reduced pressure, the residue was purified by preparative TLC using
hexanes−EtOAc (8:2) to afford the corresponding esters (27−36).
Preparation of Compounds 27 and 34. To a solution of betulin
CH Cl −EtOAc (9:1) to afford the new compounds 1 (7.3 mg), 2
2
2
(
3.2 mg), 3 (3.1 mg), and 4 (10.8 mg), in addition to the known
compounds 7 (6.4 mg), 8 (8.5 mg), 10 (6.7 mg), 18 (6.5 mg), 25 (5.3
mg), and 26 (30.8 mg). The root bark of M. cuzcoina (0.9 kg) was
(
2
2
extracted with hexanes−Et O in a Soxhlet apparatus. The extract (25.5
2
g) was chromatographed on Sephadex LH-20 (hexanes−CHCl −
3
MeOH, 2:1:1) to afford 64 fractions, which were combined into nine
fractions (A−I) on the basis of their TLC profiles. Preliminary NMR
studies revealed that six fractions (B−G) were rich in lupane
triterpenoids and were further investigated. Flash chromatography of
fraction G (1.6 g) on silica gel, using a gradient elution with hexanes−
EtOAc (20−100% EtOAc), yielded 18 fractions (G1−G18) following
combination on the basis of TLC profiles. The new compounds 5 (3.5
mg) and 6 (2.3 mg) were isolated from fractions G12 (14.8 mg) and
G7 (15.1 mg), respectively, by preparative TLC, using hexanes−
acetone (6:4 and 7:3, respectively) as eluent. In this way, after several
(
150.0 mg, 0.3 mmol) was added Ac O (0.03 mL, 0.3 mmol), and the
2
reaction mixture was stirred for 4 h. The residue was purified by flash
chromatography on silica gel (eluting with hexanes−EtOAc from 5−
0%) to give 27 (127.2 mg, 77%) and 34 (40.3 mg, 9%). Their NMR
5
and MS data were identical to those of 28-O-acetylbetulin and 3,28-di-
14
O-acetylbetulin.
chromatographies on Sephadex LH-20 (hexanes−CHCl −MeOH,
3
Preparation of Compound 28. A solution of betulin (22.7 mg,
.05 mmol) was treated with pivaloyl chloride (0.04 mL, 39.2 mg, 0.32
mmol) and stirred for 5 min. The residue was purified by flash
chromatography on silica gel using hexanes−EtOAc (10:0 to 7:3) as
eluent to yield 28 (14.6 mg, 54%) as an amorphous solid.
Spectroscopic and spectrometric data of the known derivative 28-O-
2
:1:1), silica gel (CH Cl −Et O of increasing polarity), and
2
2
2
0
preparative TLC, the known compounds 9 (7.0 mg), 11 (40.0 mg),
2 (10.0 mg), 13 (200.0 mg), 14 (75.0 mg), 15 (12.3 mg), 16 (10.0
1
mg), 17 (7.3 mg), 19 (7.0 mg), 20 (2.3 mg), 21 (20.5 mg), 22 (220.0
mg), 23 (30.0 mg), and 24 (16.0 mg) were afforded.
6
β,30-Dihydroxylup-20(29)-en-3-one (1): colorless, amorphous
29
pivaloylbetulin (28) are given in the Supporting Information (S26),
25
solid; [α] −38 (c 0.5, CHCl ); IR (film) ν 3462, 2937, 2864,
701, 1461, 1385, 1058, 1027, 760 cm ; for H (400 MHz, CDCl )
and C NMR (100 MHz, CDCl ) data, see Tables 1 and 2; EIMS m/z
%) 456 (M , 95), 438 (37), 425 (85), 407 (8), 397(4), 329 (21), 234
D
3
max
as these data have not been previously reported.
−1
1
1
3
Preparation of Compounds 29 and 35. To a solution of betulin
(41.0 mg, 0.09 mmol) was added 0.4 mL of a previously prepared
solution of octanoyl chloride (0.1 mL, 0.6 mmol, in 2 mL of dry
CH Cl ), and the mixture was stirred for 4.5 h. The mixture was
purified to afford 29 (4.5 mg, 9%) and 35 (22.5 mg, 35%) as
amorphous solids. NMR and MS data of 29 were identical with those
13
3
+
(
(
(
81), 221 (53), 205 (51), 203 (100), 121 (78), 107 (86), 95 (95), 81
2
2
75), 67 (44), 55 (90); HREIMS m/z 456.3595 (calcd for C H O
30
48
3
+
[
M ] 456.3603).
β-Hydroxy-3-oxolup-20(29)-en-30-al (2): colorless, amorphous
2
0
6
reported for 28-O-octanoylbetulin.
2
5
3β,28-Di-O-octanoylbetulin (35): [α]2 +1 (c 2.2, CHCl ); IR
(film) νmax 2928, 2858, 1733, 1461, 1257, 1168, 978 cm ; H NMR
5
D
solid; [α] −7 (c 0.1, CHCl ); UV (EtOH) λ (log ε) 225 (4.18)
nm; IR (film) νmax 3526, 2926, 2856, 1695, 1520, 1458, 1379, 1084,
71, 937, 470 cm ; for H (400 MHz, CDCl ) and C NMR (100
MHz, CDCl ) data, see Tables 1 and 2; EIMS m/z (%) 454 (M , 90),
D
3
max
3
−1 1
−1
1
13
9
3
(CDCl , 400 MHz) δ 2 × Oct [2.34 (4H, t, J = 7.7 Hz, H-2′, H-2″),
3
+
3
1.69 (4H, m, H-3′, H-3″), 1.26 (16H, m, H-4′, H-4″, H-5′, H-5″, H-6′,
4
2
39 (10), 436 (27), 421 (21), 357 (10), 339 (10), 232 (15), 219 (31),
03 (100), 189 (21), 185 (12), 175 (28), 161 (24), 149 (23), 135
H-6″, H-7′, H-7″), 0.90 (6H, t, J = 6.6 Hz, H-8′, H-8″)], for other
signals, see Table 3; 13C NMR (CDCl , 100 MHz) δ 2 × Oct [174.4
3
(
27), 119 (22), 109 (24), 95 (35), 81 (27), 69 (22), 55 (29);
(C, C-1′), 173.8 (C, C-1″), 34.9 (2 × CH , C-2′, C-2″), 31.7 (2 ×
2
+
HREIMS m/z 454.3434 (calcd for C H O [M ] 454.3447).
CH , C-3′, C-3″), 29.8 (CH , C-4″), 29.6 (CH , C-4′), 25.1 (2 × CH ,
30
46
3
2
2
2
2
3
-Oxolup-20(29)-en-30-oic acid (3): colorless, amorphous solid;
C-5′, C-5″), 23.7 (2 × CH , C-6′, C-6″), 22.6 (2 × CH , C-7′, C-7″),
2
2
25
[
α] +9 (c 0.3, CHCl ); UV (EtOH) λ (log ε) 226 (4.09) nm; IR
D
15.0 (CH , C-8″), 14.0 (CH , C-8′)], for other signals, see Table 4;
3
max
3
3
+
(
1
film) νmax 3470−2640, 3393, 2928, 2859, 1702, 1620, 1458, 1382,
EIMS m/z (%) 694 [M ] (3), 550 (100), 507 (23), 407 (9), 393 (9),
333 (4), 229 (8), 203 (43), 189 (88), 135 (41), 95 (34); HREIMS m/
−1
1
13
272, 1140, 940, 670 cm ; for H (400 MHz, CDCl ) and C NMR
3
+
+
(
100 MHz, CDCl ) data, see Tables 1 and 2; EIMS m/z (%) 454 (M ,
z 694.5935 [M ] (calcd for C H O , 694.5900).
3
46 78
4
1
00), 439 (10), 436 (14), 426 (4), 421 (8), 408 (7), 382 (4), 313
Preparation of Compound 30. A betulin solution (40.6 mg, 0.09
mmol) was treated with benzoyl chloride (0.1 mL, 0.86 mmol), and
the mixture was stirred for 3 h. The crude product was purified to
(
(
20), 248 (17), 205 (58), 189 (32), 149 (44), 95 (59), 81 (57), 55
68); HREIMS m/z 454.3456 (calcd for C H O [M ] 454.3447).
+
30
46
3
H
DOI: 10.1021/np501025r
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
afford 28-O-benzoylbetulin (30, 31.8 mg, 63%) as an amorphous solid:
room temperature for 3 days and then diluted with a saturated solution
of NaHCO and extracted with CH Cl repeatedly. The combined
25
[
α] +1 (c 3.2, CHCl ); UV (EtOH) λ (log ε) 201 (3.6), 229 (3.4),
D 3 max
3
2
2
2
76 (2.0) nm; IR (film) ν 3516, 2945, 2870, 1715, 1454, 1274,
organic layer was dried with MgSO and concentrated to give an oil,
max
4
−1 1
1
115, 757, 712 cm ; H NMR (CDCl , 400 MHz) δ Bz [8.08 (2H,
which was purified by flash chromatography and preparative TLC on
silica gel (eluting with hexanes−EtOAc, 7.5:2.5) to afford 33 (13.6 mg,
19%). Spectroscopic and spectrometric data of the known derivative
3
dd, J = 8.4, 1.1 Hz, H-2′, H-6′), 7.58 (1H, m, H-4′), 7.46 (2H, t, J =
1
3
7
1
1
.5 Hz, H-3′, H-5′)], for other signals, see Table 3; C NMR (CDCl ,
3
3
0
00 MHz) δ Bz [166.9 (C, COO), 132.9 (CH, C-4′), 130.5 (C, C-1′),
28-O-tosylbetulin (33) are provided in the Supporting Information
(S26), as these data have not been previously reported.
29.6 (2 × CH, C-2′, C-6′), 128.3 (2 × CH, C-3′, C-5′)], for other
+
signals, see Table 4; EIMS m/z (%) 546 [M ] (16), 528 (33), 513 (5),
Preparation of Compound 37. A solution of betulin (39.2 mg,
0.09 mmol) was treated with imidazole (6.7 mg, 0.10 mmol), DMAP
(11.3 mg, 0.09 mmol), and TBDMSCl (28.7 mg, 0.19 mmol) at 0 °C.
The resulting mixture was stirred at room temperature for 3.5 h under
4
85 (7), 424 (25), 411 (20), 409 (5), 381 (4), 220 (10), 216 (19), 207
(
35), 189 (60), 135 (40), 105 (100), 81 (40); HREIMS m/z 546.4064
+
[
M ] (calcd for C H O , 546.4073).
37 54 3
Preparation of Compounds 31 and 36. To a solution of betulin
16.5 mg, 0.04 mmol) was added 1-naphthoyl chloride (0.1 mL, 0.7
a N atmosphere until TLC showed complete conversion. The mixture
2
(
was diluted with H O (10 mL) and extracted with CH Cl repeatedly.
2
2
2
mmol), and the mixture was stirred for 3 h. Et O (5 mL) and KF (128
The organic layer was dried with MgSO , filtered, and concentrated in
2
4
mg, 2.2 mmol) were added and stirred at room temperature for 3 h
and filtered on Celite. The crude was purified to afford 31 (9.5 mg,
vacuo. The crude product was purified by preparative TLC on silica gel
(eluting with 40% EtOAc in hexanes) to yield 37 (25.8 mg, 33%) as an
1
13
4
3%) and 36 (7.0 mg, 25%) as amorphous solids.
amorphous solid. The complete H and C NMR assignments of the
8-O-(1-Naphthoyl)betulin (31): [α]2 +1 (c 1.0, CHCl ); UV
5
known derivative 28-O-(tert-butyldimethylsilyl)betulin (37) are
31
2
D
3
(
(
1
EtOH) λmax (log ε) 211 (4.4), 221 (4.5), 237 (4.1), 294 (3.7), 325
given in the Supporting Information (S27).
Preparation of Compound 38. To a solution of betulin (11.0
3.0) nm; IR (film) ν 3438, 2933, 2872, 1715, 1463, 1284, 1247,
max
−
1 1
201, 1140, 787 cm ; H NMR (CDCl , 400 MHz) δ Nap [8.95 (1H,
mg, 0.02 mmol) in dry CH Cl (2 mL) at 0 °C was added PCC (17.3
3
2
2
d, J = 8.5 Hz, H-8′), 8.19 (1H, d, J = 7.2 Hz, H-2′), 8.03 (1H, d, J = 8.2
mg, 0.08 mmol). The mixture was stirred for 90 min until TLC
showed complete conversion. The organic layer was filtrated through
Florisil and concentrated to give an oil, which was purified by
preparative TLC (hexanes−EtOAc, 8.5:1.5) to yield 38 (6.7 mg, 62%)
as an amorphous solid. The NMR and MS data of 38 were identical to
Hz, H-4′), 7.89 (1H, d, J = 8.3 Hz, H-5′), 7.62 (1H, t, J = 7.9 Hz, H-
7
′), 7.54 (1H, t, J = 7.6 Hz, H-6′), 7.51 (1H, t, J = 7.8 Hz, H-3′)], for
13
other signals, see Table 3; C NMR (CDCl , 100 MHz) δ Nap [168.0
3
(
1
C, COO), 133.9 (C, C-4a′), 133.3 (CH, C-4′), 131.4 (C, C-8a′),
3
1
30.1 (CH, C-2′), 128.5 (CH, C-5′), 128.1 (C, C-1′), 127.7 (CH, C-
those reported for 3-oxolup-20(29)-en-28-al (betulonal).
7
′), 126.2 (CH, C-6′), 125.9 (CH, C-8′), 124.5 (CH, C-3′)], for other
Preparation of Compound 39. To a solution of 28-O-
acetylbetulin (27, 17.7 mg, 0.04 mmol) in dry DCM (5 mL) was
added PCC (25.4 mg, 0.11 mmol), and the mixture was stirred at 0 °C
for 4 h. Filtration through Florisil and concentration in vacuo gave an
oil, which was purified by flash chromatography on silica gel
(hexanes−EtOAc of increasing polarity 10−50%) to yield 39 (17.7
mg, 92%) as an amorphous solid. NMR and MS data of 39 were
+
signals, see Table 4; EIMS m/z (%) 596 [M ] (17), 578 (22), 535 (5),
4
24 (30), 409 (4), 393 (2), 270 (2), 216 (15), 203 (17), 189 (28), 172
(
12), 155 (100), 127 (21), 95 (15), 81 (15); HREIMS m/z 596.4251
+
[
M ] (calcd for C H O , 596.4229).
41 56 3
2
5
3
β,28-Di-O-(1-naphthoyl)betulin (36): [α] −1 (c 0.7, CHCl );
D
3
UV (EtOH) λmax (log ε) 203 (3.8), 219 (3.8), 233 (3.6), 294 (3.0)
nm; IR (film) νmax 2927, 2868, 1711, 1241, 1134, 1011, 781 cm ; H
−1
1
32
identical to those reported for 28-O-acetyl-3-oxobetulin.
NMR (CDCl , 400 MHz) δ 2 × Nap [8.96 (1H, d, J = 8.4 Hz, H-8′),
Preparation of Compound 40. To a mixture of 27 (16.0 mg,
0.03 mmol) and N-methylmorpholine N-oxide (11.6 mg, 0.09 mmol)
in 2 mL of THF−H O (1:1) was added a catalytic amount of OsO .
3
8
8
8
.95 (1H, d, J = 8.5 Hz, H-8″), 8.20 (1H, dd, J = 7.4, 1.3 Hz, H-2′),
.17 (1H, dd, J = 7.2, 1.3 Hz, H-2″), 8.03 (1H, d, J = 8.1 Hz, H-4′),
.01 (1H, d, J = 8.0 Hz, H-4″), 7.89 (1H, d, J = 7.7 Hz, H-5′), 7.88
2
4
The resulting mixture was stirred at room temperature for 54 h,
diluted with brine, and extracted with EtOAc repeatedly. The organic
layers were combined, washed with NaHSO , dried over MgSO , and
(
1H, d, J = 7.6 Hz, H-5″), 7.61 (2H, m, H-7′, H-7″), 7.52 (4H, m, H-
13
3
′, H-3″, H-6′, H-6″)], for other signals, see Table 3; C NMR
3
4
(
CDCl , 100 MHz) δ 2 × Nap [167.3 (2 × C, 2 × COO), 133.9 (2 ×
concentrated to give an oil, which was purified by flash
3
C, C-4a′, C-4a″), 133.3 (CH, C-4′), 133.1 (CH, C-4″), 131.4 (2 × C,
chromatography on silica gel (hexanes−EtOAc, 6:4) to yield 28-O-
C-8a′, C-8a″), 130.1 (CH, C-2′), 129.8 (CH, C-2″), 128.6 (CH, C-
acetyl-3β,20,29-trihydroxylupane (40) (12.5 mg, 73%) as an
amorphous solid: [α]2 −7 (c 1.2, CHCl ); IR (film) ν
5
3416,
5
′), 128.5 (CH, C-5″), 128.0 (C, C-1′), 127.7 (CH, C-7′), 127.6 (CH,
D
3
max
−1
1
C-7″), 127.4 (C, C-1″), 126.2 (CH, C-6′), 126.1 (CH, C-6″), 125.9
2940, 2873, 1730, 1459, 1387, 1245, 1032, 756 cm ; H NMR
(
CH, C-8′), 125.8 (CH, C-8″), 124.5 (2 × CH, C-3′, C-3″)], for other
(CDCl , 400 MHz) δ Ac [2.08 (3H, s, H-2′)], for other signals, see
3
+
13
signals, see Table 4; EIMS m/z (%) 750 [M ] (2), 578 (18), 565 (2),
35 (4), 423 (2), 406 (3), 393 (2), 269 (1), 216 (4), 189 (16), 155
Table 3; C NMR (CDCl , 100 MHz) δ Ac [171.3 (C, C-1′), 20.8
3
5
(CH , C-2′)], for other signals, see Table 4; EIMS m/z (%) 500 [M −
3
+
+
(
100), 127 (17); HREIMS m/z 750.4617 [M ] (calcd for C H O ,
H O] (12); 482 (21), 469 (14), 427 (31), 409 (26), 371 (14), 353
52
62
4
2
7
50.4648).
(7), 273 (5), 245 (8), 219 (15), 207 (90), 189 (100), 135 (80), 95
+
Preparation of Compound 32. A solution of betulin (44.1 mg,
(65), 81 (61); HREIMS m/z 500.3848 [M − H O] (calcd for
2
0.1 mmol) and 4-bromobenzoyl chloride (29.3 mg, 0.13 mmol) was
C H O , 500.3866).
32
52
4
stirred for 5 min. The mixture was purified to afford 28-O-(4-
bromobenzoyl)betulin (32, 15.2 mg, 24%) as an amorphous solid:
Preparation of Compound 41. To an ice-cold solution of
compound 27 (15.5 mg, 0.04 mmol) in CH Cl (2 mL) was added a
2
2
25
[
α] +3 (c 0.3, CHCl ); UV (EtOH) λ (log ε) 202 (4.5), 244 (4.3)
suspension of NaHCO (130 mg, 1.55 mmol) and MCPBA (10.0 mg,
D
3
max
3
nm; IR (film) ν 3432, 2927, 2868, 1721, 1591, 1459, 1270, 1104,
0.06 mmol) in DCM (2 mL). The resulting mixture was stirred at 0 °C
max
−1 1
7
56 cm ; H NMR (CDCl , 400 MHz) δ pBrBz [7.92 (2H, d, J = 8.5
under an Ar atmosphere for 7 h and diluted with Et O (10 mL). KF
3
2
Hz, H-2′, H-6′), 7.61 (2H, d, J = 8.5 Hz, H-3′, H-5′)], for other
(30 mg, 0.52 mmol) was added, and the mixture was stirred at room
temperature for 24 h and filtered on Celite. The filtrate was diluted
signals, see Table 3; 13C NMR (CDCl , 100 MHz) δ pBrBz [165.6 (C,
3
COO), 131.8 (2 × CH, C-3′, C-5′), 131.1 (2 × CH, C-2′, C-6′), 129.4
with H O and extracted with DCM repeatedly. The organic layer was
2
(
(
C, C-1′), 128.0 (C, C-4′)], for other signals, see Table 4; EIMS m/z
dried (Na SO ), filtered, and concentrated in vacuo. The crude
2
4
+
%) 627 [M ] (4), 626 (12), 624 (12), 608 (24), 425 (9), 424 (26),
product was purified by preparative TLC on silica gel (hexanes−
EtOAc, 7:3) to afford 41 (9.5 mg, 54%) as an amorphous solid. NMR
and MS data of 41 were identical to those reported for 28-O-acetyl-
4
1
11 (32), 393 (7), 257 (8), 216 (26), 207 (62), 189 (100), 184 (54),
35 (64), 95 (54); HREIMS m/z 627.3190 [M ] (calcd for
+
3
3
C H BrO , 627.3236).
20R,29-epoxy-3β-hydroxylupane.
37
54
3
Preparation of Compound 33. To a solution of betulin (54.9
mg, 0.12 mmol) were added p-toluenesulfonyl chloride (70.0 mg, 0.37
Preparation of Compound 42. To a solution of compound 27
(70.0 mg, 0.14 mmol) in EtOH (6 mL) was added SeO (18.0 mg,
2
mmol) and a catalytic amount of DMAP. The mixture was stirred at
0.16 mmol). The resulting mixture was heated under reflux and an Ar
I
DOI: 10.1021/np501025r
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
atmosphere for 32 h and then diluted with H O (20 mL). The mixture
Plasmids and Virus. The vector pNL4.3-Luc was generated by
cloning the luciferase gene in the HIV-1 proviral clone pNL4.3.
Plasmid pNL4.3-Ren was generated by cloning the renilla gene in the
2
was extracted with EtOAc (3 × 20 mL), and the organic layers were
combined, dried, and concentrated to give an oil, which was purified
by flash chromatography on silica gel (hexanes−EtOAc of increasing
polarity 10−50%) to yield 42 (28-O-acetyl-3β-hydroxylup-20(29)-en-
3
9
Luc site of pNL4.3-Luc.
Recombinant Virus Assay. The anti-HIV activity of compounds
was performed as follows: infectious supernatants were obtained from
Ca (PO ) transfection on 293t cells of plasmid pNL4.3-Ren. These
34
3
0-al) (9.3 mg, 13%) as an amorphous solid.
Preparation of Compounds 43 and 44. To a solution of
3
4 2
compounds 8 (7.4 mg, 0.02 mmol) or 18 (5.1 mg, 0.01 mmol) and a
supernatants were used to infect cells in the presence or absence of the
compounds to be evaluated. Anti-HIV activity quantification was
performed 48 h postinfection. Briefly, cells were lysed with 100 μL of
buffer provided by the Renilla Assay System (Promega). Relative
luminescence units (RLUs) were obtained in a luminometer (Berthold
Detection Systems) after the addition of substrate to cell extracts.
Viability was performed in parallel treated cells with the same
concentrations of compound. After 48 h, cell viability was evaluated
with the CellTiter Glo (Promega) assay system following the
catalytic amount of DMAP in CH Cl (2 mL) were added Et N (0.1
2
2
3
mL, 0.7 mmol) and Ac O (0.1 mL, 1.0 mmol). After being stirred for
2
3
0 min at room temperature, the mixture was quenched with EtOH (1
mL) and stirred for 30 min. The mixture was evaporated to dryness,
and the residue was purified by flash chromatography on silica gel
(
7
hexanes−EtOAc of increasing polarity 20−40%) to yield 43 (6.7 mg,
1 13
0%) or 44 (5.0 mg, 95%) as amorphous solids. The H and C NMR
assignments of the known derivative 30-O-acetyl-3-oxo-lup-20(29)-ene
35
(
43) are provided in the Supporting Information (S28), as these data
manufacturer’s specifications. Inhibitory concentrations 50% (IC50)
have not been previously reported. NMR and MS data of 44 were
identical to those reported for 3β,30-di-O-acetyllup-20(29)-ene.
and cytotoxic concentrations 50% (CC50) were calculated using
GraphPad Prism software.
1
6
Preparation of Compound 45. To a solution of compound 9
22.0 mg, 0.05 mmol) in dry THF (2 mL) at 0 °C and under an Ar
atmosphere was added PTAB (21.0 mg, 0.06 mmol). The resulting
(
ASSOCIATED CONTENT
■
*
S
Supporting Information
mixture was stirred at 0 °C for 12 min and diluted with 10 mL of H O.
2
The aqueous phase was extracted with EtOAc (3 × 10 mL), and the
organic phases were combined and washed with brine, dried (MgSO4),
and concentrated in vacuo. The crude product was purified by flash
chromatography using hexanes−EtOAc (9:1) as eluent to provide 45
NMR spectra for natural pentacyclic lupane triterpenes (1−6)
and new derivatives (30−32, 35, 36, 40, and 48), experimental
data of derivatives 28, 33, 37, and 43, computational modeling
of two possible stereoisomers of compound 1, and screening of
compounds 1−48 as HIV replication inhibitors are available
free of charge on the ACS Publications website at DOI:
(
19.0 mg, 75%). NMR and MS data of 45 were identical to those
36
reported for 2-bromo-3-oxolup-20(29)-ene.
Preparation of Compound 46. To a solution of compound 13
1
0.1021/np501025r.
(
81.1 mg, 0.18 mmol) in CH Cl (3 mL) were added a catalytic
2
2
amount of DMAP, Et N (0.1 mL, 0.7 mmol), and Ac O (0.1 mL, 1.0
mmol). After being stirred for 30 min at room temperature, until TLC
showed complete conversion, the mixture was quenched with EtOH
3
2
AUTHOR INFORMATION
■
*
Corresponding Author
Tel: +34-922-318594. E-mail: ilopez@ull.es.
(
1 mL) and stirred for 30 min. The mixture was evaporated to dryness,
and the residue was purified by flash chromatography on silica gel
hexanes−EtOAc of increasing polarity 20−40%) to yield 46 (80.7
mg, 91%), whose NMR and MS data were identical to those of the
(
Notes
The authors declare no competing financial interest.
14
reported 11α-O-acetyl-3-oxolup-20(29)-ene.
Preparation of Compounds 47 and 48. A solution of
compound 46 (76.0 mg, 0.16 mmol) and PhSeCl (76.0 mg, 0.4
mmol) in EtOAc (30.0 mL) was stirred at room temperature for 12 h.
To the stirred mixture was added a saturated aqueous NaHCO3
solution. After most of the aqueous layer was removed, pyridine (0.15
mL, 0.4 mmol) and MCPBA (50−60%) (284.0 mg, 0.4 mmol) were
added to the organic layer. The mixture was washed with 5% aqueous
ACKNOWLEDGMENTS
■
This study was supported by the FP7-REGPOT-2012-CT2012-
316137-IMBRAIN project and by CHAARM and AIM-HIV
UE projects of the Seventh Framework program, AIDS
Network ISCIII-RETIC (RD06/0006), and Fondo de Inves-
tigaciones Sanitarias (FIS PI12/00506). We also thank the
NaOH (3 × 10 mL), saturated aqueous NH Cl, and brine, dried over
4
Servicio de Parques Nacionales y Vida Silvestre, Direccion
Recursos Renovables, Ministerio de Agricultura y Ganaderia
(MAG), and Fundacion Ecologica de El Salvador (SALVANA-
́
de
MgSO , and filtered. The mixture was evaporated to dryness, and the
4
́
residue was purified by flash chromatography on silica gel (hexanes−
EtOAc of increasing polarity 40−70%) to yield 47 (21.8 mg, 25%) and
́
́
4
8 (8.2 mg, 7%) as amorphous solids. NMR and MS data of 47 were
TURA) for supplying the speciesC. xylocarpa and Prof. A.
Tupayachi for kindly providing the species M. cuzcoina. A.A.O.
would like to thank the MAEC-AECID for a fellowship.
coincident with those reported for 11α-O-acetyl-30-chloro-3-oxolup-
1
4
2
0(29)-ene.
1α-O-Acetyl-30-chloro-3-oxolup-1,20(29)-diene (48): [α] 2D 5 +45
c 0.4, CHCl ); IR (film) ν 2927, 2856, 1735, 1673, 1459, 1380,
1
(
1
(
3
max
−1
1
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■
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