Dihydro-β-agarofuran sesquiterpenes isolated from Celastrus vulcanicola as potential anti-Mycobacterium tuberculosis multidrug-resistant agents

Article abstract of DOI:10.1016/j.bmc.2011.02.034

In the present study, we report four new dihydro-β-agarofuran sesquiterpenes (1-4), which were isolated from the leaves of Celastrus vulcanicola, in addition to five derivatives (5-9). Their stereostructures were elucidated on the basis of spectroscopic a

Full text of DOI:10.1016/j.bmc.2011.02.034

Bioorganic & Medicinal Chemistry 19 (2011) 2182–2189
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dihydro-b-agarofuran sesquiterpenes isolated from Celastrus vulcanicola
as potential anti-Mycobacterium tuberculosis multidrug-resistant agents
David Torres-Romero a,b, Ignacio A. Jiménez a, Rosario Rojas c, Robert H. Gilman c, Matías López a,
Isabel L. Bazzocchi a,
⇑
a Instituto Universitario de Bio-Orgánica Antonio González y, Departamento de Química Orgánica, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2, 38206
La Laguna, Tenerife, Spain
b Departamento de Bioquímica y Contaminación Ambiental, Facultad de Quıímica y Farmacia, Universidad de El Salvador, Final 25 Avenida Norte, El Salvador
c Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, we report four new dihydro-b-agarofuran sesquiterpenes (1–4), which were isolated
from the leaves of Celastrus vulcanicola, in addition to five derivatives (5–9). Their stereostructures were
elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, X-ray studies,
chemical correlations and biogenetic means. Compounds 1–9 and the previously reported sesquiterpenes
10–25 have been tested as potential antimycobacterial agents against sensitive and resistant Mycobacte-
Received 4 October 2010
Revised 18 February 2011
Accepted 21 February 2011
Available online 26 February 2011
rium tuberculosis strains. 1
berculosis activity against the MDR TB strain with a MIC value of 6.2
than isoniazid or rifampin, two of the best first-line drugs commonly used in the treatment of TB. The
structure–activity relationship is discussed.
a
-Acetoxy-6b,9b-dibenzoyloxy-dihydro-b-agarofuran (20) exhibited antitu-
Keywords:
Celastrus vulcanicola
Sesquiterpenes
Mycobacterium tuberculosis
Multidrug-resistant
l
g/mL, comparable to or better
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
tures and wide range of biological properties, including P-glypro-
tein dependent multidrug resistance modulation in Leishmania
It is estimated that there are approximately 9 million new
tuberculosis (TB) cases every year. Two major obstacles to global
tuberculosis control are due to the high prevalence of HIV among
TB patients and the deteriorating socio-economic conditions of
the most underprivileged members of society.1 Although the exist-
ing standard regimen is very effective against TB, the length of
treatment (6 months), the toxicity and the potential for drug–drug
interactions, particularly in the setting of antiretroviral treatment,
are factors that highlight the need for new antitubercular drugs.2,3
In addition, the advent of strains of Mycobacterium tuberculosis
(MTB) resistant to, at least, the two first-line drugs (MDR TB), iso-
niazid and rifampin or resistant to three of the six classes of sec-
ond-line drugs (XDR TB) is of great concern.4 Thus, there is a
great need to develop new therapeutic agents to treat tuberculosis,
particularly MDR TB which has severely limited the number of
effective treatment options.5
tropica6 and cancer cells.7 On the basis of these properties sesqui-
terpenes have been selected as ‘privileged structures’.8 Recently,
the antituberculosis activity of this type of metabolites on M. tuber-
culosis sensitive strains has been reported.9–11
In the present study, and as a continuation of our research on
Celastrus vulcanicola,7,12 we describe the isolation, structure eluci-
dation and bioactivity of four new (1–4) sesquiterpenes with a
dihydro-b-agarofuran skeleton, in addition to five derivatives
(5–9). Their structures were elucidated by means of 1H and 13C
NMR spectroscopic studies, including homonuclear (COSY and
ROESY) and heteronuclear correlation (HSQC and HMBC) experi-
ments. The absolute configurations of these compounds were
established by biogenetic means, chemical correlations and X-ray
crystallographic analysis. In the search for potential antimycobac-
terial agents, sesquiterpenes 1–9 and those previously isolated
from C. vulcanicola, 10–25,7,12 were evaluated against the MTB
The sesquiterpene polyesters with a dihydro-b-agarofuran skel-
eton are the most widespread and characteristic group of second-
ary metabolites isolated from Celastraceae family. These
compounds have attracted considerable attention from synthetic
organic chemists and pharmacologists due to their complex struc-
H37Rv and MDR strains.
2. Results and discussion
The dichloromethane extract of the leaves of C. vulcanicola was
subjected to repeated chromatography on Sephadex LH-20 and sil-
ica gel, affording four new sesquiterpenes (1–4) (Fig. 1). Compound
1 was isolated as a colorless amorphous solid and showed the
⇑
Corresponding author. Tel.: +34 922 318594; fax: +34 922 318571.
E-mail address: ilopez@ull.es (I.L. Bazzocchi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.034

D. Torres-Romero et al. / Bioorg. Med. Chem. 19 (2011) 2182–2189
2183
AcO
OBz
AcO
OBz
OR
AcO
OBz
15
OAc
OR2
OR2
9
1
3
7
11
5
13
O
O
O
HO
HO
14
OR1
OR1
R1
R2
R1 R2
R
4
1
2
Ac
Ac
H
Nic Ac
H
Bz
3
b)
a)
19
H
Ac
tCin
tCin
H
Nic
13
14
15
16
17
18
10
Bz Ac
AcO
OR2
11
12
H
H
H
tCin
cCin
H
cCin
Ac
Bz
Nic
O
Ac
R1
AcO
OBz
O
R1
R2
15
O
OR2
HO
OBz
OAc
OH
H
Bz
20
Bz =
Ac =
tCin
tCin
tCin
21
22
23
O
O
HO
CH=CH
O
OR1
Cin =
Nic =
R2
R1
Bz
H
24
25
Ac
a)
b)
NicCl, Et3N
tCinCl, Pyridine
tCin
N
Figure 1. Structure of the natural sesquiterpenes assayed for their anti-Mycobacterium tuberculosis activity.
molecular formula C32H37NO9 by EI-HRMS, 1H and 13C NMR data.
The IR spectrum showed an absorption band for ester (1729
cmꢀ1) group. The EI-MS exhibited peaks consistent with loss of
methyl (m/z 564 [M+ꢀMe]), acetic acid (m/z 519 [M+ꢀCH3CO2H]),
benzoic acid (m/z 458 [M++1ꢀPhCO2H], m/z 105 [PhCO]+) and nic-
otinic acid (m/z 456 [M+ꢀC5H4NCO2H], m/z 106 [C5H4NCO]+). Its 1H
NMR data (Table 1) included signals for two acetyl groups at d 1.66
(3H, s) and 1.91 (3H, s) and nine protons in the aromatic region [d
7.50 (3H, m), 7.60 (1H, m), 8.11 (2H, m), 8.33 (1H, m) 8.85 (1H, m)
and 9.26 (1H, s)], corresponding to benzoyl and nicotinoyl groups.
In addition, resonances for six methine protons, three forming an
ABX system at d 5.68 (dd, J = 3.0, 6.3 Hz, H-8), 5.35 (d, J = 6.3 Hz,
H-9) and 2.66 (d, J = 3.0 Hz, H-7), two oxymethine protons at d
5.60 (s, H-6) and 5.50 (dd, J = 4.0, 11.7 Hz, H-1) and one methine
proton at d 2.43 (m, H-4), were observed. Two methylene systems
at d 1.47, 2.04 (2H, m) and 1.72, 2.27 (2H, m) were assigned to pro-
tons H-2, and H-3, respectively. Also singlets from three tertiary
methyl groups at d 1.43, 1.46 and 1.58, and a doublet for one sec-
ondary methyl group at d 1.03 (d, J = 7.4 Hz) were observed. All
these data were confirmed by the 13C NMR spectrum (Table 1),
and the chemicals shifts for the carbons attached to protons were
assigned according to a 2D heteronuclear HSQC experiment. These
data indicate that 1 is a polyester sesquiterpene with a 1,6,8,9-tet-
rasubstituted-dihydro-b-agarofuran skeleton.
Table 1
1H and 13C NMR (d, CDCl3, J in Hz in parentheses) data of compounds 1 and 2
Position
1
2
a
a
dH
dC
dH
dC
The regiosubstitution of 1 was determined by an HMBC exper-
iment, showing a three-bond correlation between the nicotinoyl-
oxy carbonyl resonance at dC 163.9 and the proton at dH 5.60
that corresponds to H-6. The attachment of the benzoyloxy group
to C-9 was defined by the cross-peak between the carboxyl reso-
nance at dC 165.4 and the signal at dH 5.35 (H-9), whereas the acet-
oxy groups were located at positions C-1 and C-8 by correlation of
the carboxyl resonances at dC 169.7 and dC 169.0 with signals as-
signed to H-1 (dH 5.50) and H-8 (dH 5.68), respectively. The relative
configuration of 1 was established on the basis of the coupling con-
stants and confirmed by 2D homonuclear experiments. Therefore,
the coupling constants of H1–H2 (J1–2 = 4.0, 11.7 Hz), H6–H7
(J6–7 = 0 Hz) and H8–H9 (J8–9 = 6.3 Hz), observed in the COSY exper-
iment, indicated an axial orientation for H-1, an equatorial one for
H-6 and a cis-relationship between H-8 and H-9. The ROESY exper-
iment showed correlations between H-1 and H-3 and between Me-
15 and H-6, H-8, H-9 and Me-14 (Fig. 2). Thus, the structure of
1
2
3
4
5
6
7
8
5.50 dd (4.0, 11.7)
1.47 m, 2.04 m
1.72 m, 2.27 m
2.43 m
73.0 d
22.5 t
26.5 t
5.45 dd (4.2, 11.8)
1.64 m, 1.91 m
1.45 m, 2.18 m
2.31 m
73.4 d
21.4 t
26.5 t*
34.4 d
90.7 s
76.3 d
55.3 d
69.6 d
72.6 d
48.4 s
82.6 s
31.3 q
26.5 q*
17.7 q
18.6 q
20.6 q,
169.6 s
20.5 q,
169.7 s
34.1 d
89.6 s
78.4 d
53.6 d
68.9 d
72.3 d
48.8 s
82.7 s
31.3 q
26.3 q
17.2 q
18.5 q
20.5 q*,
169.7 s
20.5 q*,
169.0 s
5.60 s
4.42 s
2.66 d (3.0)
5.68 dd (3.0, 6.3)
5.35 d (6.3)
2.40 d (2.7)
5.41 dd (2.7, 6.2)
5.23 d (6.2)
9
10
11
12
13
14
15
OAc-1
1.46 s
1.58 s
1.03 d (7.4)
1.43 s
1.66 s
1.51 s
1.53 s
1.18 d (7.3)
1.34 s
1.63 s
OAc-8
1.91 s
1.90 s
a
compound 1 was established as 1
a,8b-diacetoxy-9b-benzoyloxy-
Data are based on DEPT, HSQC, and HMBC experiments.
Overlapping signals.
*
6b-nicotinoyloxy-dihydro-b-agarofuran. Its absolute configuration

2184
D. Torres-Romero et al. / Bioorg. Med. Chem. 19 (2011) 2182–2189
169.5, 170.0 and 170.4, assigned to a benzoyl and two acetyl
groups. In addition, two signals at dH 2.88 (1H, s, OH-4) and 4.35
(1H, s, OH-8) interchangeable with D2O, indicating the presence
of two hydroxyl groups, a methyl group at dC 1.34 attached to an
oxygen bearing carbon at dC 72.4, and three methyls at dH 1.55,
1.49 and d 1.41, were also observed. The regiosubstitution was
established by the long-range 1H–13C HMBC couplings observed
between the dH 5.36 (dd, J = 4.5, 12.0 Hz, H-1) and 5.72 (s, H-6) pro-
ton resonances and the signals of the acetate groups at dC 170.0
and 170.4, respectively, whereas the signal at dH 5.29 (d, J =
6.2 Hz, H-9) was coupled to the signal of the benzoate group (dC
169.5). The observed cross-peaks between H-1 and H-3b and be-
tween Me-15 and H-6, H-8, H-9 and Me-14 in a ROESY experiment
13
H
1
H
O
OBz
OAc
12
3
2
9
H
4
8
NicO
6
OAc
14
15
H
H
assigned the relative configuration of 3 as 1a, 4b, 6b, 8b and 9b. The
Figure 2. ROE effects for compound 1.
absolute configuration was established as (1S,4S,5S,6R,7R,8S,9R,
10S)-1,6-diacetoxy-9-benzoyloxy-4,8-dihydroxy-dihydro-b-aga-
rofuran through chemical correlation, as treatment of 3 with trans-
cinnamoyl chloride yielded a compound with spectroscopic data
identical to the known compound 13, whose absolute stereochem-
istry has been previously established.7
was assumed based on it having the same polyhydroxy sesquiter-
pene core as compound 11, whose absolute stereochemistry has
been previously established by dichroism circular.7
The EI-HRMS of compound 2 gave a molecular ion at m/z
474.2261, corresponding to the molecular formula C26H34O8. Its
1H and 13C NMR data (Table 1) were closely related to those of 1,
except for the absence of the signals assigned to the nicotinoyl
group at C-6 and the shift of the signal corresponding to the H-6
proton from dH 5.60 in 1 to dH 4.42 in 2. Detailed assignments of
the 1H and 13C NMR data and the relative configuration were deter-
mined on the basis of COSY, HSQC, ROESY and HMBC experiments.
The absolute configuration of 2 was established by chemical corre-
lation. When compound 2 was treated with nicotinoyl chloride
yielded a compound whose spectroscopic data were identical to
those of 1.
Compound 3 was obtained as a colorless lacquer. Its molecular
formula was established as C26H34O9 by EI-HRMS and 13C NMR
data. The IR absorption bands at 3466 and 1731 cmꢀ1 indicated hy-
droxyl and ester functions, and the UV spectrum revealed the
occurrence of aromatic ring absorptions at 224 and 274 nm. This
was confirmed by the 1H and 13C NMR spectra (Table 2), which in-
cluded signals of five aromatic protons (dH 7.48–8.10), two acetate
methyl groups (dH 1.80, 2.16) and three carboxyl groups at dC
Compound 4 had the same molecular formula and substitution
patterns as 1712 as shown by their spectroscopic data. Thus, careful
comparison of their 1H NMR data (Table 2) indicated the main dif-
ferences were the chemical shift, multiplicity and coupling con-
stants assigned to the methine protons H-8 (dH 5.34 d, J7,8 3.0 Hz
in 4 instead of dH 5.74 dd, J7,8 2.8 Hz, J8,9 6.3 Hz in 17) and H-9
(dH 5.15 s in 4 instead of dH 5.39 d, J8,9 6.3 Hz in 17). The multiplic-
ity of the H-9 resonance in compound 4 indicated a dihedral angle
very close to 90°, which is only possible for a H-8b, H-9a relative
stereochemistry in the dihydro-b-agarofuran skeleton. This was
confirmed by molecular mechanic analysis13 and the ROE correla-
tion observed between H-8 and Me-13 in a ROESY experiment.
Even so, it should be noted that a complete set of 2D NMR spectra
(COSY, HSQC, HMBC) was acquired in order to gain the complete
and unambiguous assignment of the 1H and 13C NMR resonances
as listed in Table 2. Taking into account biosynthetic consider-
ations, the absolute configuration of compound 4 can be proposed
because the only difference with compound 1712 is the epimeriza-
tion at C-8.
Derivatives 5–9 were obtained, partly to provide support for
their chemical structures, as well as to learn about the bioactivity
changes resulting from altering the parent compound. The natural
sesquiterpene 147 was used as the starting material and was trans-
formed into derivative 5 by stereoselective Sharpless dihydroxyla-
Table 2
1H and 13C NMR (d, CDCl3, J in Hz in parentheses) data of compounds 3 and 4
Position
3
4
tion, using AD-mix-a as the oxidant. Furthermore, the absolute
configurations at C-20 and C30 were assigned as R and S, respec-
tively, by applying the Sharpless mnemonic device for asymmetric
dihydroxylation.14 The basic hydrolysis of 14 with K2CO3 in
MeOH–Me2CO resulted in the formation of polyol 6. Acetylated
derivatives 7–9 were prepared by treatment of 6 with acetic anhy-
dride or acetyl chloride (Scheme 1).
a
a
dH
dC
dH
dC
1
2
3
4
5
6
7
8
5.36 dd (4.5, 12.0)
1.45 m*, 1.97 m
1.64 m, 1.91 m
75.1 d
22.9 t
38.3 t
72.4 s
90.9 s
77.8 d
54.8 d
70.2 d
75.0 d
49.4 s
84.5 s
30.4 q
26.2 q
23.7 q
19.7 q
21.3q*,
170.0 s
21.3q*,
170.4 s
5.35 dd (3.9, 12.1)
1.46 m, 1.90 m
1.73 m, 1.98 m
72.4 d
22.4 t
38.5 t
70.4 s
91.8 s
76.4 d
52.8 d
75.4 d
76.2 d
50.7 s
83.4 s
29.8 q
25.2 q
23.7 q
19.2 q
20.5 q,
169.6 s
5.72 s
2.46 d (3.1)
4.60 m
6.07 s
The structures of these derivatives were established on the
basis of detailed spectroscopic analysis and comparison with those
reported for 14.7 Furthermore, the structure of 6 was confirmed by
a single-crystal X-ray diffraction experiment of a crystal obtained
from CDCl3 (Fig. 3). Subsequent structure elucidation and refine-
ment led to an unambiguous determination of the absolute config-
uration of 6. Its crystallographic data reveal that the torsion angles
obtained correspond to a decalin system with a trans union be-
tween rings A and B, which adopt a chair conformation, whereas
the furan ring presented an envelope conformation as expected.
Thus, the stereostructure of this semisynthetic compound was
established as (1S,4S,5S,6R,7R,8S,9R,10S)-1,4,6,8,9-pentahydroxy-
dihidro-b-agarofuran, which confirms the absolute configuration
2.66 d (3.0)
5.34 d (3.0)
5.15 s
9
5.29 d (6.2)
10
11
12
13
14
15
OAc-1
1.55 s
1.49 s
1.34 s
1.41 s
1.80 s
1.59 s
1.61 s
1.42 s
1.60 s
1.64 s
OAc-6
OAc-8
2.16 s
2.33 s
21.0 q,
169.3 s
a
Data are based on DEPT, HSQC, and HMBC experiments.
Overlapping signals.
of those sesquiterpenes with
a
4b,8b-dihydroxycelorbicol
*
polyhydroxy sesquiterpenoid core15 previously established by CD

D. Torres-Romero et al. / Bioorg. Med. Chem. 19 (2011) 2182–2189
2185
AcO
OBz
AcO
OH
OH
HO
OH
15
OtCin
9
1
OAc
OH
K2CO3
5
3
7
11
AcCl, Et3N
MeOH-Me2CO
13
12
DMAP, CH2Cl2
O
O
O
HO
14
OH
HO
HO
OH
14
6
9
O
AD-Mix-α, CH3SO2NH2
t-BuOH-H2O
Ac2O, Et3N
DMAP, CH 2Cl2
Ac =
Bz =
O
AcO
OAc
OAc
AcO
OH
AcO
OBz
O
ODPP
OAc
OAc
CH=CH
Cin =
+
OH
OH
O
O
O
O
CH-CH
1'
HO
HO
3' 2'
HO
OAc
OH
DPP =
8
5
7
Scheme 1. Preparation of derivatives 5–9 from the natural sesquiterpene 14.
MIC values as quickly and accurately as the more expensive MABA
procedure.19 The clinically used antituberculosis agents, isoniazid
and rifampicin, were used as positive controls. The antituberculosis
activity data are shown in Table 3. All the assayed sesquiterpenes
were inactive against the MTB H37Rv drug-sensitive strain with
MIC values over 25
the compounds showed moderate activity against the MDR TB
strain, with MIC values of 12.5 g/mL. Moreover, compound 20
showed the more potent antimycobacterial activity against the
resistant strain with a MIC value of 6.2 g/mL (11.9 M) compara-
ble or better than the positive controls used, isoniazid (MIC 4 g/
mL, 29.1 M) and rifampicin (MIC >16.0 g/mL, >19.4 M).
lg/mL (data not shown). Surprisingly, six of
l
l
l
l
l
l
l
In general, we observed some trends on the preliminary struc-
ture–activity relationship of this series of sesquiterpenes that seem
to be important for high anti-MDR-TB activity in this class of com-
pounds: (a) the overall esterification level of the compound; (b) the
presence of two aromatic ester moieties; (c) the regiosubstitution of
Table 3
Anti-Mycobacterium tuberculosis activitya of compounds 1–25b
Compound
MDR-TB
Compound
MDR-TB
MIC
M)
Figure 3. A view of the molecular structure of 6. The ellipsoids are drawn at the 30%
probability level.
MICc
g/mL)
MIC
M)
MICc
g/mL)
(l
(
l
(
l
(l
1
2
3
4
5
6
7
8
25
43.1
>60
51.0
21.0
>60
>60
>60
>60
>60
21.6
22.2
44.4
20.1
21.6
15
16
17
18
19
20
21
22
>25
>25
>25
>25
>25
6.2
12.5
25
>25
>25
>25
4
>60
>60
>60
>60
>60
11.9
25.8
56.6
>60
>60
>60
29.1
>19.4
studies.7 This result has a special significance, as to date, there are
few examples of the absolute configuration of related sesquiter-
penes determined by X-ray, the most recent being those given
for boariol16 and a 3,12-oxygenated dihydro-b-agarofuran.17 How-
ever, this is the first X-ray report of a 4b,8b-dihydroxy-celorbicol-
related sesquiterpenoid.
So far, few reports concerning the biological evaluation of dihy-
dro-b-agarofuran sesquiterpenes on M. tuberculosis drug-sensitive
strains have been published.9–11 Moreover, there are not any re-
ports on the activity of this type of compounds against drug-resis-
tant M. tuberculosis strains.
The biological activity in vitro of the isolated compounds (1–4)
and their derivatives (5–9), along with the previously isolated ses-
quiterpenes 10–257,12 (Fig. 1 and Scheme 1), was determined by
their ability to inhibit the growth of M. tuberculosis (MTB) drug-
sensitive and multidrug-resistant (MDR TB) strains using the tetra-
zolium microplate assay (TEMA) method.18 This assay determines
>25
>25
12.5
>25
>25
>25
>25
>25
12.5
12.5
25
9
23
24
25
10
11
12
13
14
isoniazidd
rifampicind
12.5
12.5
>16
a
Data were means of 3 or 4 replicates.
Compounds 1–25 showed a MIC value >25 lg/mL against the sensitive H37Rv
strain.
MIC values is the minimum inhibitory concentration of the tested compounds
effecting 100% of inhibition.
Isoniazid and rifampicin were used as positive controls, with MICs values
against the H37Rv strain of 0.125 and 0.063 lg/mL, respectively.
b
c
d

2186
D. Torres-Romero et al. / Bioorg. Med. Chem. 19 (2011) 2182–2189
the molecule. Thus, compounds which contain an ester group on C-
6 were more potent than those with an hydroxyl group (21 vs 22).
Moreover, the presence of a benzoyl group instead of a nicotynoyl
group at position C-6 significantly improve the activity, as can be
seen in compounds 4 versus 19 or 10 versus 1, while the absence
of functionalization at C-6 causes a loss of activity (21 vs 23). In
addition, the presence of a hydroxyl group at C-2 produces a de-
crease in activity, as seen from the lower activity of compound 25
compared to 14. The substituent at C-8 also proved to be important
for the activity. Thus, compounds with a trans-cinnamate ester
group (13 and 14) are more active than those with a dihydroxy-
phenyl-propanoate (5), a cis-cinnamate (16) or a hydroxyl group (3).
We can conclude that compound 20, with the basic polyhy-
droxy skeleton of celorbicol,15 is structurally quite different from
known antitubercular agents, and its activity on the MDR-TB strain
falls into the range of the clinically used anti-TB drugs, isoniazid
and rifampicin. Thus, our results warrant further investigation to
improve the observed activity and elucidate the mechanism of
the anti-MDR-TB potential of this class of compounds.
3.2.1. (1S,4R,5S,6R,7R,8S,9R,10S)-1,8-Diacetoxy-9-benzoyloxy-6-
nicotinoyloxy-dihydro-b-agarofuran (1)
Colorless lacquer; ½a D25
ꢀ10.8 (c 0.1, CHCl3); UV kmax (EtOH)
ꢁ
(log e) 264 (1.4), 225 (5.1) nm; IR mmax (film) 2923, 2853, 1729,
1456, 1368, 1275, 1222, 1106, 1025, 772, 670 cmꢀ1 1H NMR
;
(CDCl3) d OBz, ONic [7.50 (3H, m), 7.60 (1H, m), 8.11 (2H, m),
8.33 (1H, m), 8.85 (1H, m), 9.26 (1H, s)], for other signals, see Table
1; 13C NMR (CDCl3) d OBz, ONic [123.4 (d) 125.4 (s), 128.1 (2ꢂ d),
129.1 (s), 130.0 (2ꢂ d), 133.1 (d), 136.9 (d), 150.6 (d), 153.8 (d)],
163.9 (s, ONic-6), 165.4 (s, OBz-9), for other signals, see Table 1;
EI-MS m/z (%) 579 [M]+ (15), 564 (13), 519 (5), 458 (14), 456 (2),
400 (7), 339 (3), 245 (13), 106 (38), 105 (100), 77 (14); EI-HRMS
m/z 579.2457 (calcd for C32H37NO9, 579.2468).
3.2.2. (1S,4R,5S,6R,7S,8S,9R,10S)-1,8-Diacetoxy-9-benzoyloxy-6-
hydroxy-dihydro-b-agarofuran (2)
Colorless lacquer; ½a D25
ꢀ21.4 (c 0.2, CHCl3); UV kmax (EtOH)
ꢁ
(log e) 274 (1.0), 230 (9.8) nm; IR mmax (film) 3504, 2923, 2852,
1721, 1456, 1367, 1279, 1232, 1104, 1028, 960, 712, 670 cmꢀ1
;
1H NMR (CDCl3) d OBz [7.45 (2H, m), 7.56 (1H, m), 8.08 (2H, m)],
for other signals, see Table 1; 13C NMR (CDCl3) d OBz-9 [128.0
(2ꢂ d), 129.2 (s), 130.0 (2ꢂ d), 133.0 (d), 165.6 (s)], for other sig-
nals, see Table 1; EI-MS m/z (%) 474 [M]+ (3), 459 (16), 414 (6),
399 (8), 353 (4), 339 (8), 251 (12), 149 (13), 105 (100), 83 (30),
77 (18); EI-HRMS m/z 474.2261 (calcd for C26H34O8, 474.2254).
3. Experimental
3.1. General procedures
Optical rotations were measured on a Perkin–Elmer 241 auto-
matic polarimeter in CHCl3 at 25 °C and the [a] values are given
D
3.2.3. (1S,4S,5S,6R,7R,8S,9R,10S)-1,6-Diacetoxy-9-benzoyloxy-
4,8-dihydroxy-dihydro-b-agarofuran (3)
in 10ꢀ1 deg cm2 gꢀ1. UV spectra were obtained in absolute EtOH
on a JASCO V-560 instrument. IR (film) spectra were measured in
CHCl3 on a Bruker IFS 55 spectrophotometer. 1H (400 MHz) and
13C (100 MHz) NMR spectra were recorded on a Bruker Avance
400 spectrometer; chemical shifts were referred to the residual sol-
vent signal (CDCl3: dH 7.26, dC 77.0); DEPT, COSY, TOCSY (spin lock
field 8000 Hz), ROESY (spin lock field 2500 Hz), HSQC and HMBC
(optimized for J = 7.7 Hz) experiments were carried out with the
pulse sequences given by Bruker. EI-MS and EI-HRMS were re-
corded on a Micromass Autospec spectrometer. Silica gel 60 (15–
Colorless lacquer; ½a D25
ꢁ
ꢀ7.5 (c 0.1, CHCl3); UV kmax (log
e)
(EtOH) 274 (0.6), 224 (3.8) nm; IR mmax (film) 3466, 2928, 1731,
1369, 1276, 1234, 1101, 1029, 966, 756, 716 cmꢀ1; 1H NMR (CDCl3)
d 2.88 (1H, s, OH-4), 4.35 (1H, s, OH-8), OBz [7.48 (2H, m), 7.60 (1H,
m), 8.10 (2H, m)], for other signals, see Table 2; 13C NMR (CDCl3) d
OBz-9 [128.1 (2ꢂ d), 130.0 (s), 130.6 (2ꢂ d), 133.4 (d), 169.5 (s)],
for other signals, see Table 2; EI-MS m/z (%) 475 [M+ꢀ15] (6),
457 (3), 430 (3), 353 (17), 293 (4), 251 (4), 233 (5), 202 (10), 166
(8), 105 (100), 77 (15), EI-HRMS m/z 475.1958 [MꢀCH3]+ (calcd
for C25H31O9, 475.1968).
40 lm) for column chromatography, silica gel 60 F254 for TLC, and
nanosilica gel 60 F254 for preparative HPTLC were purchased from
Macherey-Nagel, and Sephadex LH-20 was obtained from Pharma-
cia Biotech. The spots were visualized by UV light and heating silica
gel plates sprayed with H2O/H2SO4/AcH (1:4:20). All solvents used
were analytical grade from Panreac. Reagents were purchased from
Sigma-Aldrich and used without further purification.
3.2.4. (1S,4S,5S,6R,7R,8S,9R,10S)-1,8-Diacetoxy-9,6-
dibenzoyloxy-4-hydroxy-dihydro-b-agarofuran (4)
Colorless lacquer; ½a D25
ꢀ4.9 (c 0.4, CHCl3); UV kmax (EtOH)
ꢁ
(log e) 230 (13.9) nm; IR mmax (film) 3562, 2921, 2852, 1741,
1721, 1271, 1232, 1095, 1028, 713 cmꢀ1 1H NMR (CDCl3) d 3.08
;
(1H, s, OH-4), OBz [7.49 (4H, m), 7.60 (2H, m), 8.08 (2H, m), 2.22
(2H, m)], for other signals, see Table 2; 13C NMR (CDCl3) d OBz
[128.2 (2ꢂ d), 128.4 (2ꢂ d), 128.6 (s), 129.8 (s), 129.9 (4ꢂ d),
133.0 (d), 133.5 (d)], 164.6 (s, OBz-9), 165.6 (s, OBz-6), for other
signals, see Table 2; EI-MS m/z (%) 594 [M]+ (9), 579 (1), 534 (5),
472 (3), 457 (2), 384 (1), 270 (5), 167 (1), 149 (4), 105 (100), 77
(11); EI-HRMS m/z 594.2437 (calcd for C33H38O10, 594.2465).
3.2. Plant material
C. vulcanicola J. Donnell Smith, collected in June 2004 at the
Montecristo National Park, province of Santa Ana, El Salvador,
was identified by Jorge Monterrosa, and a voucher specimen
(J. Monterrosa & R. Carballo 412) is deposited in the Herbarium
of Missouri Botanical Garden, USA. The dried leaves (1.5 kg) of C.
vulcanicola were sliced into chips, extracted with EtOH in a Soxhlet
apparatus, and concentrated under reduced pressure. The EtOH ex-
tract (311.0 g) was partitioned into CH2Cl2/H2O (1:1, v/v) solution.
Removal of the CH2Cl2 from the organic fraction under reduced
pressure yielded 74.8 g of residue, which was chromatographed
by vacuum liquid chromatography (VLC) on a silica gel column
chromatography using mixtures of n-hexane–EtOAc of increasing
polarity as eluent to afford seven fractions (A–G). Fraction D was
subjected to column chromatography over Sephadex LH-20 using
mixtures of CH2Cl2/MeOH (1:1) to provide two subfractions (D1
and D2). Fraction D2 was further purified by silica gel column chro-
matography, and preparative TLC developed with CH2Cl2/Me2CO
(9:1) to yield the new compounds 1 (8.8 mg), 2 (21.1 mg), 3
(12.1 mg) and 4 (6.0 mg).
3.3. Chemical correlations
A mixture of nicotinoyl chloride hydrochloride (50 mg, 0.28
mmol), triethylamine (6 drops), compound 2 (3.5 mg, 0.07 mmol)
and a catalytic amount of 4-dimethylamino-pyridine in dry dichlo-
romethane (2 mL) was refluxed for 16 h. The mixture was evapo-
rated to dryness, and the residue was purified by preparative TLC
using n-hexane/EtOAc (1:1) to give compound 1 (2.0 mg, 47%).
Moreover, compound 3 (2.9 mg, 0.06 mmol) was dissolved in dry
pyridine (0.5 mL), and trans-cinnamoyl chloride (35 mg, 0.23
mmol) and some crystals of 4-dimethylamino-pyridine were
added under argon atmosphere. The mixture was heated to 60 °C
for 15 h, poured over H2O and extracted with EtOAc. The organic
phases were combined, dried over anhydrous MgSO4 and

D. Torres-Romero et al. / Bioorg. Med. Chem. 19 (2011) 2182–2189
2187
evaporated under reduced pressure. The residue was purified on
preparative TLC with a mixture of n-hexane/EtOAc (1:1) to give
compound 137 (1.8 mg, 48%).
(d, C-8), 73.0 (s, C-4), 74.9 (d, C-9), 78.1 (d, C-6), 83.9 (s, C-11),
91.4 (s, C-5); EI-MS m/z (%) 287 [M+ꢀ15] (100), 233 (11), 205
(10), 191 (10), 166 (32), 151 (21), 137 (12), 125 (19), 109 (27),
98 (39), 85 (38); EI-HRMS m/z 287.1505 [MꢀCH3]+ (calcd for
3.4. Preparation of 5
C14H23O6, 287.1495).
AD-mix-
a
(51 mg) and methanesulfonamide (6.3 mg, 0.07
3.6. X-ray crystal structure analysis
mmol) were dissolved in a solvent mixture of t-BuOH (1.5 mL)
and H2O (1.5 mL). The resulting mixture was stirred at room tem-
perature for 5 min, then it was cooled to 0 °C and a solution of
compound 147 (20 mg, 0.04 mmol) in dichloromethane was added.
Subsequently, a 10% Na2S2O3 solution was added to quench the
reaction, and the mixture was extracted with EtOAc. The organic
phases were combined, dried (MgSO4) and evaporated. The residue
was purified by preparative TLC using n-hexane/EtOAc (1:1) to af-
ford 5 (6.3 mg, 25%).
Compound 6 crystallizes as a hemihydrate with the oxygen of
the water molecule lying on a binary crystallographic axis of sym-
metry. Intensity data were collected at room temperature on an
Enraf-Nonius
jCCD diffractometer with Mo Ka radiation (k =
0.71707 Å). Cell refinement and data reduction were performed
20
21
with COLLECT and DENZO
.
The structure was determined by direct
methods using SIR97.22 Refinements were performed with SHELXL
-
97,23 using full-matrix least squares with anisotropic displacement
parameters for all the non-hydrogen atoms.24 All the H-atoms
were located in successive difference-Fourier synthesis and iso-
tropically refined, with the exception of those corresponding to
O4, O5 and the oxygen of the water co-crystallization molecule,
3.4.1. (1S,4S,5S,6R,7S,8S,9R,10S)-1-Acetoxy-9-benzoyloxy-8-
[2R,3S-dihydroxy-3-phenyl]-propanoil-oxy-4,6-dihydroxy-
dihydro-b-agarofuran (5)
Colorless lacquer; ½a D25
ꢁ
ꢀ8.8 (c 0.5, CHCl3); UV kmax (log
e
)
which were added as a fixed isotropic contribution. Molecular
25
(EtOH) 230 (2.0) nm; IR mmax (film) 3351, 2926, 1734, 1453,
graphics were computed with PLATON.
1369, 1320, 1280, 1152, 1103, 1037, 984, 772, 717 cmꢀ1
;
1H
X-ray crystal data: C15H26O6. 0.5 (H2O), Mw = 311.4, ortho-
NMR (CDCl3) d 1.42 (3H, s, Me-15), 1.48 (1H, m, H-2b), 1.55 (6H,
s, Me-12, Me-13), 1.62 (3H, s, Me-14), 1.65 (3H, s, OAc-1), 1.74
rhombic, space group P21212, Z = 4, a = 10.748(2), b = 17.444(5),
c = 7.787(7) Å, V = 1460.0(14) Å3, ) = 0.09 mmꢀ1
l (Mo Ka , qcalcd =
(1H, m, H-3
a
), 1.95 (1H, m, H-3b), 2.02 (1H, m, H-2
a
), 2.40 (1H,
1.42 g cmꢀ3; S = 1.07, final R indices: R1 = 0.034 and Rw = 0.089
d, J = 3.0 Hz, H-7), 2.84 (1H, s, OH-30), 3.05 (1H, s, OH-20), 3.18
(1H, s, OH-4), 4.09 (1H, m, H-20), 4.49 (1H, d, J = 5.2 Hz, H-6),
4.81 (1H, m, H-30), 5.13 (1H, d, J = 5.2 Hz, OH-6), 5.33 (1H, d,
J = 6.4 Hz, H-9), 5.38 (1H, dd, J = 4.1, 11.9 Hz, H-1), 5.51 (1H, dd,
J = 3.0, 6.4 Hz, H-8), OBz, ODPP [7.15 (2H, dd, J = 1.7, 6.5 Hz), 7.28
(2H, m), 7.51 (3H, m), 7.65 (1H, m), 8.08 (2H, m)]; 13C NMR (CDCl3)
d 19.5 (q, C-15), 20.4 (q, OAc-1), 22.8 (t, C-2), 23.5 (q, C-14), 26.5 (q,
C-13), 30.2 (q, C-12), 36.8 (t, C-3), 48.6 (s, C-10), 54.5 (d, C-7), 70.6
(d, C-8), 71.8 (d, C-1), 72.1 (d, C-9), 72.8 (s, C-4), 73.9 (d, C-30), 74.9
(d, C-20), 77.9 (d, C-6), 84.4 (s, C-11), 90.7 (s, C-5), ODPP, OBz [125.9
(2ꢂ d), 127.8 (d), 128.2 (2ꢂ d), 128.3 (2ꢂ d), 128.4 (s), 130.0 (2ꢂ d),
133.7 (d), 139.3 (s)], 166.1 (s, OBz-9), 169.6 (s, OAc-1), 171.2 (s, C-
10); EI-MS m/z (%) 597 [M+ꢀ15] (1), 579 (3), 506 (8), 475 (5), 446
(15), 249 (4), 233 (3), 202 (9), 191 (6), 105 (100), 83 (10), 77
for 1668 observed from 1781 independent and 8031 measured
reflections (hmax = 27.0, I > 2
r (I) criterion and 291 parameters);
maximum and minimum residues are 0.28 and ꢀ0.17 e Åꢀ3
,
respectively.
Crystallographic data (excluding structure factor tables) for the
structure reported has been deposited with the Cambridge Crystal-
lographic Data Centre as supplementary publications no. 778506.
Copies of the data can be obtained free of charge on application
to the Director, CCDC, 12 Union Road, Cambridge CB 1EZ, UK
(fax: Int. +44 1223 336 033); e-mail: deposit@ccdc.cam.ac.uk.
3.7. Acetylation of 6
A mixture of acetic anhydride (55 mg, 0.79 mmol), triethyl-
amine (0.3 mL), compound 6 (64.5 mg, 0.21 mmol), and 4-dimeth-
ylamino-pyridine (12.0 mg) in dichloromethane (12.0 mL) was
stirred at rt for 16 h. The mixture was evaporated to dryness, and
the residue was purified by preparative TLC using CH2Cl2-Me2CO
(9:1) to give compounds 7 (21.3 mg, 24%) and 8 (15.6 mg, 19%).
Furthermore, a mixture of acetyl chloride (0.13 mL, 1.92 mmol),
triethylamine (0.13 mL, 0.96 mmol), compound 6 (71.0 mg, 0.24
mmol), and 4-dimethylamino-pyridine (12 mg) in dichlorometh-
ane (5.0 mL) was stirred at rt for 24 h. Then an aqueous solution
of HCl 0.1 N (2 mL) was added, and the residue was extracted three
times with EtOAc (2 mL). The collected organic layers were dried
over anhydrous MgSO4 and evaporated under reduced pressure
giving a thick oil, which was purified by preparative TLC using n-
hexane/diethylether (1:3) to yield 9 (46.2 mg, 50%).
(15); EI-HRMS m/z 597.2330 [MꢀCH3]+ (calcd for C32H37O11
,
597.2336).
3.5. Preparation of 6
A mixture of 14 (205 mg, 0.4 mmol) and K2CO3 (300 mg) in a
solvent mixture of MeOH (20 mL) and acetone (12 mL) was re-
fluxed for 33 h, until TLC showed complete conversion. The mix-
ture was filtered and the solution was concentrated under
reduced pressure. The residue was further purified by flash column
chromatography on silica gel (2 ꢂ 30 cm, 10 g) using n-hexane/
EtOAc (1:4) to give a crystalline compound, 6 (71.1 mg, 59%).
3.5.1. (1S,4S,5S,6R,7R,8S,9R,10S)-1,4,6,8,9-Pentahydroxy-
dihydro-b-agarofuran (6)
Colorless orthorhombic crystals; ½a D25
ꢁ
ꢀ20.1 (c 0.4, CHCl3);
3.7.1. (1S,4S,5S,6R,7R,8S,9R,10S)-1,6,8,9-Tetra-acetoxy-4-
hydroxy-dihydro-b-agarofuran (7)
recrystallization solvent: CDCl3; mp 180.8–181.2 °C; IR mmax (film)
3399, 2924, 2853, 1458, 1389, 1216, 1140, 1039 cmꢀ1
;
1H NMR
Colorless lacquer; ½a D25
ꢁ
ꢀ32.4 (c 0.15, CHCl3); IR mmax (film)
(CDCl3) d 1.13 (3H, s, Me-15), 1.54 (3H, s, Me-14), 1.56 (3H, s,
Me-12), 1.62 (2H, m, H-2), 1.70 (2H, m, H-3), 1.73 (3H, s, Me-13),
2.48 (1H, d, J = 3.2 Hz, H-7), 3.07 (1H, d, J = 5.4 Hz, OH-9), 3.18
(1H, s, OH-4), 3.71 (1H, t, J = 5.4 Hz, H-9), 4.09 (1H, dd, J = 3.2,
5.4 Hz, H-8), 4.22 (1H, d, J = 5.2 Hz, H-6), 4.37 (1H, dd, J = 4.0,
12.0 Hz, H-1), 5.00 (1H, d, J = 5.2 Hz, OH-6); 13C NMR (CDCl3) d
18.2 (c, C-15), 23.6 (c, C-14), 26.2 (t, C-2), 27.0 (c, C-13), 30.5 (c,
C-12), 37.5 (t, C-3), 49.0 (s, C-10), 56.7 (d, C-7), 68.9 (d, C-1), 70.4
3554, 2928, 1739, 1368, 1234, 1175, 1054, 968, 875 cmꢀ1 1H
;
NMR (CDCl3) d 1.34 (3H, s, Me-14), 1.38 (3H, s, Me-15), 1.43 (1H,
m, H-2 ), 1.55 (3H, s, Me-12), 1.65 (1H, m, H-3 ), 1.66 (3H, s,
a
a
Me-13), 1.82 (1H, m, H-3b), 1.84 (1H, m, H-2b), 1.95 (3H, s, OAc-
1), 2.00 (3H, s, OAc-8), 2.07 (3H, s, OAc-9), 2.14 (3H, s, OAc-6),
2.41 (1H, d, J = 3.0 Hz, H-7), 2.80 (1H, s, OH-4), 5.05 (1H, d,
J = 6.1 Hz, H-9), 5.29 (1H, dd, J = 4.0, 12.1 Hz, H-1), 5.47 (1H, dd,
J = 3.0, 6.1 Hz, H-8), 5.48 (1H, s, H-6); 13C NMR (CDCl3) d 19.0 (q,

2188
D. Torres-Romero et al. / Bioorg. Med. Chem. 19 (2011) 2182–2189
C-15), 20.5 (q, OAc-8), 20.8 (q, OAc-9), 20.9 (q, OAc-1), 21.2 (q,
OAc-6), 23.4 (q, C-14), 23.5 (t, C-2), 25.6 (q, C-13), 30.1 (q, C-12),
38.1 (t, C-3), 49.1 (s, C-10), 53.4 (d, C-7), 68.8 (d, C-8), 70.1 (s, C-
4), 71.3 (2ꢂ d, C-1, C-9), 77.1 (d, C-6), 84.2 (s, C-11), 91.0 (s, C-5),
168.9 (s, OAc-8), 169.8 (s, OAc-6), 169.9 (s, OAc-9), 170.2 (s, OAc-
1); EI-MS m/z (%) 470 [M]+ (1), 455 (36), 428 (11), 410 (39), 395
(35), 368 (34), 353 (20), 308 (22), 248 (41), 208 (54), 191 (50),
166 (56), 140 (89), 109 (43), 98 (75), 83 (100); EI-HRMS m/z
470.2176 (calcd for C23H34O10, 470.2152).
solutions were further diluted with appropriate volumes of
7H9GC broth to yield final concentrations of 0.1–50 g/mL. Final
drug concentration ranges of standard antibiotics used as positive
controls were 0.125–32 g/mL for isoniazid and 0.063–16 g/mL
for rifampin (Sigma Chemical Co., St. Louis, MO). The standard
drugs or compounds (100 L) were mixed in the wells with
100
l
l
l
l
l
L of bacterial inoculum, resulting in a final bacterial concen-
tration of ca. 1.2 ꢂ 106 CFU/mL. Solvent (DMSO) was included in
every experiment as a negative control. The plates were sealed in
plastic bags and then incubated at 37 °C for 5 days. On day 5,
3.7.2. (1S,4S,5S,6R,7R,8S,9R,10S)-1,6,8-Triacetoxy-4,9-dihydroxy-
dihydro-b-agarofuran (8)
50 lL of the MTT/Tween 80 mixture (1.5 mL of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Al-
drich Chemical Co., Milwaukee, WI) at a dilution of 1 mg/mL in
absolute EtOH and 1.5 mL of 10% Tween 80) was added to the
wells, and the plate was incubated at 37 °C for 24 h. After this incu-
bation period, the growth of the microorganism was visualized by
the change in colour of the dye from yellow to purple. The tests
were carried out in triplicate. MIC value is defined as the lowest
drug concentration that prevents the aforementioned change in
colour.
Colorless lacquer; ½a D25
ꢁ
ꢀ58.0 (c 0.8, CHCl3); IR
1H NMR (CDCl3) d 1.34
),
), 1.88
mmax (film) 3535,
2940, 1737, 1368, 1235, 1046, 966 cmꢀ1
;
(6H, s, Me-14, Me-15), 1.56 (3H, s, Me-12), 1.68 (1H, m, H-3
1.74 (3H, s, Me-13), 1.78 (1H, m, H-3b), 1.81 (1H, m, H-2
a
a
(1H, m, H-2b), 2.04 (3H, s, OAc-1), 2.12 (3H, s, OAc-8), 2.13 (3H,
s, OAc-6), 2.49 (1H, d, J = 3.0 Hz, H-7), 2.55 (1H, d, J = 10.3 Hz,
OH-9), 2.63 (1H, s, OH-4), 3.76 (1H, dd, J = 5.4, 10.3 Hz, H-9), 5.23
(1H, dd, J = 3.0, 5.4 Hz, H-8), 5.31 (1H, dd, J = 4.1, 11.9 Hz, H-1),
5.46 (1H, s, H-6); 13C NMR (CDCl3) d 18.8 (q, C-15), 20.8 (q, OAc-
1), 21.1 (q, OAc-6), 21.2 (q, OAc-8), 23.4 (q, C-14), 23.5 (t, C-2),
26.2 (q, C-13), 29.9 (q, C-12), 38.3 (t, C-3), 49.1 (s, C-10), 53.6 (d,
C-7), 70.1 (d, C-4), 71.3 (d, C-1), 71.7 (d, C-8), 72.8 (d, C-9), 77.3
(d, C-6), 85.2 (s, C-11), 92.8 (s, C-5), 169.6 (s, OAc-8), 169.8 (s,
OAc-6), 170.0 (s, OAc-1); EI-MS m/z (%) 413 [M+ꢀ15] (20), 386
(12), 368 (17), 353 (31), 308 (35), 207 (42), 166 (70), 149 (45),
109 (78), 83 (100); EI-HRMS m/z 413.1840 (calcd for C20H29O9,
413.1812).
Acknowledgments
This work was supported by the Agencia Canaria de Investiga-
ción, Innovación y Sociedad de la Información (C200801000049),
and PCI-Iberoamérica (A/023081/09, AECID) projects, and the anti-
mycobacterial study was financed by project SIDISI50989. D.T.R.
thanks the Gobierno Autónomo de Canarias for the ‘‘Antonio
González’’ fellowship and the Servicio de Parques Nacionales y
Vida Silvestre, Dirección de Recursos Renovables del Ministerio
de Agricultura y Ganadería (MAG) and Fundación Ecológica de El
Salvador (SALVANATURA) for supplying the plant material.
3.7.3. (1S,4S,5S,6R,7S,8S,9R,10S)-1,8,-Diacetoxy-4,6,9-trihydroxy-
dihydro-b-agarofuran (9)
Colorless lacquer; ½a D25
ꢁ
ꢀ35.0 (c 0.2, CHCl3); IR
m
max (film) 3434,
2934, 1728, 1438, 1368, 1244, 1106, 1037, 965 cmꢀ1
;
1H NMR
Supplementary data
(CDCl3) d 1.24 (3H, s, Me-15), 1.55 (6H, s, Me-13, Me-14), 1.52
(1H, m, H-2 ), 1.63 (1H, m, H-3 ), 1.73 (3H, s, Me-12), 1.77 (1H,
a
a
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.034.
m, H-3b), 1.88 (1H, m, H-2b), 2.02 (3H, s, OAc-1), 2.10 (3H, s,
OAc-8), 2.40 (1H, d, J = 7.4 Hz, OH-9), 2.49 (1H, d, J = 2.9 Hz, H-7),
3.10 (1H, s, OH-4), 3.69 (1H, dd, J = 5.6, 7.4 Hz, H-9), 4.39 (1H, d,
J = 5.4 Hz, H-6), 4.98 (1H, d, J = 5.4, OH-6), 5.03 (1H, dd, J = 2.9,
5.6 Hz, H-8), 5.34 (1H, dd, J = 4.4, 12.0 Hz, H-1); 13C NMR (CDCl3)
d 19.0 (q, C-15), 20.9 (q, OAc-8), 21.1 (q, OAc-1), 23.3 (q, C-14),
23.4 (t, C-2), 27.1 (q, C-13), 30.5 (q, C-12), 36.7 (t, C-3), 48.2 (s,
C-10), 54.7 (d, C-7), 71.2 (d, C-1), 72.1 (d, C-8), 72.7 (s, C-4), 73.0
(d, C-9), 77.9 (d, C-6), 85.0 (s, C-11), 92.1 (s, C-5), 169.7 (s, OAc-
8), 170.0 (s, OAc-1); EI-MS m/z (%) 371 [M+ꢀ15] (40), 353 (6),
311 (23), 308 (31), 293 (24), 233 (20), 207 (22), 191 (23), 156
(27), 109 (100), 98 (94), 83 (76); EI-HRMS m/z 371.1710 (calcd
for C18H27O8, 371.1706).
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