Tropane alkaloids from erythroxylum caatingae plowman

Article abstract of DOI:10.1002/cbdv.200900400

Three tropane alkaloids, 1-3, were isolated from Erythroxylum caatingae, i.e., 6β-benzoyloxy-3α-[(4-hydroxy-3,5-dimethoxybenzoyl)oxy]tropane (1), a new tropane alkaloid, along with the known alkaloids 3α,6β- dibenzoyloxytropane (2) and 6β-benzoyloxy-3α-[(3,4,5- trimethoxybenzoyl)oxy]tropane (catuabine B; 3). Their structures were determined by 2D- (¹H and ¹³C) NMR. By LC/ESI-MS/MS analysis of the fractions of alkaloids 1-3, it was possible to detect five more alkaloids, 4-8, two of these, 4 and 8, possibly being new natural products. X-Ray crystallography of the chloride derivate of 1, i.e., 6β-benzoyloxy- 3α-(4-hydroxy-3,5-dimethoxybenzoyloxy)tropane hydrochloride (1a) confirmed the structure of 1. Cytotoxicity was tested against the cell lines HEp-2, NCI-H292, and KB for the MeOH extract and alkaloid 3, and antitumor activity was tested against Sarcoma 180 only for the MeOH extract. Copyright

Full text of DOI:10.1002/cbdv.200900400

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
155
Tropane Alkaloids from Erythroxylum caatingae Plowman
by Steno L. de Oliveiraa), Josean F. Tavaresa), Marianna V. S. Castello Brancoa),
Hellane F. S. Lucenaa), Jose´ M. Barbosa-Filhoa), Maria de F. Agraa), Silene C. do Nascimentob),
Jaciana dos S. Aguiarb), Teresinha G. da Silvab), Carlos A. de Simonec), Joa˜o X. de Araujo-Juniorc)d),
´
´
and Marcelo S. da Silva*a)
a
ˆ
ˆ
´
ˆ
) Departamento de Ciencias Farmaceuticas, Laboratorio de Tecnologia Farmaceutica, Universidade
˜
Federal da Paraꢀba, C. P. 5009, 58051-970, Joao Pessoa, Paraꢀba, Brazil
(phone: þ558332167427; fax: þ558332167365; marcelosobral@ltf.ufpb.br)
b
c
´
) Departamento de Antibioticos, Universidade Federal de Pernambuco, 50670-901, Recife, PE, Brazil
´
) Instituto de Quꢀmica e Biotecnologia, Universidade Federal de Alagoas, 57072-970, Maceio, AL, Brazil
d
´
) ESENFAR, Universidade Federal de Alagoas, 57072-970, Maceio, AL, Brazil
Three tropane alkaloids, 1–3, were isolated from Erythroxylum caatingae, i.e., 6b-benzoyloxy-3a-
[(4-hydroxy-3,5-dimethoxybenzoyl)oxy]tropane (1), a new tropane alkaloid, along with the known
alkaloids 3a,6b-dibenzoyloxytropane (2) and 6b-benzoyloxy-3a-[(3,4,5-trimethoxybenzoyl)oxy]tropane
(catuabine B; 3). Their structures were determined by 2D- (1H and 13C) NMR. By LC/ESI-MS/MS
analysis of the fractions of alkaloids 1–3, it was possible to detect five more alkaloids, 4–8, two of these,
4 and 8, possibly being new natural products. X-Ray crystallography of the chloride derivate of 1, i.e., 6b-
benzoyloxy-3a-(4-hydroxy-3,5-dimethoxybenzoyloxy)tropane hydrochloride (1a) confirmed the struc-
ture of 1. Cytotoxicity was tested against the cell lines HEp-2, NCI-H292, and KB for the MeOH extract
and alkaloid 3, and antitumor activity was tested against Sarcoma 180 only for the MeOH extract.
Introduction. – The family Erythroxylaceae comprises four genera and ca. 240
species with pantropical distribution, with its main centers of diversity and endemism in
Venezuela, Brazil, and Madagascar. The Erythroxylaceae consist of the genera
Aneulophus, Erythroxylum, Nectaropetalum, and Pinacopodium, where Erythroxylum
is the largest and most representative genus, with ca. 230 species and wide distribution
in the tropical regions of the world, and South America is the center of diversity and
endemism. Erythroxylum caatingae is a species with distribution restricted to the
Northeast Brazil, being only recorded for the states of Bahia, Ceara, Paraꢀba,
Pernambuco, and Rio Grande do Norte [1].
Tropane alkaloids occur frequently in the families Convolvulaceae, Erythroxyla-
ceae, Proteaceae, Rhizophoraceae, and Solanaceae and have occasionally been
reported in plants of the families Brassicaceae, Euphorbiaceae, and Oleaceae [2].
Recently, 17 tropane alkaloids were reported from E. vacciniifolium, collected in
Paraꢀba (Brazil) [3][4]. Studies with the CHCl3 extract of E. pervillei and E.
rotundifolium revealed cytotoxicity against the multidrug-resistant (MDR) cell line
KB-V1 in the presence of vinblastine [2–5]. Recently, we described a new trachylobane
diterpene and its cytotoxicity against V79 cells and rat hepatocytes [6]. As part of a
continuing investigation of new bioactive molecules from plants of Paraꢀba (Brazil), we
describe here the isolation and structural identification of three tropane alkaloids,
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
among which 6b-benzoyloxy-3a-(4-hydroxy-3,5-dimethoxybenzoyloxy)tropane (1) is a
new tropane alkaloid, whereas 3a,6b-dibenzoyloxytropane (2) and 6b-benzoyloxy-3a-
(3,4,5-trimethoxybenzoyloxy)tropane (catuabine B; 3) are known compounds. The
hydrochloride of 1, i.e., 1a, was obtained by acidification with HCl. Furthermore, it was
possible to detect, in the fractions of alkaloids 1–3, five additional alkaloids, 4–8,
partially characterized by LC/ESI-MS/MS. Cytotoxicity of the MeOH extract and
alkaloid 3 against HEp-2, NCI-H292, and KB cells, and in vivo antitumor activity of the
MeOH extract of E. caatingae in the presence of methotrexate were determined.
Results and Discussion. – Compound 1 was isolated as an amorphous solid, with a
melting point of 220–2238. HR-MS exhibited a molecular ion at m/z 442.1848 ([Mþ
H]þ ; calc. 442.1866), compatible with the molecular formula C24H27NO7. The IR
spectrum showed absorptions at 3425 (OH), and 1724 and 1277 cmꢀ1 characteristic of
an ester group. The 13C-NMR spectrum of 1 showed signals at d(C) 60.1 (C(1)), 65.7
(C(5)), and 40.1 (MeN), characteristics of a tropane skeleton. In addition, the signals at
d(C) 67.3 and 79.7 are compatible with the tropane skeleton substituted at C(3) and
C(6) [7]. The HSQC spectrum showed direct correlations between d(C) 60.1 (C(1))
and d(H) 3.45 (HꢀC(1)), and 65.7 (C(5)), and 3.43 (HꢀC(5)). The presence of two
1
COO groups was supported by the signals at d(C) 165.5 and 166.0. The H-NMR

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
157
spectra revealed signals of two O-bearing CH groups at d(H) 5.34 (HbꢀC(3)) and 5.93
(HaꢀC(6)), two O-bearing CH groups at d(H) 3.45 (HꢀC(1)) and 3.43 (HꢀC(5)),
three O-bearing CH2 groups at d(H) 2.29 and 2.37 (HaxꢀC(2) and HaxꢀC(4), resp.),
1.82 and 2.03 (HeqꢀC(2) and HeqꢀC(4), resp.), and 2.33 and 2.82 (HaꢀC(7) and
HbꢀC(7), resp.), and a MeN group at d(H) 2.46. The complete assignment of all the C-
and H-atoms is compiled in Table 1. The relative configuration of 1 was established by
the signal at d(H) 5.34 (br. t, HꢀC(3)) with a coupling constant of J ¼ 5.0 Hz indicating
the a-orientation of the substituent at C(3) [3]. On the other hand, the relative
configuration of the substituent at C(6) was established by the analysis of multiplicity
and of the coupling constants of HꢀC(6). HꢀC(6) (d(H) 5.93) showed two couplings
(dd, J¼3.0, 7.5) with the two H-atoms with signals at d(H) 2.82 and 2.33 (HaꢀC(7) and
HbꢀC(7), resp.) and did not show any coupling with the signal of HꢀC(5) (d(H) 3.43).
This observation is in line with the b-orientation of the substituent, and a dihedral angle
of 908 between H-atoms HꢀC(5) and HaꢀC(6) [3]. These data were corroborated by X-
ray crystallography of 1a (Figs. 1 and 2). The chemical shifts d(H) 8.01 (dd, J ¼ 1.0, 7.5,
HꢀC(2’’,6’’)), 7.43 (t, J ¼ 7.5, HꢀC(3’’,5’’)), and 7.55 (t, J¼7.5, HꢀC(4’’)) are character-
istic of a benzoyloxy (BzO) group. The chemical shifts at d(H) 7.32 (s, HꢀC(2’,6’)) and
Table 1. NMR Data for Compound 1a)
Position
d(C)
d(H)
HMBC
1H,1H-COSY
1
60.12
34.59
3.45 (m)
2.29 (m)
1.82 (br. d, J¼15.0)
5.34 (br. t, J¼5.0)
2.37 (m)
Me
HꢀC(7)
2ax
2eq
3b
4ax
4eq
5
6a
7a
7b
HꢀC(1)
67.28
33.25
HꢀC(2)/HꢀC(4)
2.03 (br. d, J¼15.0)
65.74
79.69
36.73
3.43 (m)
Me/HꢀC(3)/HꢀC(7)
HꢀC(7)
HꢀC(4)
5.93 (dd, J¼3.0, 7.5)
2.82 (dd, J¼7.5, 14.0)
2.33 (m)
1’
2’
3’
4’
5’
6’
7’
1’’
2’’
3’’
4’’
5’’
6’’
7’’
MeN
MeO
121.15
106.55
146.97
139.60
146.97
106.55
165.46
130.28
129.46
128.34
132.92
128.34
129.46
166.00
40.07
HꢀC(2’)/HꢀC(6’)
HꢀC(6’)
7.32 (s)
MeO/HꢀC(2’)
MeO/HꢀC(6’)
HꢀC(2’)
7.32 (s)
HꢀC(3)
HꢀC(3’’)/HꢀC(5’’)
HꢀC(4’’)/HꢀC(6’’)
HꢀC(5’’)
8.01 (dd, J¼1.0, 7.5)
7.43 (t, J¼7.5)
HꢀC(3’)
HꢀC(2’)
7.55 (t, J¼7.5)
HꢀC(2’’)/HꢀC(6’’)
HꢀC(3’’)
7.43 (t, J¼7.5)
HꢀC(6’)
HꢀC(5’)
8.01 (dd, J¼1.0, 7.5)
HꢀC(2’’)/HꢀC(4’’)
HꢀC(2’’)/HꢀC(6’’)
2.46 (s)
4.00 (s)
56.50
a) CDCl3; at 500 MHz for 1H-NMR and 125 MHz for 13C-NMR.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 1. An ORTEP3 projection of the molecule 1a, showing the atom numbering and displacement
ellipsoids at the 50% probability level
Fig. 2. H-Bonding interactions in the crystal packing

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
159
4.00 (2 MeO) are characteristic of a 3’,5’-dimethoxybenzoyloxy (Hdmb) group. The
HMBC spectrum showed correlations between C(3’) and C(5’) (d(C) 147.0) and MeO
groups (d(H) 4.00). Therefore, compound 1 was identified as 6b-benzoyloxy-3a-(4-
hydroxy-3,5-dimethoxybenzoyloxy)tropane, a new tropane alkaloid.
Compound 1a was obtained as white crystals, with a melting point of 196–1988.
1
Analysis of the H- and 13C-NMR data showed chemical shifts compatible with a 3,6-
disubstituted tropane skeleton. Unlike alkaloid 1, derivative 1a showed chemical shifts
for HꢀC(1) at d(H) 4.11 and HꢀC(5) at d(H) 4.12. These chemical shifts are
compatible with those of cocaine hydrochloride [8] (Table 2). The structure of 1a was
established by X-ray crystallography (Figs. 1 and 2).
Table 2. NMR Data for Compounds 1a, 2, and 3a)
Position 1a
d(C) d(H)
2
3
d(C) d(H)
d(C) d(H)
1
67.18 4.11 (br. s)
33.02 2.40 (br. d, J¼15.5)
59.92 3.42 (m)
34.65 2.26 (m)
60.03 3.43 (m)
34.66 2.26 (m)
2ax
2eq
3b
4ax
4eq
5
6a
7a
7b
1.82 (br. d, J¼15.0)
1.83 (br. d, J¼15.0)
64.48 5.46 (br. t, J¼4.5)
34.14
67.57 5.34 (br. t, J¼5.0)
33.16 2.29 (m)
67.65 5.34 (br. t, J¼5.0)
33.31 2.33 (m)
2.20 (br. d, J¼16.0)
63.10 4.12 (br. s)
35.40 6.09 (dd, J¼3.0, 8.0)
2.06 (br. d, J¼15.0)
2.03 (br. d, J¼15.0)
65.85 3.39 (br. s)
65.74 3.42 (m)
80.10 5.87 (dd, J¼3.0, 7.5)
79.84 5.92 (dd, J¼3.0, 7.0)
74.85 3.19 (dd, J¼8.0, 15.0) 36.16 2.78 (dd, J¼7.5, 14.5) 36.69 2.80 (dd, J¼7.0, 15.0)
2.51 (m)
2.33 (m)
2.30 (m)
Hdmb
Bz
Tmb
1’
119.75
130.41
125.38
2’, 6’
3’, 5’
4’
106.58 7.35 (s)
147.12
140.25
129.50 8.11 (dd, J¼1.5, 8.0) 106.63 7.39 (s)
128.58 7.49 (t, J¼8.0)
133.00 7.58 (m)
165.70
153.13
153.13
165.32
7’
164.98
Bz
Bz
Bz
1’’
128.51
130.37
130.31
2’’, 6’’
3’’, 5’’
4’’
129.44 7.98 (br. d, J¼7.5)
128.67 7.49 (t, J¼7.5)
133.89 7.63 (t, J¼7.0)
165.10
129.48 8.03 (dd, J¼1.5, 8.0) 129.48 8.02 (dd, J¼1.5, 8.5)
128.32 7.43 (t, J¼8.0)
132.89 7.53 (m)
166.37
128.35 7.42 (t, J¼8.0)
132.94 7.55 (t, J¼7.5)
166.05
7’’
MeN
m-MeO
p-MeO
40.34 3.10 (s)
40.11 2.60 (s)
40.16 2.60 (s)
56.31 3.98 (s)
60.89 3.92 (s)
56.41 4.00 (s)
a) CDCl3; at 500 MHz for 1H-NMR and 125 MHz for 13C-NMR.
Compound 2 was isolated as white crystals, with a melting point of 119–1218. The
MS showed a molecular-ion peak of m/z 366.1 ([MþH]þ ), compatible with the
molecular formula C22H23NO4. The 1H- and 13C-NMR spectra exhibited chemical-shift
values similar to those for 1 (Table 1), the chemical shifts of which are in line with a
tropane skeleton disubstituted at C(3) and C(6). The principal difference between the

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
NMR data of compounds 1 and 2 is the type of substituent at C(3). The signals at d(H)
8.11 (dd, J ¼ 1.5, 8.0, HꢀC(2’,6’)), 7.49 (t, J ¼ 8.0, HꢀC(3’,5’)), and 7.58 (t, J¼7.5,
HꢀC(4’)) are compatible with a BzO group at C(3) (Table 2). The HMBC spectrum
showed long-range correlations between signals at 165.7 with 5.34 (HbꢀC(3)), and
166.4 with 5.87 (HꢀC(6)), defining the chemical shifts of the CO groups C(7’) and
C(7’’), respectively. Thus, compound 2 was identified as 3a,6b-dibenzoyloxytropane.
Compound 3 was isolated as white crystals, with a melting point of 154–1578. The
MS showed a molecular-ion peak of m/z 456.1 ([MþH]þ ), compatible with the
1
molecular formula C25H29NO7. The H- and 13C-NMR spectra of this alkaloid also
displayed chemical-shift values similar to those for alkaloids 1 and 2, but the signals at
7.39 (s, 2 H), 3.98 (s, 6 H), and 3.92 (s, 3 H) indicated that the substituent at C(3) is a
3,4,5-trimethoxybenzoyloxy group (Table 2). The HMBC spectrum showed correla-
tions between the signals of C(7’) (d(C) 165.3) and HꢀC(3) (d(H) 5.34), and between
the signals of C(7’’) (d(C) 166.1) and HꢀC(6) (d(H) 5.92). The relative configuration of
3 was assigned as in compound 1. Thus, the structure of compound 3 is 6b-benzoyloxy-
3a-(3,4,5-trimethoxybenzoyloxy)tropane (catuabine B).
The 1H- and 13C-NMR spectra of the fractions of alkaloids 1–3 showed the presence
of other constituents in smaller proportions. In an attempt to identify these
constituents, these fractions were analyzed by LC/ESI-MS/MS (Table 3). Supported
by the fragmentation model of alkaloid 1 (Scheme), it was possible to partially identify
alkaloids 4–8. The selective MS2 of the three fractions analyzed showed ions at m/z
140.1 and 122.2, suggesting the presence of disubstituted tropane alkaloids [9]. The
chromatogram of total ions of fraction of 1, showed three peaks, with the molecular-ion
peaks at m/z 420.1, 442.1, and 366.1, where the largest component ion, with the peak at
m/z 442.1, was identified as alkaloid 1. The MS2 experiment of the ion with the peak at
m/z 420.1 resulted in the formation of ions with peaks at m/z 320.2 and 222.1, among
others. These fragments [420.1!320.2] and [420.1!222.1] are compatible with the
loss of the Me2CCHCO and Hdmb substituents from the tropane ring, respectively,
suggesting the molecular formula C22H29NO7 for compound 4. Similarly, for fraction of
2, the chromatogram of total ions showed three peaks with molecular-ion peaks at m/z
278.1, 262.1, and 366.1, where the largest component ion, with the peak at m/z 366.1, was
identified as alkaloid 2. The MS2 experiment of the ion with the peak at m/z 278.1
resulted in the formation of ions with peaks at m/z 156.1 and 138.1, among others. These
fragments [278.1!156.1!138.1] are compatible with the loss of the BzO and OH
substituents, respectively, suggesting the molecular formula C15H19NO4 for compound
5. The MS2 experiment of ion with the peak at m/z 262.1 resulted in the formation of
ions with peaks at m/z 140.1 and 122.2, among others. These fragments [262.1!140.1!
122.1] are compatible with the loss of the BzO and OH substituents, suggesting the
molecular formula C15H19NO3 for compound 6. The chromatogram of total ions of
fraction of 3, showed four peaks with molecular-ion peaks at m/z 262.1, 352.1, 426.1, and
456.1, where the largest component ion, with the peak at m/z 456.1, was identified as
alkaloid 3. The MS2 experiment of the ion with the peak at m/z 352.1 resulted in the
formation of ions with peaks at m/z 195.0 and 140.2, among others. The fragment
[352.1!140.2] is compatible with the loss of the substituent Tmb, thus suggesting the
molecular formula C10H11NO4 for compound 7. The MS2 experiment of the ion with the
peakt at m/z 426.1, resulted in the formation of ions with peaks at m/z 304.1 and 122.2,

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
161
among others. These fragments [426.1!304.1!122.2] are compatible with the loss of
the BzO and Dmb substituents, suggesting the molecular formula C24H27NO6 for
compound 8. Thus, of the alkaloids only partially characterized, 4 and 8 are possibly
new natural products.
Scheme. Proposed Fragmentation of Alkaloid 1
Table 3. LC/MS/MS Data for Each Ion Analyzed and Its Fragmentations
Alkaloid
Peak No.
Mþ
MS2 Fragments
1
1
2
3
420
442
366
320, 222, 181, 140, 122
320, 244, 181, 140
244, 122
2
3
1
2
3
278
262
366
156, 138, 94
140, 122, 91
244, 122
1
2
3
4
262
352
426
456
140, 105
195, 140, 122
244, 165, 140, 122
244, 195, 167

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
The cytotoxic activities of the MeOH extract of Erythroxylum caatingae stems and
alkaloid 3 were evaluated against human cancer cell lines (HEp-2, NCI-H292, and
KB). Alkaloid 3 exhibited significant cytotoxicity only against NCI-H292 (IC50 ¼50 mg/
ml), and the MeOH extract of E. caatingae stems was inactive against all cell lines
tested (IC50 >50 mg/ml).
The effects of MeOH extract of E. caatingae stems on mice transplanted with
Sarcoma 180 are shown in Table 4. There was a significant reduction in tumor weight in
animals treated with different doses. These reductions gave tumor inhibition
percentages of 59.4 to 66.4%. The MeOH extract tested showed IC50 values greater
than 10 mg/ml for all tumor cell lines tested, suggesting that the in vivo anticancer
properties were not related to direct antiproliferative effects.
Table 4. In vivo Antitumor Activity against Sarcoma 180 Using MeOH Extract of Erythroxylum
caatingae Stemsa)
Compound
Dose [mg/kg]
Weight of tumor [g]
Inhibition [%]
MeOH extract of stems
100
200
400
2.5
–
0.698ꢂ0.05
0.577ꢂ0.10
0.614ꢂ0.11
0.659ꢂ0.08
1.718ꢂ0.20
59.4
66.4
64.3
61.7
–
Methotrexate
Control
a) Data are presented as meansꢂSEM for seven animals. *: P<0.05 compared with control group.
´
The authors thank the Conselho Nacional de Desenvolvimento Cientꢀfico e Tecnologico (CNPq),
CoordenaÅa˜o de AperfeiÅoamento Pessoal de Nꢀvel Superior (CAPES) for financial support, Vicente
ˆ
Carlos de O. Costa (UFPB) for the NMR spectra, and Norberto P. Lopes (Faculdade de Ciencias
ˆ
˜
Farmaceuticas da USP de Ribeirao Preto) for recording the mass spectra. The Escola de Enfermagem e
´
Farmacia/Instituto de Quꢀmica e Biotecnologia (UFAL) helped carry out the X-ray crystallography. Mr.
´
P. Wehrung of the Plateforme de Chimie Biologique Integrative de Strasbourg (PCBiS) IFR 85 and Mrs.
´
M. Schmitt, Laboratoire de Pharmacochimie, Faculte de Pharmacie, 74 route du Rhin, F-67400 Illkirch,
helped to perform the LC/ESI-MS/MS.
Experimental Part
General. M.p.: Microquimica digital melting-point apparatus, model MQAPF-302, with the Pt block
in a REICHERT Kofler-type light microscope, model R3279, with a temp. that varies from 0 to 3508;
values uncorrected. IR Spectra: BOMEM SERIE 100 MB spectrometer, in the range of 4000–400 cmꢀ1
,
with KBr pellets (0.5 mg of the sample/100 mg of KBr). NMR Spectra: VARIAN-NMR SYSTEM
1
spectrometer, operating at 500 MHz for H and at 125 MHz for 13C; recorded in CDCl3, with TMS as
internal standard. LC/ESI-MS/MS: HPLC Agilent 1200RRLC and Bruker HCT Ultra mass spectrom-
eter. X-Ray crystallography: Enraf-Nonius Kappa-CCD diffractometer.
Plant Material. The stems of Erythroxylum caatingae were collected in Picui, Paraꢀba, Brazil. The
botanical material was identified by M. F. A. A dried specimen was deposited with the Herbario Prof.
L. P. Xavier (JPB), Universidade Federal da Paraꢀba, under the identification label AGRA 5666.
Extraction and Isolation. The crude MeOH extract (500 g) was dissolved in H2O and defatted with
hexane. The defatted aq. extract was acidified with 3% HCl by mechanical mixing and filtered through
Celite, yielding a residue that was discarded and an acidic soln. This acidic soln. was extracted with CHCl3
(3ꢁ500 ml) resulting in an acidic CHCl3 phase and an aq. phase that was neutralized to pH 7.0 with
NH4Cl. The aq. phase at pH 7.0 was then extracted with CHCl3 to yield an aq. phase and basic CHCl3

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
163
phase FA1 (4.0 g), which was submitted to column chromatography (CC) utilizing SiO2 as the stationary
phase, and CHCl3 and MeOH as the eluents alone or in binary mixtures with increasing polarity. The
result were 55 fractions of 100 ml. The 55 fractions were monitored by TLC, eluted with various solvent
systems (CHCl3 and/or CHCl3/MeOH in order of increasing polarity) in chambers pre-saturated with
NH3 vapor, revealed with Dragendorffꢃs reagent, and placed in 21 groups based on their Rf values. Frs. 25,
45, and 46 were submitted to repeated recrystallization with acetone and Et2O, to yield compounds 1, 2,
and 3. Compound 1 was acidified with HCl to yield the alkaloid hydrochloride, referred to as 1a.
6b-Benzoyloxy-3a-(4-hydroxy-3,5-dimethoxybenzoyloxy)tropane (¼(3S,6S)-8-Methyl-6-[(phenyl-
carbonyl)oxy]-8-azabicyclo[3.2.1]oct-3-yl 4-Hydroxy-3,5-dimethoxybenzoate; 1). White crystals. IR
1
(KBr): 3425 (OH), 1724 (C¼O), 1277. H- and 13C-NMR: see Table 1. ESI-MS: 442 ([MþH]þ ), 320
([MꢀBzOH]þ ), 244 ([MꢀHdmbOH]þ ), 181 [MꢀBzOHꢀC8H13ON]þ ), 140 ([MꢀHdmbOHꢀ
C7H5O]þ ).
3a,6b-Dibenzoyloxytropane (¼(3S,6S)-8-Methyl-8-azabicyclo[3.2.1]octane-3,6-diyl Dibenzoate; 2).
White crystals. IR (KBr): 1724 (C¼O), 1600–1585 (C¼C). 1H- and 13C-NMR: see Table 2. ESI-MS: 366
([MþH]þ ), 244 ([MꢀBzOH]þ ), 122 ([MꢀBzOHꢀC8H14N]þ ).
Catuabine B (¼6b-Benzoyloxy-3a-(3,4,5-trimethoxybenzoyloxy)tropane¼(3S,6S)-8-Methyl-6-
[(phenylcarbonyl)oxy]-8-azabicyclo[3.2.1]oct-3-yl 3,4,5-Trimethoxybenzoate; 3). White crystals. IR
(KBr): 1709 (C¼O), 1600–1585 (C¼C), 1281. 1H- and 13C-NMR: see Table 2. ESI-MS: 456 ([MþH]þ ),
334 ([MꢀBzOH]þ ), 244 ([MꢀBzOHꢀ3 MeO]þ ), 195 ([MꢀBzOHꢀC8H14N]þ ).
X-Ray Crystallographic Analyses1). X-Ray diffraction data collections were performed on an Enraf-
Nonius Kappa-CCD diffractometer (95-mm CCD camera on k-goniostat) using graphite monochro-
mated MoKa radiation (0.71073 ꢄ), at r.t. Data collections were carried out using the COLLECT
software [10] up to 508 in 2q. Final unit-cell parameters were based on 9048 reflections. Integration and
scaling of the reflections, and correction for Lorentz and polarization effects were performed with the
HKL DENZO-SCALEPACK system of programs [11]. The structure of the compound was solved by
direct methods with SHELXS-97 [12]. The models were refined by full-matrix least squares on F2 using
SHELXL-97 [13]. The program ORTEP-3 [14] was used for graphic representation and the program
WINGX [15] to prepare materials for publication. All H-atoms were located by geometric considerations
placed (d(CꢀH)¼0.93–0.98 ꢄ) and refined as riding with Uiso(H)¼1.5 Ueq(Cꢀmethyl) or 1.2 Ueq-
(other). An ORTEP-3 diagram of the molecule is shown in Fig. 1, and Table 5 contains the main
crystallographic parameters.
The compound crystallized with one Cl-atom that forms NꢀH1N· · ·Cl1i and O3’ꢀH3· · ·Cl1ii H-
bonding interactions, where: [i¼xþ1/2, yþ1/2, z; ii¼x, y, z] and H1N· · ·Cl1i ¼2.065(2) ꢄ ; NꢀH1N· · ·
Cl1i ¼1788 and H3· · ·Cl1ii ¼2.477(1) ꢄ; O3’ꢀH3· · ·Cl1ii ¼1408 (Fig. 2).
Cytotoxicity Assay. NCI-H292 (human lung mucoepidermoid carcinoma cell line), HEp-2 (human
larynx epidermoid carcinoma cell line), and KB (human mouth epidermoid carcinoma cell line) cells
˜
were obtained from the Adolph Lutz Institute (Sao Paulo, Brazil) and were maintained in DMEM (¼
Dulbeccoꢃs Modified Eagleꢃs Medium) supplemented with 10% fetal bovine serum (FBS), 1000 IU/ml of
penicillin, and 250 mg/ml of streptomycin, and 1% of 200 mm glutamine at 378 with 5% CO2. Cytotoxicity
was evaluated with the colorimetric MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide) assay [16][17]. Cell suspensions were diluted to 105 cells/ml and were distributed in 96-well
culture plates (220 ml in each well), which were incubated for 24 h at 378 in a humidified incubator with
5% CO2. After 24 h, 22 ml of MeOH extract of Erythroxylum caatingae stems and alkaloid 3 were added,
and the plates were incubated again at 378. At the end of this period, the culture medium with excess
MTT was removed, and 100 ml of DMSO were added to each well to dissolve the formazan crystals. The
optical density (OD) of the wells was measured at 595 nm with an ELISA plate reader and compared to
the control.
1
)
Crystallographic data have been deposited with the Cambridge Crystallographic Data Center as
supplementary publication No. CCDC-711367. Copies of available material can be obtained, free of
charge, upon request through the Director, CCDC, 12 Union Road, Cambridge CH21EZ, UK (fax:
þ44-1223-336-033 or e-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).

164
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 5. Crystal Data and Structure Refinement for Compound 1a
Empirical formula
Formula weight [g molꢀ1
Temp. [K]
C24H28ClNO7
477.92
295(2)
]
Crystal dimensions [mm]
Crystal system
Space group
0.19ꢁ0.17ꢁ0.13
Monoclinic
C2
Unit cell dimensions
a [ꢄ]
22.5640(13)
b [ꢄ]
14.8870(5)
c [ꢄ]
8.3820(5)
b [8]
V [ꢄ3]
97.135(2)
2793.8(2)
Z
4
L(MoKa) radiation [ꢄ]
0.71073
1.136
0.174
pcalc [Mg mꢀ3
]
m(MoKa) [mmꢀ1
]
F000
1008
q Range for data collection [8]
Index range
Reflections collected
2.5–27.5
ꢀ23ꢃhꢃ29, ꢀ15ꢃkꢃ19, ꢀ8ꢃlꢃ10
9939
Independent reflections [Rint
Reflections with I>2s(I)
]
5809 [0.024]
4374
Number of parameters refined
299
0.085
1.07
R [F2 >2s(F2)]
Goodness-of-fit on F2
Residual electron density [e Aꢀ3
]
0.93
Animals. Female Swiss albino mice (Mus musculus), 60-d-old and weighing 25ꢂ5 g, were obtained
from the animal house of the Departamento de Antibioticos, UFPE, Brazil. The animals were housed
under standard environmental conditions of temp., humidity, and under a light-dark cycle of 12 h. The
mice were fed animal house diet (LABINE Purina, Brazil) and given water ad libidum. The animals were
treated according to the ethics principles of animal experimentation of COBEA (Colegio Brasileiro de
˜
Experimentacao Animal), Brazil. The Animal Studies Committee of the Universidade Federal de
Pernambuco approved the experimental protocols (No. 23076.012173/2007-77).
Antitumor Assay. Sarcoma 180 tumor cells were maintained in the peritoneal cavities of the Swiss
mice in the Laboratorio de Bioensaios para Pesquisa de Farmacos from the Departamento de
Antibioticos, UFPE, Brazil. Ascitic tumor cells (suspension of 5ꢁ106 cells) were injected subcutaneously
in the axillary region of healthy animals previously weighed, and the mice were divided into experimental
groups (n¼7) [18]. Twenty-four hours after inoculation, MeOH extract of E. caatingae stems (100, 200,
and 400 mg/kg) was dissolved in normal saline soln. with cremophor EL (2%) and administered
intraperitoneally for 7 d in mice transplanted with Sarcoma 180 tumor. Methotrexate (2.5 mg/kg) was
used as the positive control. The negative control received the vehicle only. On day 8, the mice were
weighed and euthanized. The tumors were dissected and weighed. The percentage of tumor inhibition
was calculated in accordance with [19] as: TWI¼CꢀT/Cꢁ100, where C is the weight of the control
tumor and T is the weight of the treated tumor.
Statistical Analysis. Data are reported as meansꢂstandard error of the mean (SEM) of n
experimental animals. Statistical differences were compared by analysis of variance (ANOVA) followed
by Tukeyꢃs multiple comparison test. Differences were considered statistically significant if P<0.05.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
165
REFERENCES
[1] M. I. B. Loiola, M. F. Agra, G. S. Baracho, R. T. de Queiroz, Acta Bot. Bras. 2007, 21, 473.
´
[2] D. Chavez, B. Cui, H.-B. Chai, R. Garcꢀa, M. Mejꢀa, N. R. Farnsworth, G. A. Cordell, J. M. Pezzuto,
A. D. Kinghorn, J. Nat. Prod. 2002, 65, 606.
[3] B. Zanolari, D. Guilet, A. Marston, E. F. Queiroz, M. Q. Paulo, K. Hostettmann, J. Nat. Prod. 2003,
66, 497.
[4] B. Zanolari, D. Guilet, A. Marston, E. F. Queiroz, M. Q. Paulo, K. Hostettmann, J. Nat. Prod. 2005,
68, 1153.
´
[5] G. L. Silva, B. Cui, D. Chavez, M. You, H.-B. Chai, P. Rasoanativo, S. M. Lynn, M. J. OꢃNeill, J. A.
Lewis, J. M. Besterman, A. Monks, N. R. Farnsworth, G. A. Cordell, J. M. Pezzuto, A. D. Kinghorn,
J. Nat. Prod. 2001, 64, 1514.
[6] J. F. Tavares, K. F. Queiroga, M. V. B. Silva, M. F. F. M. Diniz, J. M. B. Filho, E. V. L. da-Cunha,
´
C. A. de Simone, J. X. de A. Junior, P. S. Melo, M. Haun, M. S. da Silva, J. Nat. Prod. 2006, 69, 960.
[7] S. L. Oliveira, M. S. da Silva, J. F. Tavares, J. G. Sena-Filho, H. F. S. Lucena, M. A. V. Romero, J. M.
Barbosa-Filho, Chem. Biodiversity 2010, 7, 302.
[8] J. F. Muhtadi, A. A. Al-Badr, in ꢅAnalytical Profiles of Drug Substancesꢃ, Ed. K. Florey, Academic
Press, New York, 1986, Vol. 15, pp. 151–229.
[9] B. Zanolari, J.-L. Wolfender, D. Guilet, A. Marston, E. F. Queiroz, M. Q. Paulo, K. Hostettmann, J.
Chromatogr. A 2003, 1020, 75.
[10] Enraf-Nonius COLLECT, Nonius BV, Delft, The Netherlands, 1997–2000.
[11] Z. Otwinowski, W. Minor, in ꢅMethods in Enzymologyꢃ, Eds. C. W. Carter, R. M. Sweet, Academic
Press, New York, 1997, Vol. 276, pp. 307–326.
[12] G. M. Sheldrick, SHELXS-97, Program for Crystal Structure Resolution, University of Gçttingen,
Gçttingen, 1997.
[13] G. M. Sheldrick, SHELXL-97, Program for Crystal Structure Refinement, University of Gçttingen,
Gçttingen, 1997.
[14] L. J. Farrugia, J. Appl. Crystallogr. 1997, 30, 565.
[15] L. J. Farrugia, J. Appl. Crystallogr. 1997, 32, 837.
[16] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
[17] M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J. Czerwinski, D. L. Fine, B. J. Abbott, J. G.
Mayo, R. H. Shoemaker, M. R. Boyd, Cancer Res. 1988, 48, 589.
[18] R. M. Ribeiro-Costa, A. J. Alves, N. P. Santos, S. C. Nascimento, E. C. P. GonÅalves, N. H. Silva,
˜
N. K. Honda, N. S. Santos-Magalhaes, J. Microencapsul. 2004, 21, 371.
´
´
[19] Z. Machon, L. Kuczynski, J. Giełdanowski, Z. Wieczorek, M. Zimecki, B. Błaszczyk, M. Mordarski,
J. Wieczorek, L. Fiszer-Maliszewska, Arch. Immunol. Ther. Exp. 1981, 29, 217.
Received December 1, 2009