Antibacterial cyclopeptide alkaloids from the bark of Condalia buxifolia.

Article abstract of DOI:10.1016/S0031-9422(02)00287-X

The cyclopeptide alkaloid, named condaline-A, was isolated from the root bark of Condalia buxifolia Reissek (Rhamnaceae), along with the known compounds adouetine-Y', scutianine-B, and scutianine-C. Their structures were determined by spectroscopic analys

Full text of DOI:10.1016/S0031-9422(02)00287-X

Phytochemistry 61 (2002) 561–566
www.elsevier.com/locate/phytochem
Antibacterial cyclopeptide alkaloids from the bark of
Condalia buxifolia
Ademir F. Morela,*, Carla A. Araujoa, Ubiratan F. da Silvaa, Solange C.S.M. Hoelzela,
Renato Zachiab, Nelci R. Bastosb
a
´
Departamento de Quımica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
bDepartamento de Biologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
Received in revised form 3 July 2002
Abstract
The cyclopeptide alkaloid, named condaline-A, was isolated from the root bark of Condalia buxifolia Reissek (Rhamnaceae),
along with the known compounds adouetine-Y0, scutianine-B, and scutianine-C. Their structures were determined by spectroscopic
analyses, with their antibacterial activities being evaluated by use of a direct bioautography method.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Condalia buxifolia; Rhamnaceae; Cyclopeptide alkaloid; Condaline-A; Antimicrobial activity
1
1. Introduction
LSIMS mass spectrometry, Hand 13C NMR spectro-
scopy, and some chemical transformations indicated
structure 1 for condaline-A. Peptide alkaloids possess a
variety of biological activities, including sedative (Lee et
al., 2001; Han et al., 1989), antifungal (Tschesche et al.,
1974; Tschesche and Ammermann, 1974; Gournelis et
al., 1997), and antibacterial (Tschesche et al., 1974;
Tschesche and Ammermann, 1974; Gournelis et al.,
1997) properties. In this work, the antibacterial activity
of each alkaloid was determined by direct bioauto-
graphy (Rahalison et al., 1991), against Gram-positive
(Staphylococcus aureus, Staphylococcus epidermidis and
Micrococcus luteus) Gram-negative (Klebsiela pneumo-
niae, Salmonella setubal and Escherichia coli) bacteria.
As a continuation of investigations on peptide alka-
loids of plants belonging to the family Rhamnaceae
(Morel et al., 1979, 1984, 1985, 1998; Machado et al.,
1995; Menezes et al., 1995), the isolation of peptide
alkaloids from the methanol extract of a plant of the
genus Condalia is reported. The genus Condalia includes
18 American species distributed all over the continent,
from North to South America (Johnston, 1962). Five of
them are found in South America, one in Brazil (John-
ston and Soares, 1972). Condalia buxifolia Reissek is a
tree up to 4 m high that grows in the wild in southern
Brazil, Uruguay, and Argentina, where it is used by
local populations as
a febrifuge, for its antiin-
flammatory properties, and against dysentery (Bastos,
1989). Since its chemistry has not previously been
investigated, the root bark of the plant was examined to
afford a new peptide alkaloid, condaline-A (1), together
with three known peptide alkaloids, adouetine-Y0
(=myrianthine-B, 2) (Marchand and Monseur, 1968),
scutianine-B (3) (Tschesche et al., 1971, 1974), and scu-
tianine-C (4) (Sierra et al., 1974). A combination of
2. Results and discussion
The methanol extract of the root bark of C. buxifolia
was fractionated as described in the Experimental sec-
tion. The main component, condaline-A (1) was sepa-
rated by conventional chromatographic methods as a
colorless crystalline material. Its LSIMS-mass spectrum
displayed a prominent [M+H]+ at m/z 555 which, in
combination with 13C NMR spectroscopy and ele-
mental analysis data, suggested that 1 had the molecular
formula C33H38N4O4.
* Corresponding author. Tel.: +55-55220-8869; fax: +55-55220-
8031.
E-mail address: afmorel@base.quimica.br (A.F. Morel).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00287-X

562
A.F. Morel et al. / Phytochemistry 61 (2002) 561–566
1
The HNMR spectrum of 1 showed a methyl triplet
at ꢀ 0.83 (J32,31=6.5 Hz) and a methyl doublet at ꢀ 0.84
(J33,30=6.5 Hz), which were assigned to the C-32 and C-
33 methyl protons, respectively. In the COSY (H–H)
spectrum, the doublet at ꢀ 0.84 showed a cross peak
with the signal at ꢀ 1.40 (1H, m) which corresponds to
H-30, whereas the triplet at ꢀ 0.83 showed a cross peak
with the resonance at ꢀ 0.86/1.29 (2H, m) which corre-
sponds to H2-31. H-30 had a cross peak with the reso-
nance at ꢀ 3.90 (dd, J7,6=9.0; J7,30=4.4 Hz), which
corresponds to H-7. This spin-system confirms iso-
leucine as the a-amino acid of the ring. ꢁ-Phenylserine,
which is the hydroxylated amino acid of the macrocyclic
ring, was identified from a cross peak between H-3,
resonating at ꢀ 5.81 (1H, br s), and H-4, which reso-
nated at ꢀ 4.58 (1H, d, J4,21=9.2 Hz). The latter also
exhibited a cross peak with H-21, which resonated at d
8.03 (1H, d, J21,4=9.2 Hz).
assignments of 1, along with the proton coupling con-
stants, are summarized in Table 1.
The 13C NMR spectrum (100.6 MHz, CDCl3) pro-
vided strong support for the proposed structure. The
13C NMR chemical shifts of 1 (Table 1) were assigned
from the analysis of DEPT spectra and 2D hetero-
nuclear correlated spectra (HMQC and HMBC), toge-
ther with a previous assignment for known cyclopeptide
alkaloids (Morel et al., 1998, 1999).
The absolute stereochemistry of the C-7 amino acid
(isoleucine) and of the N-methyl phenylalanine side-
chain of 1 was determined by chiral phase gas chroma-
tography (CPGC) using 3-pentyl-2,6-dimethyl-b-cyclo-
dextrine (Konig et al., 1990) as stationary phase. The N-
trifluoroacetylated methyl ester of the D, L-mixture and
the pure L-form were used as CPGC standards. By
comparing the Rts of these standards with those of the
corresponding amino acid from the hydrolysate of
dihydrocondaline-A, it was possible to assign the abso-
lute configuration unambiguously. In condaline-A, N-
methyl phenylalanine and isoleucine both have the
L(S)-configurations. The relative stereochemistry of the
ꢁ-phenylserine unit (not found in the hydrolyzed alka-
1
loid) was determined by analysis of HNMR coupling
constants and NOESY interactions (see Fig. 1). The H-3
proton was observed at ꢀ 5.81 (1H, br s) and showed a
COSY cross peak with H-4 which resonated at ꢀ 4.58
(1H, d, J4,21=9.2 Hz). The vicinal coupling constant of
near zero of the methine protons of the ꢁ-phenylserine
(H-3/H-4) indicates a threo-configuration (ꢂ ca. 90ꢀ) for
this residue (Morel et al., 1998). The coupling constant
between H-3 and H-4 in the erythro configuration was
ca. 14 Hz (ꢂ ca. 180o) (Morel et al., 1998). The NOESY
spectrum exhibited NOE cross peaks between H-3 and
H-4. In turn, H-4 exhibits a cross peak with NH-6,
while the latter did not show a cross peak with H-7.
This evidence suggests that the b-phenylserine of con-
daline-A has an L-threo (3R*/4S*) configuration. The
1Hand 13C spectral data and optical rotation of
The C-24 methylene protons were observed as two
0
double doublets at ꢀ 2.38 (J24 ,23=5.6; J24,24 =8.4 Hz)
0
0
and 2.52 (J24, 23=13.6; J24 , 24=8.4 Hz), whereas the C-
23 methine proton appeared as a double doublet at ꢀ
0
3.02 (J23,24=5.6 Hz; J23,24 =13.6 Hz). In the COSY
spectrum, H2-24 and H-23 showed internal cross-peaks.
In addition, a singlet at ꢀ 2.08 (3H) was assigned to the
protons of the N-methyl group (H-29). This spin system
confirms N-methyl phenylalanine as the side-chain
amino acid. The C-10 methine proton showed a double
doublet at ꢀ 6.03 (J10,9=9.0; J10,11=7.6 Hz), due to
coupling with C-11 and NH-9, while the C-11 methine
proton was observed at ꢀ 6.98 (J11,12=7.6 Hz). NH-9
was difficult to assign, due to the superimposition of this
resonance with the aromatic protons. The 1HNMR
Fig. 1. Structure and selected NOESY correlations in condaline-A (1).

A.F. Morel et al. / Phytochemistry 61 (2002) 561–566
563
Table 1
1Hand 13C NMR spectral data for condaline-A (1) (in DMSO-d6, 400/100 MHz)a
1
H/C
ꢀ H (J, H z)
ꢀ 13C ppm
HMBC correlations
2JCH
3JCH
01
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
–
5.81(br s)
4.58 (d) J4,21=9.2
158.6
86.3
55.3
167.0
–
–
C-17
–
C-1
–
C-5
–
–
8.03 (d) J6,7=9.0
H-7, H-1
–
C-8
–
C-5
3.90 (dd) J7,6=9.0; J7,30=4.4
–
56.8
169.2
–
–
–
–
6.03 (d) J10,11=7.6; J10,9=9.0
6.98 (d) J11,10=7.6
–
C-11
–
C-12
131.4
126.9
132.4
129.4
124.4
119.8
121.7
139.7
126.0
127.6
127.0
–
C-10, C-12
–
–
–
6.99
–
C-1
–
6.93
7.15
–
C-12
C-12
–
6.81
–
C-1
–
7.58
–
C-19, C-20
-
C-3
7.36
7.25
C-18
–
C-17
C-18
8.03 (d) J21,4=9.2
–
3.02(dd) J23,24=5.6; J23,24 =13.6
–
C-5
176.6
65.5
39.2
–
–
C-22, C-24
–
C-25
0
0
2.52 (dd) J24,23=13.6; J24,24 =8.4
C-23, C-25
–
C-22, C-26
–
0
2.38 (dd) J24 ,23=5.6; J24,24 =8.4
0
25
26
27
28
29
30
31
32
33
138.2
128.0
129.9
–
–
C–24
C–25
7.0–7.5
–
–
–
–
C–23
2.08 (s)
1.40 (m)
37.6
34.2
24.4
15.2
10.8
–
–
–
1.29/0.86 9 (m)
0.83 (t) J32,31=6.5
0.84 (d) J33,30=6.5
–
–
C–30
C–7, C–31
–
C–30
Assignments were obtained by 2D H–1HCOSY, NOESY, DEPT 135 ꢀ and 2D 1H–13C COSY (HMQC, HMBC) experiments.
a
1
condaline-A suggest that it is a stereoisomer of aralio-
line-B, isolated as the minor alkaloid from the leaves of
Araliorhamnus vaginatus (Tschesche et al., 1970).
limits of alkaloids 1 and 3 are shown in Table 2, and the
highest sample amount tested was 50 mg. Amoxicillin
(0.5 mg) was used as a control.
Adouetine-Y0 (2), scutianine-B (3), and scutianine-C
(4) were identified by direct comparison (TLC) with
authentic samples and by comparison of their NMR
spectral data with analogous alkaloids (Haslinger, 1978;
Pais et al., 1979; Morel et al., 1997, 1999). This is the
first isolation of these alkaloids from this plant.
3. Experimental
3.1. General
The antibacterial activity of the methanol extract and
of alkaloids 1–4 was evaluated by means of direct
bioautography using a TLC bioassay (Rahalison et al.,
1991), against standard bacterial strains S. aureus, S.
epidermidis, M. luteus, K. pneumoniae, S. setubal, and E.
coli. The methanol extract showed modest activity
(>100 mg), while condaline-A (1) and scutianine-B (3)
were much more active (12.5–3.12 mg) against the
strains, than the methanol extract. Alkaloids 2 and 4
were found to be inactive against these bacteria. Detection
Mps are uncorr. IR spectra were recorded in KBr
pellets on a Bruker IFS 28 FT spectrophotometer.
LSIMS MS were obtained on a Quattro LC instrument
from Micromass (UK) equipped with a mega flow elec-
trospray probe. Optical rotations were taken on a Per-
1
kin Elmer 341 digital polarimeter. Hand 13C NMR
spectra were recorded at 400 and 100.6 MHz, on a
Bruker DPX-400 spectrometer. Chemical shifts are given
as ꢀ (ppm) using TMS as an internal standard. Chiral
GLC analyses were carried out with a Varian 3800 Gas

564
A.F. Morel et al. / Phytochemistry 61 (2002) 561–566
Table 2
Bioautography assay results of condaline-A (1) and scutianine-B (3), against bacteriaa
Micro-organismsb
Antibacterial activityc (mg)
1
3
Staphylococcus aureus
Staphylococcus efidermidis
Micrococcus luteus
Klebsiella pneumoniae
Salmonella setubal
ATCC 6538p
ATCC 12228
ATCC 9341
ATCC 10031
ATCC 19196
ATCC 11103
12.5
3.12
12.5
3.12
6.25
6.25
3.12
6.25
12.5
6.25
6.25
6.25
Escherichia coli
a
Positive control: amoxicillin (0.5 mg).
ATCC (American type culture collection).
Minimum amount required for inhibition of bacterial growth on TLC plates.
b
c
Chromatograph with a flame ionization detector using
an 0.25 i.d. fused silica capillary coated with 3-Pe-2,6-
Me-b-CD (Konig et al., 1990), diluted with the poly-
siloxane OV 1701. Thin layer chromatography was per-
formed on pre coated TLC plates (Merk, silica 60 F-254).
3.4. Condaline-A (1)
25
ꢀ
Needles from CHCl3–MeOH, mp 115–116 C; [ꢃ]D :
À73ꢀ (c 0.08, MeOH); IR ꢄmax cmÀ1: 3400–3200 (NH),
2800 (C–O-arylether), and 1685–1635 (C¼O); LSIMS
(positive) m/z: 555 [C33H38N4O4]; (Found: C, 71.38; H,
6.89; N,10.06. Calcd. for C33H38N4O4: C, 71.46; H, 6.91;
N,10.10); 1Hand 13C NMR spectral data: see Table 1.
3.2. Plant material
Condalia buxifolia was collected in December, 1998 in
a suburb of Piratini, State of Rio Grande do Sul, Brazil.
Nelci R. Bastos identified the plant, and a voucher spe-
cimen (3296) is deposited in the Herbarium SMDB of
the University of Santa Maria.
3.5. Dihydrocondaline-A
The hydrogenation of condaline-A (10 mg), under the
conditions described for peptide alkaloids (Morel et al.,
1979) (MeOH, 5 ml; 10% Pd/C, 5 mg; 15 h; 1 atm)
yielded the corresponding dihydroalkaloid (ca. 8 mg).
3.3. Extraction and isolation
ꢀ
Mp: 236–238 C. IR ꢄmax cmÀ1: 3400–3200 (NH), 2805
Dried ground root bark of C. buxifolia (920 g) was
exhaustively extracted with MeOHin a Soxhlet appa-
ratus for 12 h. The resulting MeOHextract was filtered
and concentrated in vacuum to obtain a crude residue
(181 g). This residue was dissolved in H2O (50 ml) and
acidified to pH2–3. The acidic solution was exhaus-
tively extracted with Et2O (5Â50 ml) to yield the acidic
ether extract (10 g). The aqueous solution was then
made basic (pH8–9) to yield the basic ether extract (2.5
g), mainly consisting of one alkaloid. A portion of the
basic ether extract (1.0 g) was applied to a silica gel
column (80 g) and eluted with CHCl3 containing
increasing amounts of MeOH(up to 20%) to give 10
fractions. Fractions 2–3 (CHCl3:MeOH, 99:1) were
combined (20 mg) and submitted to preparative TLC
(CHCl3:MeOH, 99:1, two elutions) to yield 2 (12 mg).
Fractions 4–5 (CHCl3:MeOH, 99:2) were recombined
and concentrated in vacuo to give a yellow solid mate-
rial (50 mg) which was applied to a silica gel column (2.0
g) eluted with CHCl3 containing increasing amounts of
MeOH(up to 5%) to give 3 (15 mg), and 4 (10 mg).
Fractions 7–8 (CHCl3:MeOH, 95:5), consisting of one
alkaloid, were combined and concentrated in vacuo to
give 1 (850 mg).
(C–O-arylether), and 1680–1630 (C¼O). 1HNMR
(DMSO-d6): ꢀ 0.63 (3H, d, J=6.8 Hz, CH3-33), 0.73
(3H, t, J=6.8 Hz, CH3-32), 1.29 (2H, m, H-31), 1.31
(1H, m, H-30), 2.09 (3H, s, CH3-29), 2.40/2.83 (2H, m,
H-24), 2.71 (2H, m, H-11), 3.63 (1H, dd, J=10.0; 5.0
Hz, H-7), 3.89 (2H, m, H-10), 4.57 (1H, d, J=9.0 Hz,
H-4), 5.75 (1H, br s, H-3), 6.75 (1H, dd, J=8.4; 2.2 Hz,
H-16), 6.85 (1H, dd, J=8.0; 2.2 Hz, H-14), 6.95 (1H, dd,
J=8.0; 2.2 Hz, H-13), 7.05 (1H, dd, J=8.4; 2.2, H-15),
6.50–7.60 (10H, superimposition of aromatic hydrogens
of ꢁ-phenylserine and N-methyl phenylalanine), 7.11
(1H, m, H-9), 7.90 (1H, d, J=10.0 Hz, H-6), 8.12 (1H,
br, H-21); 13C (DMSO-d6): ꢀ 11.1 (C-33), 15.2 (C-32),
24.4 (C-31), 34.0 (C-30), 34.2 (C-11), 34.4 (C-24), 38.7
(C-29), 39.3 (C-10), 56.3 (C-4), 56.9 (C-7), 68.1 (C-23),
85.5 (C-3), 119.0 (C-16), 120.9 (C-14), 130.0 (C-15),
131.3 (C-13), 133.6 (C-12), 139.6 (C-17), 157.8 (C-1),
166.6 (C-8), 169.8 (C-5), 169.9 (C-22), 120.0–129.0 (10
unidentified carbons).
3.6. Hydrolysis
Hydrolysis of dihydrocondaline-A (5 mg) was per-
formed in a sealed tube at 110ꢀ with 6 N HCl for 12 h,

A.F. Morel et al. / Phytochemistry 61 (2002) 561–566
565
the aq. acidic solvent was concentrated and the residue
was used to identify the absolute stereochemistry of
isoleucine (C-7 aminoacid) and of the N-methylpheny-
lalanine side-chain as previously described (Silva et al.,
1996).
ichia coli (ATCC 11103), were evaluated using a bioau-
tography technique (Rahalison et al., 1991). Methanolic
crude extract of the root bark of Condalia buxifolia (10
ml of solution corresponding to 100 mg) was used as
reference crude plant extract. The microorganisms used
in the antibacterial assay are maintained at the Depar-
tamento de Quımica of the Universidade Federal de
Santa Maria, RS, Brazil. For the antimicrobial assay of
1–4, samples of 50.0, 25.0, 12.5, 6.25, 3.12, 1.06 mg was
applied to pre-coated TLC plates. TLC plates were
developed with CHCl3:MeOH(95:5) and dried for
complete removal of solvents. The inoculum was pre-
pared by culturing each organism in tryptone soya
broth (TSb, Oxoid) at 37 ꢀC to a turbidity equivalent to
McFarland 0.5 standard (1.5Â108 CFU/ml). One
microliter of each diluted inoculum (104–106 CFU/ml)
was added in 10 ml of Mueller–Hinton Agar medium
(MHA-DIFCO), and distributed over developed TLC
plates (5Â5 cm). After solidification of the media, the
3.7. N-Methyl amino acids
The methyl amino acids were prepared by methyla-
tion of the corresponding N-benzyloxycarbonyl deriva-
tives (MacDermott and Benoiton, 1973). Z-D,L-Phe
and Z-L-Phe (596 mg; 2 mmol) and methyl iodide (1 ml,
16 mmol) were dissolvꢀed in THF (10 ml) and the solu-
tion were cooled to 0 C. A sodium hydride dispersion
(264 mg; 6 mmol) was added cautiously with gentle
stirring. The suspension was stirred at room temp. for
24 h, and the product was isolated by the general pro-
cedure of McDermott and Benoiton (1973). Finally, the
benzyloxycarbonyl group was removed by catalytic
hydrogenation in the presence of palladium on carbon,
yielded 469.5 mg (ca. 75%) of D,L-MePhe and 407.0 mg
(ca. 60%) of L-MePhe.
ꢀ
TLC plates were incubated overnight at 37 C (Saxena
et al., 1995). Subsequently, bioautograms were stained
with an aqueous solution of 2,3,5-triphenyltetrazolium
chloride (TCC, 5 mg/ml). Amoxicillin (0.5 mg) was used
as positive control.
3.8. Amino acid derivatization
Acid catalyzed esterifications were carried out by
addition of a 1.6 N anhydrous solution of HCl (gas) in
MeOHand leaving the mixture at room temp. for 30
min (Bayer and Konig, 1969). After removal of the
reagents in a stream of dry nitrogen, the samples were
taken up in CH2Cl2 (200 ml) and trifluoroacetic anhy-
dride (50 ml), the mixture was allowed to stand at room
temp. for 30 min, and the reagent was removed in a
stream of dry nitrogen.
Acknowledgements
The authors thank FAPERGS (Fundacao de Amparo
a Peaquisa do Rio Grande do Sul) and CNPq (Con-
selho Nacional de Pesquisa e Tecnologia) for financial
support.
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