Alkamides from the leaves of Zanthoxylum syncarpum

Article abstract of DOI:10.1021/np0580558

Three alkamides (1-3) were isolated from the leaves of Zanthoxylum syncarpum. The structures of the new compounds 1 and 2 were established by spectroscopic data and chemical conversion, and by the X-ray crystallography of 1. Compound 3, the racemic form of the known compound syncarpamide, showed moderate antiplasmodial activity, with IC₅₀ values of 4.2 and 6.1 μM against Plasmodium falciparum D6 clone and W2 clone, respectively.

Full text of DOI:10.1021/np0580558

J. Nat. Prod. 2005, 68, 1297-1299
1297
Alkamides from the Leaves of Zanthoxylum syncarpum
Samir A. Ross,*,†,‡ Mounirah A. Al-Azeib,‡ Kesanapalli S. Krishnaveni,† Frank R. Fronczek,§ and
Charles L. Burandt†
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and Department of
Pharmacognosy, School of Pharmacy, The University of Mississippi, University, Mississippi 38677-1848, and Department of
Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804
Received April 18, 2005
Three alkamides (1-3) were isolated from the leaves of Zanthoxylum syncarpum. The structures of the
new compounds 1 and 2 were established by spectroscopic data and chemical conversion, and by the
X-ray crystallography of 1. Compound 3, the racemic form of the known compound syncarpamide, showed
moderate antiplasmodial activity, with IC50 values of 4.2 and 6.1 µM against Plasmodium falciparum
D6 clone and W2 clone, respectively.
Zanthoxylum, commonly called “prickly ash”, is the
largest genus in the family Rutaceae and comprises about
200 species of trees and shrubs, with a worldwide, but
predominantly tropical, distribution.1,2 Zanthoxylum spe-
cies are reported to have many medicinal properties,3 and
phytochemical studies on the genus have shown it to be a
rich source of coumarins, lignans, and alkaloids, with
febrifuge, sudorific, and diuretic properties.4-16
Zanthoxylum syncarpum Tul. is an evergreen tree oc-
curring in both North and South America.13 Although there
are several reports on the chemical constituents of various
Zanthoxylum species,5-11 there are only four previous
reports on Z. syncarpum.12-15 In view of the potential
biological properties of Zanthoxylum species, we have
investigated a methanol extract of the leaves of Z. syncar-
pum, since it exhibited potent in vitro bioactivity (81%)
against a chloroquine-sensitive (D6, Sierra Leone) strain
of Plasmodium falciparum, and have isolated two new
alkamides (1 and 2) and the racemic form of syncarpamide
(3),12 which are the subject of the present paper.
protonated molecular ion [M + H]+ peak at m/z 328.1546
(calcd 328.1543) in the HRESIMS, and the resulting
molecular formula was determined to be C19H21NO4, rep-
resenting 10 degrees of unsaturation. The IR bands at
3337, 1713, 1656, 1615, and 1516 cm-1, respectively,
indicated the presence of hydroxyl, carbonyl, olefinic, and
amide functionalities in 1. The 1H and 13C NMR spectra of
1 gave signals including five quaternary carbons, 11
methines, one methylene, and two methyl carbons. Two
olefinic signals at δ 7.57 (141.8, C-7′) and 6.39 (120.4, C-8′),
each integrating for one proton with a J value of 15.6 Hz,
indicated that they were trans coupled, and these assign-
1
ments were confirmed using the H-1H COSY spectrum.
The corresponding olefinic carbons were assigned by their
HMQC correlations. The proton signals at δ 6.92 (1H, d, J
) 1.2 Hz), 6.85 (1H, dd, J ) 1.6, 8.0), and 6.75 (1H, d, J )
8.4 Hz), and signals at δ 7.40 (2H, dd, J ) 1.6, 6.8 Hz) and
7.30 (3H, m), indicated the presence of 1,3,4- and mono-
substituted benzene rings in 1. Two singlets, each integrat-
ing for three protons at δ 3.80 and 3.79, suggested the
presence of two methoxy groups, which were placed at C-3
and C-4, on the basis of HMBC correlations (Table 1). A
deshielded secondary carbon at δ 48.1 and a methine
carbon at δ 73.6 supported the presence of nitrogen- and
oxygen-bearing carbons in 1. These spectroscopic features
revealed that compound 1 has the general structural
features of aegeline.16,17 The NMR data of both these
compounds showed similar patterns, except for the pres-
ence of an additional methoxy group in 1, which was placed
at C-3 on the basis of HMBC correlations. Thus, compound
1 was characterized as a methoxy derivative of aegleine,
and data from the 1H-1H-COSY, HMQC, and HMBC NMR
spectra (Table 1) provided additional support to justify the
gross structure shown for 1. Compound 1 (3-methoxyae-
geline) did not show any optical rotation, indicating that
it was obtained as a racemic mixture. Recrystallization
from EtOAc-MeOH afforded racemic crystals, and un-
equivocal evidence for the assigned structure was obtained
from an X-ray diffraction analysis (Figure 1). Interestingly,
the crystals are kryptoracemic; that is, the space group does
not contain symmetry elements that invert the handedness,
but the asymmetric unit is a pair of enantiomers. Such
crystallization behavior from a racemic solution is uncom-
mon.
The leaves of Z. syncarpum were collected in Lara State,
Venezuela, and were air-dried, ground, and exhaustively
extracted with methanol at room temperature, with the
extract concentrated under reduced pressure. The concen-
trate (80 g) was chromatographed over a silica gel column,
and further workup as described in the Experimental
Section resulted in the isolation and purification of three
compounds (1-3).
Compound 1 was obtained as white crystalline ag-
gregates from EtOAc-MeOH (mp 138-139 °C) and gave
a positive test with Dragendorff’s reagent. It showed a
* To whom correspondence should be addressed. Tel: +1-662-915 -1031.
Fax: +1-662-915-7989. E-mail: sross@olemiss.edu.
† National Center for Natural Products Research.
‡ Department of Pharmacognosy.
Compound 2 was obtained as a light yellow, amorphous
powder and showed a molecular ion peak [M + Na]+ at
m/z 392.1462 (calcd for C21H23NO5Na, 392.1474) in the
§ Louisiana State University.
10.1021/np0580558 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/05/2005

1298 Journal of Natural Products, 2005, Vol. 68, No. 8
Notes
Table 1. 13C, 1H, and HMBC NMR Spectroscopic Data for Compounds 1 and 2 (CDCl3)a
1
2
position
δC
δH
HMBC (H to C)
3,6,7
δC
δH
HMBC (H to C)
1
2
3
4
5
6
7
8
134.8 s
109.3 d
149.3 s
148.8 s
111.4 d
118.3 d
73.6 d
130.2
6.92 d (1.2)
109.8 d
149.2 s
149.1 s
111.2 d
119.1 d
74.7 d
44.5 t
6.94 d (1.2)
6,7,3,4
6.75 d (8.4)
3
6.86 d (8.4)
1,3,4
2
1,2,6,7
6.85 dd (1.6, 8.0)
4.82 dd (3.2, 8.4)
3.73 Ha, dd (3.2, 4.8)
3.45 Hb, m
1,2,4,7
6.97 dd (1.6, 8.0)
5.94 dd (4.8, 8.0)
3.75 dd (3.2, 4.4)
48.1 t
7,9′
9
170.2 s
23.32 q
134.2 s
128.2 d
128.9 d
130.6 d
128.9 d
128.2 d
145.8 d
117.6 d
166.5 s
55.9 q
10
2.03 s
10
1′
134.8 s
128.1 d
129.0 d
130.1 d
129.0 d
128.1 d
141.8 d
120.4 d
167.6 s
56.1 q
2′
7.40 dd (1.6, 6.8)
7.30 m
1′,3′,7′
7.54 t (3.6, 6.4)
7.41 m
3′
3′
4′
7.30 m
7.41 m
5′
7.30 m
7.41 t (3.6, 6.4)
7.54 m
7.73 d (16.0)
6.50 d (16.0)
5′
8′,9′
1′,7′,9′
6′
7.40 dd (1.6, 6.8)
7.57 d (15.6)
6.39 d (15.6)
1′,5′,7′
2′,5′,9′
1′,9′
7′
8′
9′
OCH3-3
OCH3-4
NH-8
3.80 s
3,4
3,4
3.92 s
3.89 s
3,4
3,4
7
56.1 q
3.79 s
55.9 q
6.63 br s
5.80 br s
a 400 MHz for 1H and 100 MHz for 13C NMR, carbon multiplicities were determined by DEPT experiments. s ) C, d ) CH, t ) CH2.
Figures in parentheses denote J values (Hz).
derivative with physicochemical properties closely compa-
rable to 2.
The identification of 3 was based on analogies made with
1. Thus, comparison of the 1H and 13C NMR spectroscopic
data of 1 and 3 revealed identical spin systems except for
an additional cinnamoyl group. Literature data indicated
that 3 has the same structural features as syncarpamide,12
having similar physical properties with the exception of
25
its optical rotation value. Syncarpamide showed [R]D
+12.5° (c 0.08, CHCl3), while compound 3 gave [R]D25 0° (c
0.13), using the same solvent. These data supported the
identification of compound 3 as 3-methoxy-7-cinnamoylae-
geline (syncarpamide), which we have recently reported
from the stems of the same plant.12 Compound 3 is the
racemic form of this substance.
Figure 1. ORTEP diagram of two independent, enantiomeric mol-
As the MeOH crude extract of Z. syncarpum showed
ecules of 1.
potent in vitro bioactivity against a chloroquine-sensitive
(D6, Sierra Leone) strain of P. falciparum, the antimalarial
activity of the isolated compounds (1-3) was evaluated
against chloroquine-sensitive (D6, Sierra Leone) and -re-
sistant (W2, Indochina) strains of P. falciparum. Com-
pounds 1 and 2 were inactive, whereas compound 3 showed
moderate activity, with IC50 values of 4.2 and 6.1 µM
positive HRESIMS, corresponding to the molecular formula
C21H23NO5. IR absorption bands at 3368 and 3281 cm-1
indicated the presence of a secondary amide group, and a
band at 1713 cm-1 suggested the occurrence of an ester
group, which were supported by observations in the 13C
NMR spectrum of an amide carbonyl signal at δ 170.2, an
against P. falciparum D6 clone, Sierra Leone, and P.
ester carbonyl signal at δ 166.5, an oxygen-attached carbon
falciparum W2 clone, Indochina, respectively. Artemisinin
signal at δ 74.7, and a nitrogen-attached carbon signal at
and chloroquine showed IC50 values of 0.04 and 0.05 µM
δ 44.5 (Table 1). Two olefinic proton doublets (J ) 16.0 Hz),
against P. falciparum D6 clone and IC50 values of 0.03 and
each integrating for one proton at δ 7.73 (1H, d, J ) 16.0,
H-7′) and 6.50 (1H, d, J ) 16.0 Hz, H-8′), were attributable
to the presence of a trans-olefinic system in 2. The 1H NMR
spectrum also showed the presence of 1,3,4-trisubstituted
and monosubstituted benzene rings and two methoxy
0.5 µM against P. falciparum W2 clone, respectively.
Cytotoxicity was evaluated at an IC50 of 4.7 µg/mL.
Experimental Section
General Experimental Procedures. Optical rotations
were measured with a JASCO DIP-310 digital polarimeter.
UV and IR spectra were obtained using a Perkin-Elmer
Lambda 3B UV/vis spectrophotometer and an AATI Mattson
1
functionalities in 2. Comparison of the H and 13C NMR
data of 2 with those of compound 1 (Table 1) indicated that
2 is an acetyl analogue of 1, and the molecular formula of
2 supported the presence of this additional group. This
group was located at the C-7 position, which was suggested
by the HMBC correlations (Table 1). From these findings,
compound 2 was characterized as 3-methoxy-7-acetylae-
geline (2). Unequivocal evidence for this structure was
obtained by acetylation of 1, which provided an acetyl
1
Genesis Series FT-IR spectrometer, respectively. The H and
13C NMR spectra as well as 2D NMR spectra (COSY, HMQC,
HMBC) were recorded in CDCl3 using Bruker, DRX NMR
spectrometers operating at 400 or 500 MHz for 1H and 100 or
125 MHz for 13C using the solvent peak as internal standard.
The HRMS were measured using a Bioapex FTESI-MS, with

Notes
Journal of Natural Products, 2005, Vol. 68, No. 8 1299
electrospray ionization. A Chromatotron 8924 (Harrison Re-
search, Palo Alto, CA) with silica gel 60 PF 254 (1 mm) plates
was used. TLC analysis was carried out on precoated silica
gel G254 or aluminum oxide ALOX-100 UV254 500 µm plates
with visualization by 5% H2SO4 in EtOH and heating with
Dragendorff’s reagent. Single-crystal-X-ray diffraction mea-
surements were made on a Nonius Kappa CCD diffractometer
with Mo KR radiation.
Plant Material. Leaves of Z. syncarpum Tul. were identi-
fied and collected in Lara State, Venezuela, by one of the
authors (C.L.B.) in February 2002. A voucher specimen, BUR
230202 (1), is deposited at the private herbarium of this author
(518 Audubon, Oxford, MS 38655, e-mail: in_the_200@
mail2farm.com).
Extraction and Isolation. The air-dried and powdered
leaves Z. syncarpum (412 g) were extracted with MeOH at
room temperature. The extract was concentrated under re-
duced pressure to yield 80 g of residue. The methanol extract
(80 g) was subjected to silica gel vacuum liquid chromatogra-
phy (750 g of silica gel, 5.25 µm) and eluted beginning with
n-hexane (100%), n-hexane-chloroform (1:1), chloroform (100%),
and finally ethyl acetate-methanol (10%-100%). Altogether,
25 major fractions (250 mL each) were collected, and the
elution was monitored by TLC.
Fractions 9-17 (6.5 g) were combined and passed over
another silica gel column (250 g), eluted with chloroform-ethyl
acetate (25%-100%), followed by step gradient elution with
ethyl acetate-methanol (10%-100%). In total, 20 fractions
were collected. 3-Methoxyaegeline (1, 30 mg), from fraction 8,
was obtained by recrystallization of an ethyl acetate-methanol
mixture at room temperature. Fraction 17 (3.5 g) was further
chromatographed over an alumina column (38 × 39 cm; 350 g
basic alumina, Sigma-Aldrich) and eluted with chloroform
(100%) and chloroform-methanol (2% to 25%). A total of 17
fractions were collected and were divided into three groups:
group A (1), group B (2-4), and group C (5-17). Group B
(fractions 2-4, 0.9 g) was further fractionated by silica gel
chromatography with chloroform-methanol (0.5%-20%) to
afford 3-methoxy-7-acetylaegeline (2, 28 mg) and 3-methoxy-
7-cinnamoylaegeline (3, 54 mg). Group C (fractions 5-17, 1.4
g) was further purified using the Chromatotron (hexane-
CHCl3-MeOH, 1:92.5:6.5), followed by recrystallization in
ethyl acetate-methanol, to obtain an additional quantity of
3-methoxyaegeline (1, 355 mg).
Conversion of Compound 1 to 2. Treatment of 1 (10 mg)
with acetic anhydride (250 µL) and pyridine (250 µL) at room
temperature for 4 days and subsequent workup gave 2 (8 mg),
with 1H NMR and 13C NMR data identical to natural com-
pound 2.
3-Methoxy-7-cinnamoylaegeline (3): light yellow, amor-
25
phous powder; [R]D 0° (c 0.13, CHCl3); UV (MeOH) λmax 242
(4.83), 252 (4.69), 278 (4.94) nm; IR (KBr) νmax 3472, 2961,
2923, 1713, 1604, 1449, 1268, 757 cm-1; NMR data, see Table
1; HRMS m/z 480.1821 (calcd for C28H27NO5, [M + Na]+,
480.1787).
Evaluation of in Vitro Antiparasitic and Anti-infective
Activities. Antimalarial activity of the compounds was de-
termined in vitro on chloroquine-sensitive (D6, Sierra Leone)
and -resistant (W2, Indochina) strains of P. falciparum. This
96-well microplate assay is based on evaluation of the effect
of the compounds on the growth of asynchronous cultures of
P. falciparum, determined by the evaluation of parasite lactate
dehydrogenase (pLDH) activity.17 The appropriate dilutions
of the compounds were prepared in RPMI 1640 medium and
added to the cultures of P. falciparum (2% hematocrit, 2%
parasitemia) set up in clear flat-bottomed 96-well plates. The
plates were placed in a modular incubation chamber, flushed
with a gaseous mixture of 90% N2, 5% CO2, and 5% O2, and
incubated at 37 °C for 48 h. Growth of the parasite in each
well was determined by the pLDH assay using Malstat
reagent. The medium and RBC controls were included on each
plate. The standard antimalarial agents, chloroquine and
artemisinin, were used as the positive controls, while DMSO
was tested as the negative control.
Acknowledgment. We thank F. T. Wiggers and C. D.
Dunbar, National Center for Natural Products Research, for
NMR and MS, and S. C. Sanders and B. G. Smiley, National
Center for Natural Products Research, for the antimalarial
bioassays.
References and Notes
(1) Zomlefer, W. B. In Guide to Flowering Plant Families; University of
North Carolina Press: Chapel Hill, NC, 1994; pp 145-148.
(2) Burrows, G. E.; Tyel, R. J. Toxic Plants of North America; Iowa State
University Press: Ames, IA, 2001; pp 1082-1085.
(3) Millspaugh, C. F. American Medicinal Plants; Dover Publications
Inc.: New York, 1974; pp 124-128.
(4) Talapatra, S. K.; Dutta, S.; Talapatra, B. B. Phytochemistry 1973,
12, 729-730.
(5) The Wealth of India: Raw Materials; PID, Council of Scientific and
Industrial Research (CSIR): New Delhi, 1976; Vol. II, pp 18-19.
(6) Stone, B. C. In A Revised Handbook to the Flora of Ceylon; Dassan-
ayake, M. D., Fosberg, R., Eds.; Oxford and IBH Publishing: New
Delhi, 1985; Vol. 5, p 406.
(7) Jayaweera, D. M. A. In Medicinal Plants Used in Sri Lanka; National
Science Council: Sri Lanka, 1982; Part 5, p 39.
(8) Weenen, H.; Nkunya, M. H. H.; Bray, D. H.; Mwasumbi, L. B.; Kinabo,
L. S.; Kilimali, V. A.; Wijnberg, J. B. Planta Med. 1990, 56, 371-
373.
(9) Kalia, N. K.; Sing, B.; Sood, R. P. J. Nat. Prod. 1999, 62, 311-312.
(10) Reyes, B.; Navarrete, A.; Sixtos, C.; Aguirre, E.; Jimenez, S.; Estrada,
E. Rev. Mex. Cienc. Farm. 1991, 21, 30-34.
(11) Ross, S. A.; Krishnaveni, K. S.; Satoshi, T.; Burandt, C. L.; Elsohly,
M. A. Phytother. Res. 2005 (communicated).
(12) Ross, S. A.; Sultana, G. N.; Burandt, C. L.; ElSohly, M. A.; Marais,
J. P. J.; Ferreira, D. J. Nat. Prod. 2004, 67, 88-90.
(13) De Morais, S. M.; Facundo, V. A.; Braz Filho, R. J. Essent. Oil Res.
2002, 14, 274-275.
(14) Facundo, V. A.; De Morais, S. M.; Machado, M. I. L.; Matos, F. J. de
A.; Da Frota, L. C. M. J. Essent. Oil. Res. 1999, 11, 426-428.
(15) Facundo, V. A.; De Morais, S. M.; Braz Filho, R.; Matos, I. J. de A;
Souza, R. T. Rev. Bras. Farm. 1997, 78, 57-59.
(16) Swinehart, J. A.; Stermitz, F. R. Phytochemistry 1980, 19, 1219-
1223.
(17) Makler, M. T.; Ries J. M.; Williams J. A.; Bancroft J. E.; Piper R. C.;
Gibbins B. L.; Hinriches D. J. Am. J. Trop. Med. Hyg. 1993, 48,
739-741.
3-Methoxyaegeline (1): white crystals (EtOAc-MeOH),
mp 138-139 °C, [R]D25 0° (c 0.11, CHCl3); UV (EtOH) λmax (log
ꢀ) 218 (4.37), 225 (4.57), 278 (4.65) nm; IR (KBr) νmax 3337,
2920, 2851, 2358, 2339, 1713, 1656, 1615, 1540, 1516, 1451,
1261, 1231, 1027, 977 cm-1; NMR data, see Table 1; HRMS
m/z 328.1546 (calcd for C19H21NO4, [M + H]+, 328.1543).
X-ray Crystal Structure Determination of 1. A suitable
crystal of 1 was obtained by slow crystallization from EtOAc-
MeOH (1:1) at room temperature for 3 days. Crystal data:
C19H21NO4, monoclinic space group P21, a ) 11.684(3) Å, b )
9.293(2) Å, c ) 15.710(5) Å, â ) 102.586(10)°, V ) 1664.8(8)
Å3, Z ) 4, R ) 0.048 (F2>2σ), Rw ) 0.103 (all F2) for 5335
unique data having 2θ < 61° and 450 refined parameters.
Crystallographic data for the structure reported in this paper
have been deposited with the Cambridge Crystallographic
Data Centre and allocated the deposition number CCDC
274925. Copies of the data can be obtained, free of charge, on
application to the Director, CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: deposit@
ccdc.cam.ac.uk).
3-Methoxy-7-acetylaegeline (2): light yellow, amorphous
25
powder; [R]D 0° (c 0.23, CHCl3); UV (MeOH) λmax (log ꢀ) 242
(4.82), 252 (4.67), 282 (4.98) nm; IR (KBr) νmax 3368, 3281,
2953, 1713, 1659, 1517, 1450, 1263, 1162, 1026, 975, 766 cm-1
;
NMR data, see Table 1; HRMS m/z 392.1462 (calcd for C21H23-
NO5, [M + Na]+, 392.1474).
NP0580558