Syncarpamide, a New Antiplasmodial (+)-Norepinephrine Derivative from Zanthoxylum syncarpum

Article abstract of DOI:10.1021/np030417t

A new (+)-norepinephrine derivative, syncarpamide (1), along with a known coumarin, (+)-S-marmesin (2), and one known alkaloid, decarine (3), have been isolated from the stem of Zanthoxylum syncarpum. The structure of compound 1 was elucidated on the basis of 1D and 2D NMR, MS, IR, optical rotation, and CD analyses. Its absolute stereochemistry was elucidated by synthesis of its enantiomer and subsequent comparison of CD data. Characterizations of compounds 2 and 3 were based on spectral analysis and comparison with reported data. Compounds 1 and 3 showed antiplasmodial activity, with IC₅₀ values of 2.04 and 1.44 μM against Plasmodium falciparum D₆ clone and 3.06 and 0.88 μM against P. falciparum W₂ clone, respectively. Compound 3 showed cytotoxicity at 56.42 μM, whereas compound 1 was not cytotoxic at 10.42 μM. Compound 1 was tested for hypotensive activity, but no activity was observed. Compound 2 showed no antiplasmodial or antimicrobial activities.

Full text of DOI:10.1021/np030417t

8
8
J . Nat. Prod. 2004, 67, 88-90
Syn ca r p a m id e, a New An tip la sm od ia l (+)-Nor ep in ep h r in e Der iva tive fr om
Za n th oxylu m syn ca r pu m
Samir A. Ross,*,†,‡ Gazi N. N. Sultana, Charles L. Burandt, Mahmoud A. ElSohly, J annie P. J . Marais, and
†
†
†,§
†
Daneel Ferreira†
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, Department of Pharmacognosy,
and Department of Pharmaceutics, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848
Received September 16, 2003
A new (+)-norepinephrine derivative, syncarpamide (1), along with a known coumarin, (+)-S-marmesin
(2), and one known alkaloid, decarine (3), have been isolated from the stem of Zanthoxylum syncarpum.
The structure of compound 1 was elucidated on the basis of 1D and 2D NMR, MS, IR, optical rotation,
and CD analyses. Its absolute stereochemistry was elucidated by synthesis of its enantiomer and
subsequent comparison of CD data. Characterizations of compounds 2 and 3 were based on spectral
analysis and comparison with reported data. Compounds 1 and 3 showed antiplasmodial activity, with
IC50 values of 2.04 and 1.44 µM against Plasmodium falciparum D
P. falciparum W clone, respectively. Compound 3 showed cytotoxicity at 56.42 µM, whereas compound
was not cytotoxic at 10.42 µM. Compound 1 was tested for hypotensive activity, but no activity was
observed. Compound 2 showed no antiplasmodial or antimicrobial activities.
6
clone and 3.06 and 0.88 µM against
2
1
The genus Zanthoxylum (Rutaceae) comprises some 200
species distributed worldwide. Plants of this genus ex-
hibit a variety of biologically active secondary metabo-
lites including alkaloids, lignans, and coumarins with
Stems of Z. syncarpum were air-dried, ground, and
sieved through a 1.0 mm mesh. An ethanolic extract of this
material was carefully chromatographed over a silica gel
column. The collected fractions were further purified by
repeated chromatography over open silica gel columns,
reversed-phase HPLC, and crystallization to afford com-
pounds 1, 2, and 3.
1
febrifuge, sudorific, and diuretic properties. The bark of
Zanthoxylum integrifoliolum (Merr.) Merr. (Rutaceae)
has been utilized in traditional medicine by Ya-Mei ab-
origines in Lanyu Island in Taiwan as a remedy for snake-
2
3
bite and dyspepsia and as an aromatic tonic in fever. In
Sri Lanka, the genus Zanthoxylum is represented by three
species, viz., Z. caudatum, Z. rhetza, and Z. tetraspermum.4
Of these, Z. tetraspermum (Sinhala-katukeena) is em-
ployed in folklore medicine for the treatment of rheuma-
5
tism and some forms of diarrhea. Pure compounds with
antimicrobial activity have been obtained from the root
6
bark of Z. gilletii. The bark is pungent, and sticks prepared
7
from it are used for preventing toothache. The stem bark
of Z. liebmanniaum (Engelm.) P. Wilson is used in Mexican
traditional medicine for the treatment of stomach aches,
amebiasis, and intestinal parasites and as a local anes-
thetic agent.8
Z. syncarpum Tul. is an evergreen tree occurring in both
North and South America.9 Although there are several
reports on the chemical constituents of different species of
Zanthoxylum,3 only two papers dealt with the constitu-
-8
ents of Z. syncarpum.1
0,11
These phytochemical studies
revealed the presence of two alkaloids, skimmianine and
cis-N-methylcanadine, two coumarins, xanthotoxin and
isopimpinellin, a diterpene, centipedic acid, the flavonoid
1
0
ternatin, the triterpene lupeol, â-sitosterol glucoside, and
an essential oil.11
This paper deals with the isolation and characterization
of a new alkaloid (1) and also the first report of the presence
of 2 and 3 in Z. syncarpum. The isolation of these types of
alkaloids and coumarins is of chemotaxonomic importance,
as structurally related alkaloids have been reported earlier
1
2,13
from Fagara and Zanthoxylum species.
Syncarpamide (1) was obtained as light brown solid, [R]
+12.5°, which gave a positive test with Dragendorff’s
reagent. The molecular formula was established as C28
NO by HRESIMS with 16 degrees of unsaturation. Analy-
D
*
To whom correspondence should be addressed. Tel: (662) 915-5928.
Fax: (662) 915-5587. E-mail: sross@olemiss.edu.
27
H -
†
National Center for Natural Products Research.
Department of Pharmacognosy.
Department of Pharmaceutics.
‡
5
§
1
1
1
sis of H NMR data, supported by H- H COSY spectra,
1
0.1021/np030417t CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/18/2003

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 1 89
revealed the presence of two pairs of trans olefinic systems
at δ 6.38 (1H, d, J ) 15.5 Hz), 7.63 (1H, d, J ) 15.5 Hz),
and δ 6.50 (1H, d, J ) 16.0 Hz), 7.72 (1H, d, J ) 16.0 Hz).
-
1
The IR absorption band at 1713 cm indicated the pres-
ence of a carbonyl group, and corresponding carbon peaks
1
3
at δ 166.0 and 166.5 in the C NMR spectra were
reminiscent of an amide and an ester bond in 1. In the
HMBC spectra, the first pair of trans olefinic protons
showed cross-peaks with the carbonyl carbon at δ 166.0
and the second pair with the carbon at δ 166.5. Analysis
1
13
of H and C NMR data also revealed that 1 contained a
,3,4-trisubstituted and two monosubstituted benzene
1
rings. HMBC connectivity linked each pair of olefinic
protons to a 1-substituted benzene ring. Two O-methyl
singlets at δ 3.87 and 3.90 of the 1, 3, 4-trisubstituted
benzene rings were observed in the 1H NMR spectrum.
Cross-peaks in the HMBC spectrum indicated that these
methoxy groups are attached to the aromatic carbons at δ
F igu r e 1. CD spectra of syncarpamide 1 and its (-)-R-norepinephrine
enantiomer 7.
1
6
49.1 and 149.2, respectively. One more spin system at δ
.00 (1H, t, J ) 6.4 Hz), δ 74.7, and δ 3.88 (2H,
44.7 was observed.
The singlet aromatic resonance of the 1,3,4-trisubstituted
benzene ring at δ 6.96 (δ 109.8) showed a cross-peak in
the HMBC spectra to the oxymethine signal at δ 6.00 of
the aliphatic spin system. The characteristic deshielded
secondary carbon at δ 44.7 of the latter spin system
indicated that it was connected to the nitrogen atom. A
deshielded oxymethine proton resonance at δ 6.00 also
H
having IC50 values of 2.04 and 1.44 µM against the D
strain and 3.06 and 0.88 µM against the W strain,
These compounds showed no cytotoxicity,
6
C
H
2
respectively.2
2,23
overlapped with methoxy signals), δ
C
except decarine at high concentration (56.42 µM). Decarine
isolated from Z. tetraspermum and Z. coudatum showed
significant antibacterial activity.20 Marmesin (2) was not
active in either the antimicrobial or antiplasmodial screen.
H
C
H
C
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured on a RUDLPH Research Autopol IV automatic
polarimeter. UV spectra were recorded using a Hewlett-
Packard 8453 instrument. IR spectra were obtained using an
H
indicated O-cinnamoylation at this position. Thus the
structure of syncarpamide was elucidated as shown in 1.
It bears some resemblance to the naturally occurring
1
13
aegline 4, which was obtained in racemic form.1
3,14
AATI Mattson Genesis Series FTIR. H and C NMR spectra
as well as 2D spectra (COSY, HMQC, and HMBC) were
recorded on a Bruker DRX 400 spectrometer operating at 400
MHz for 1H and 100 MHz for C using the solvent peaks as
internal references. EIMS was acquired on a GC/MS: Hewlett-
Packard 5890A gas chromatography (GC) interface with 5970A
selection detector (MSD). The GC/MS system was fitted with
a DB-1 column (15 m × 0.25 mm, and 0.25 µm film thickness,
J & W Scientific, Folsom, CA), and HRESIMS and ESIMS data
were acquired on a Bruker BioAPEX 30es mass spectrometer.
TLC was performed on Merck TLC plates precoated with Si60
13
The positive specific rotation of syncarpamide (1) indi-
cated that it was probably a derivative of (+)-S-norepi-
nephrine. To confirm the absolute configuration of (1), we
opted to derivatize commercially available (-)-R-norepi-
nephrine (5) with a view to comparing its chiroptical
properties with those of syncarpamide (1). Thus, methyl-
ation of the sparingly soluble (-)-R-norepinephrine (5) with
ethereal diazomethane in a large excess of MeOH afforded
F
H
254 or Si60 RP18 F254 with visualization by spraying with 5%
SO in EtOH and heating or by Dragendorff’s reagent.
2
4
HPLC was carried out on a Waters Millennium system with
a 996 photodiode array detector. CD spectra were recorded in
CHCl (ca. 0.1 mg/mL CHCl ) on a J ASCO J -715 spectrometer;
3
3
scan parameters: bandwidth (2.0 nm), sensitivity (10 mdeg),
response (4 s), scan speed (50 nm/min), step resolution (0.1
nm).
(-)-R-3,4-di-O-methylnorepinephrine (6). Treatment with
cinnamoyl chloride (2.1 equiv) in pyridine gave the N,O-
dicinnamoyl derivative (7). Its CD spectrum exhibited
sequential high-amplitude positive and negative Cotton
effects in the 290-300 and 260-270 nm regions for the
nfp* and πfp* transition of the R,â-unsaturated carbonyl
chromophores, respectively. The CD spectrum of syncar-
pamide (1) showed a mirror image type relationship to that
of 7 (Figure 1), hence unequivocally confirming the S-
absolute configuration of the new naturally occurring (+)-
norepinephrine derivative (1).
P la n t Ma ter ia l. Specimens of Z. syncarpum were identified
and collected in Lara State, Venezuela, by Dr. Charles L.
Burandt in February 2002. Dried and ground stems were used
for the isolation. A voucher specimen, BUR230202 (2), is
deposited at the private herbarium of Dr. Burandt.
Extr a ction a n d Bioa ssa y. The powdered, air-dried stems
(
1.2 kg) were exhaustively extracted with MeOH (3 × 2 L).
The combined MeOH extracts were evaporated, and the
residue (63 g, 5.3% yield of dry plant weight) was chromato-
graphed on a silica gel column (380 × 78 mm, 540 g of silica
32-63 µm, 100:0:0-0:0:100 hexanes-EtOAc-MeOH step
gradient) to give eight fractions, C
F
F
Compound 2 was identified as (+)-S-marmesin by com-
1
-F
1
, C
1
-F
2
, C
1
-F
3
, C
1
-F
4
, C
1
-
-
5
, C
to C
1
-F
1
6
, C
-F
1 7 1 8
-F , and C -F (1 L each). Combined fractions C
1
parison of IR, HREIMS, and 1D and 2D NMR spectral data
with published values.15,16
2
4
(6.23 g) were further chromatographed on silica
gel (280 × 35 mm column, 160 g of silica 32-63 µm, eluted
with 100:0-0:100 CHCl -MeOH step gradient) to give 10
fractions. Fraction C -F (1.83 g) was further chromatographed
Compound 3 was identified as decarine by comparison
3
of IR, HREIMS, and 1D and 2D NMR spectral data with
2
5
1
7,18
published values.
Decarine was also isolated from Z.
on silica gel (150 × 35 mm column, 60 g of silica 32-63 µm,
eluted with 98:2:0-0:0:100 hexane-EtOAc-MeOH step gradi-
ent) to give fractions F -1, F -2, F -3, F -4, F -5, and F -6.
decaryi18 and Z. viride.19
Syncarpamide (1) and decarine (3) showed strong in vitro
antiplasmodial activity21 against Plasmodium falciparum,
5
5
5
5
5
5
Fraction F -3 was crystallized from an EtOAc-hexane mixture
5

9
0
J ournal of Natural Products, 2004, Vol. 67, No. 1
Notes
derivative (7). Its H NMR spectrum was identical to that of
1
to afford compound 3 (44.0 mg). Fraction F -4 was crystallized
from a MeOH-EtOAc mixture to give compound 2 (8.5 mg),
and the mother liquid was subjected to reversed-phase HPLC
5
3
the natural product (1), CD (CHCl
3
) [θ]298.6 -12.5 × 10 , [θ]264.3
3
+23.5 × 10
(
Prodigy ODS-3, 5 µm, 21.2 × 250 mm column, 70:30 (v/v)
Ma r m esin (2): white crystals, [R] 25 +9.63° (c 0.166,
D
-
1
1
13
MeOH-H
2
O, 14 mL min , photodiode-array detection moni-
CHCl ); IR, UV, HREIMS, and H and C NMR data identical
3
tored at 220 nm) separation to afford compound 1 (5.9 mg).
L-Norepinephrine and cinnamoyl chloride were purchased
from Acros Organics, NJ . Diazald, 99% (N-methyl-N-nitroso-
p-toluenesulfonamide) was purchased from Aldrich Chemical
Co. Inc.
to literature value.16
Deca r in e (3): yellow crystals; UV, HREIMS, 1H, 13C NMR
17-19
data identical to reference values.
Ack n ow led gm en t. The authors acknowledge the United
States Department of Agriculture, Agriculture Research Ser-
vice Specific Cooperative Agreement No. 58-6408-2-0009, for
partial support of this work. The authors are also grateful to
Dr. D. G. Nagle and Dr. C. F. Hossain (Department of
Pharmacognosy, School of Pharmacy, University of Missis-
sippi) for allowing the use of the HPLC facility and Dr. D. C.
Dunbar (NCNPR, University of Mississippi) for the HRESIMS
analysis.
Cytotoxicity Assa y. In Vitr o Cytotoxic Activity. The
level of toxicity of each sample was determined by measuring
the effect on a fibroblast cell line from African green monkey
kidney (VERO, nontransformed). For initial (primary) evalu-
ation, extracts and fractions were screened at a single con-
centration (100 µg/mL). Follow-up secondary assays were
conducted at three concentrations (4.76, 1.58, and 0.52 µg/mL),
using a culture-treated 96-well microplate 16. For secondary
assay, IC50 values were determined from logarithmic graphs
of growth inhibition values. The cytotoxic agents doxorubicin
and 5-fluorouracil were used as positive controls, while DMSO
was used as the negative (vehicle) control.
An tim icr obia l Assa y. Preliminary antimicrobial activities
of the crude exctracts/fractions and the IC50/MIC values of
compounds were determined using a modified 96-well micro-
plate assay protocol. The test organisms used were ATCC
strain of Candida albicans B311 (#90028), Cryptococcus neo-
formans (#90113), Stephylococcus aureus (#6535), and S.
aureus (# 33591). Amphotericin B and rifampin were used as
positive controls, with DMSO as a negative control.
Refer en ces a n d Notes
(
(
(
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5
Syn ca r p a m id e (1): light brown solid, [R]
CHCl ); IR (film) νmax 3472, 2961, 2923, 1713, 1604, 1449, 1268,
3
57 cm ; UV λmax (MeOH) 242, 250, 278 nm; CD (CHCl )
D
+12.50° (c 0.08,
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7
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2
002, 14, 274-275.
(
(
(
10) Facundo, V. A.; De Morais, S. M.; Braz Filho, R.; Matos, I. J . de A;
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(
(
1H, m, H-3′′), 7.35 (1H, m, H-4′′), 7.35 (1H, m, H-5′′), 7.49
1H, m, H-2′′), 7.49 (1H, m, H-6′′), 7.49 (1H, m, H-2′), 7.49 (1H,
1
47.
m, H-6′), 7.63 (1H, d, J ) 15.5 Hz, H-7′′), 7.72 (1H, d, J ) 16.0
(13) Swinehart, J . A.; Stermitz, F. R. Phytochemistry 1980, 19, 1219-
1
3
1223.
Hz, H-7′); C NMR (CDCl
OCH -4), 56.0 (q, OCH -3), 74.7 (d, C-7), 109.8 (d, C-2), 111.2
d, C-5), 117.6 (d, C-8′), 119.1 (d, C-6), 120.3 (d, C-8′′), 127.9
3
, 100 MHz) δ 44.7 (t, C-8), 55.9 (q,
(
14) Chatterjee, A.; Bose, S.; Srimany, S. K. J . Org. Chem. 1959, 24, 687-
3
3
6
90.
(
(
(
(
(
(
(
(15) Warren, S. Can. J . Chem. 1971, 49, 2297-2301.
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d, C-2′′), 127.9 (d, C-6′′), 128.2 (d, C-2′), 128.2 (d, C-6′), 128.8
d, C-3′), 128.8 (d, C-5′), 128.9 (d, C-3′′), 128.9 (d, C-5′′), 129.8
s, C-1), 130.3 (d, C-4′), 130.6 (d, C-4′′), 134.2 (s, C-1′), 134.7
s, C-1′′), 141.6 (d, C-7′′), 145.8 (d, C-7′), 149.2 (s, C-4), 149.2
(
17) Krane, B. D.; Fagbule, M. O.; Shamma, M. J . Nat. Prod. 1984, 47,
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+
(19) Waterman, P. G. Phytochemistry 1975, 14, 843-844.
calcd for C28
H
27NO
5
Na, [M + Na] , 480.1787).
(
20) Nissanka, A. P. K.; Karunaratne, V.; Bandara, B. M. R.; Kumar, V.;
Nakanishi, T.; Nishi, M.; Inada, A.; Tillekeratn, L. M. V.; Wije-
sundara, D. S. A.; Gunatilaka, A. A. L. Phytochemistry 2001, 56, 857-
Syn th esis of (-)-R-Nor ep in ep h r in e Der iva tive (7).
Diazomethane was added to a solution of l-norepinephrine 5
100 mg) in MeOH (200 mL), and the mixture was cooled to
8
61.
(
(
21) Dou, J .; McChesney, J . D.; Sindelar, R. D.; Goins, D. K.; Walker, L.
A. J . Nat. Prod. 1996, 59, 73-76.
-
15 °C. The mixture was kept 72 h at -15 °C. A second batch
of diazomethane was added, and the mixture was kept for a
(
22) 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,
further 72 h at -15 °C. The solvent was evaporated with a
strong stream of N .
2
The crude methylated product (6) was dried and dissolved
in pyridine (2 mL). Cinnamoyl chloride (207 mg, 2.1 equiv)
was added, and the reaction mixture was gently heated (40 to
7
39-741.
23) Makler, M. T.; Hinriches, D. J . Am. J . Trop. Med. Hyg. 1993, 48, 205-
10.
(
2
(24) National Committee for Clinical Laboratory Standards. Methods for
Dilution. Antimicrobial Susceptibility Test for Bacteria that Grow
Aerobically. Approved Standard M7-A, 4th ed.; National Committee
for Clinical Laboratory Standards: Wayne, PA, 1997.
4
5 °C) for 16 h. The excess pyridine was removed by a strong
stream of N . The mixture was separated by preparative TLC
using CHCl -MeOH, 99:1, to give the (-)-R-norepinephrine
2
3
NP030417T