2011

NPC Natural Product Communications Vol. 6

No. 11
Isolation, Structure Elucidation, and Biological Activity of a 1595 - 1596
New Alkaloid from Zanthoxylum rhetsa
Karsten Krohna*, Stephan Cludius-Brandta, Barbara Schulzb, Mambatta Sreelekhac
and Pottachola Mohamed Shafic*
a
Department of Chemistry, University of Paderborn, Warburger Str. 100, 33098 Paderborn, Germany
b
Institute of Microbiology, University of Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany
c
Department of Chemistry, Calicut University, Kerala 673 635, India
k.krohn@uni-paderborn.de

Received: March 21st, 2011; Accepted: June 28th, 2011

Several biologically active alkaloids (1-4, 6), including a new quinazoline-6-carboxylic acid (1), were isolated from the medicinal plant
Zanthoxylum rhetsa, an evergreen tree, native to subtropical areas. Whereas the pharmacological properties of the plant extract and single
constituents have been widely tested, we now show that all of the metabolites have antialgal activities, all but 6 are antibacterial, and 6 and
the reduction product 5 (derived from 4) are also antifungal.

Keywords: Zanthoxylum rhetsa, alkaloids, biological activity.

Zanthoxylum rhetsa is a deciduous and evergreen tree, O 10 9 O O

1
12a 12 10a 8 N

which belongs to the Rutaceae family. The essential oil

2 N O
6a 7 Me
3 N N N O
(mulillam oil) obtained from the fruits is used to treat
4a N5 5a 6 H MeO OMe
4 H 1'
OH MeO
O OMe
cholera [1], asthma, toothache and rheumatism [2]. In the 1 2 3

present paper we report the isolation and biological OMe O OMe OH

MeO
OH
N O
activity of several alkaloids from Z. rhetsa. In addition to
MeO MeO
N N

the new quinazoline-6-carboxylic acid (1), the known 1-

O O
OMe

methoxy-7,8-dehydrorutaecarpine (2) [3a], arnottianamide

O O
4 5 6

(3) [3a,3d,3e,4a], 6-acetonyldihydro-chelerythrine (4) Figure 1: Compounds isolated from Zanthoxylum rhetsa (1-4, 6) and reduction
product 5.
[3b,3c,4b], and skimmianine (6) [3a,3e,4c,4d] were isolated.
Compounds 2, 3, 4 and 6 are known to exhibit cytotoxic Table 1: Biological activities of pure compounds 1-6 against microbial test
activity [3a]. In addition, 6-acetonyldihydrochelerythrine organisms in an agar diffusion assay.
(4) shows anti-HIV [3b] and antioxidant [3c] activity. Metabolites/ Escherichia Bacillus Microbotryum Chlorella
control substances coli megaterium violaceum fusca
1 8 pi1 6 0 5
Compound 1 was isolated as a light yellow powder (m. p. 2 7 pi 8 0 8
313°C) with the molecular formula C16H10N2O3 and a 3 0 pi 6 0 10
4 10 8 0 10
pseudomolecular ion peak at m/z 279.02437 [M+H]+ in the 5 10 0 5 7
HREIMS. The 1H NMR spectrum, in combination with the 6 0 0 8 10
acetone 0 0 0 0
data from the COSY experiment, revealed the presence of penicillin 14 +8 pi 18 0 0
two ortho-disubstituted benzene rings, one at δ 7.59 (1H, t, tetracycline
nystatin
18 + 16
0
18
0
0
20
10 pi
0
J = 7.1 Hz, H-8), 7.85 (1H, d, J = 8.3 Hz, H-10), 7.91 (1H, actidione 0 0 50 35
t, J = 6.9 Hz, H-9), and 8.17-8.19 (1H, m, H-7), and the 50 µg of test substance/test filter disc (50 µL at a concentration of 1 µg/µL) of the
pure substances and of the control substances were tested in an agar diffusion assay;
other at δ 7.28 (1H, t, J = 7.2 Hz, H-2), 7.37 (1H, t, J = 7.2 radius of zone of inhibition is given in mm. 1pi = partial inhibition, i.e. there was
Hz, H-3), 7.71 (1H, d, J = 8.2 Hz, H-4), and 8.19-8.21 (1H, some growth within the zone of inhibition.
m, H-1). The proton H-1 showed HMBC correlation with
an amido carbon (C-12, δ 161.4). A singlet at δ 12.79 Therefore, the structure of compound 1 was determined as
displayed no HMQC but NOE correlation with H-4, 5,12-dihydro-12-oxoindolo[2,1-b]quinazoline-6-carboxylic
indicating the existence of a hydroquinazolinone unit in acid, as shown in Figure 1. Compounds 2, 3, 4 and 6 were
compound 1. H-7 correlated with the olefinic carbon C-6 identified through comparison of their spectral data with
(δ 108.1), indicating the presence of an indole unit. A those known from the literature. To confirm the structure
broad singlet at δ 14.53 (1H, s, COOH) was typical for the of compound 4, its reduction with NaBH4 was performed
presence of a carboxyl group (δ 169.8) attached to C-6. affording the racemic alcohol 5. Since 5 is a hitherto
1596 Natural Product Communications Vol. 6 (11) 2011 Krohn et al.

1
unknown compound it was also subjected to the H NMR (500 MHz, DMSO-d6): 7.28 (1H, t, J = 7.2 Hz,
subsequent biological tests. Ar), 7.37 (1H, t, J = 7.2 Hz, Ar), 7.59 (1H, t, J = 7.1 Hz,
Ar), 7.71 (1H, d, J = 8.2 Hz, Ar), 7.85 (1H, d, J = 8.3 Hz,
Biological activity: All six metabolites were biologically Ar), 7.91 (1H, t, J = 6.9 Hz, Ar), 8.17-8.21 (2H, m, Ar),
active (Table 1); all but 6 were antibacterial, inhibiting the 12.79 (1H, br s, NH), 14.53 (1H, br s, COOH).
Gram-negative bacterium, Escherichia coli, and / or the 13
C NMR (125 MHz, DMSO-d6): 108.1 (C), 113.5 (CH),
Gram-positive bacterium, Bacillus megaterium. 122.1 (C), 122.9 (CH), 123.1 (CH), 125.3 (CH), 126.4
Metabolites 5 and 6 inhibited the fungal test organism, (CH), 127.5 (2 CH), 127.8 (C), 134.7 (C), 135.2 (CH),
Microbotryum violaceum, and all six substances were 136.1 (C), 148.9 (C), 161.4 (C), 169.9 (C).
active against the alga, Chlorella fusca. MS (EI, 70 eV): m/z (%) = 279 [M + H]+ (8), 200 (22), 169
(63), 114 (95), 86 (43), 70 (100), 42 (31). HREIMS: Calcd.
Experimental for C16H10N2O3: 278.06914; found: 279.02437.
General: CC, silica gel 60 (particle size 0,040-0,063 nm); Anal. Calcd for C16H10N2O3: C, 69.04; H, 3.62; N, 10.07.
Melting points, Büchi SMP-20 melting point apparatus, Found C, 68.85; H, 3.59; N, 9.98.
uncorrected; NMR, Bruker ARX 500; MS, FINNEGAN
MAT 8200 and FISON MD 800. 6-(2-Hydroxypropyl)-dihydrochelerythrine (5): A
solution of 4.0 mg of 4 in 1 mL of ethanol was treated with
Plant material: Spines on the bark of Zanthoxylum rhetsa 1 mg of NaBH4 and stirred for 1 h and worked up as usual
(Roxb.) DC. (5 kg) were collected during January 2007 to afford 4.0 mg of 5, as a white solid.
from Calicut University campus, Kerala, India. The plant MP: 210°C
was authenticated by Dr A. K. Pradeep, Calicut Univeristy. 1
H NMR (500 MHz, CDCl3): 1.01 (3H, d, J = 6.3 Hz, Me),
A voucher specimen (No.34174) is deposited at the Calicut 1.38-1.62 (2H, m, CH2), 2.69 (3H, s, NCH3), 3.93 (3H, s,
University herbarium (CALI). OMe), 3.95 (3H, s, OMe), 4.02-4.10 (1H, m, -C(OH)H-),
Extraction and isolation: The dry spines were coarsely 4.61-4.85 (1H, m, -CHCH2-), 6.05 (2H, s, -OCH2O-), 6.95
powdered and extracted thrice with 7 L each of boiling (1H, d, J = 8.5 Hz, Ar), 7.11 (1H, s, Ar), 7.47-7.54 (3H, m,
light petroleum (60-80oC) and then with acetone in the Ar), 7.70 (1H, d, J = 8.6 Hz, Ar).
13
same manner. The acetone extract was concentrated and C NMR (125 MHz, CDCl3): 23.2 (CH3), 41.5 (CH2),
chromatographed using mixtures of light petroleum – 42.9 (NCH3), 55.9 (CH3), 58.5 (CH), 61.2 (OCH3), 68.0
EtOAc. The following compounds were isolated: 8- (CH), 99.6 (CH), 101.1 (CH2), 104.6 (CH), 111.8 (CH),
acetonyldihydrochelerythrin (4): from the fraction eluting 119.2 (CH), 119.7 (CH), 123.7 (C), 123.9 (CH), 124.7 (C),
with 20% ethyl acetate (800 mg); arnottianamide (3): from 127.3 (C), 128.1 (C), 131.2 (C), 139.2 (C), 145.5 (C),
the fraction eluting with 33% ethyl acetate (15 mg); 7,8- 147.7 (C), 148.7 (C), 152.2 (C).
dihydro-1-methoxyrutaecarpine (2): from the fraction MS (EI, 70 eV): m/z (%) = 407 [M]+ (69), 348 (100), 290
eluting with 33% ethyl acetate, and after isolating (75), 185 (72), 157 (33). HREIMS: Calcd. for C24H25NO5:
arnottianamide (10 mg); skimmianine (6): from the 407.17326; found: 407.04189.
fraction eluting with 33% ethyl acetate, and after isolating Supplementary data: Spectroscopic data of compounds
7,8-dihydro-1-methoxyrutaecarpine (31 mg); 12-oxo-5- 2-4 and 6.
hydroindolo[2,1-b]quinazoline-6-carboxylic acid (1): from
the fraction eluting with 50% ethyl acetate (11 mg). Acknowledgement - P. M. Shafi is thankful to UGC (SAP
programme), New Delhi, India for financial assistance; we
5,12-Dihydro-12-oxoindolo[2,1-b]quinazoline-6- thank Monika Beltau for technical assistance.
carboxylic acid (1)
MP: 313°C.
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