Antimicrobial activity of 9-O-acyl- and 9-O-benzoyl-substituted berberrubines

Article abstract of DOI:10.1055/s-2000-8536

In the course of a structure-activity relationship study on berberrubine derivatives, a series of compounds bearing 9-O-acyl- (4-6) and 9-O-benzoyl- (7) substituents was synthesized with the expectation of increasing the antimicrobial activity. One of the

Full text of DOI:10.1055/s-2000-8536

Letters
Planta Med. 66 (2000) 361
Antimicrobial Activity of
9-O-Acyl- and 9-O-Benzoyl-
Substituted Berberrubines
Sa Weon Hong1, Sung Han Kim1, Jung Ae Jeun1,
Sang Jun Lee2, Sung Uk Kim2, Jung Han Kim1,*
1 Department of Biotechnology, Bioproducts Research Center,
Yonsei University, Seoul, Korea
2 Plant Protectants RU., Korea Research Institute of Bioscience and
Biotechnology, Taejon, Korea
Revciesiivoenda: cJucleyp2te8d,:1N99o9v;emAcbceerp2te1d, :19N9o9v;eRmebceriv2e1d,:1J9u9ly928, 1999
Fig.1 The structures of berberine, berberrubine, andberberrubine
derivatives.
Abstract: In the course of a structure-activity relationship study
on berberrubine derivatives, a series of compounds bearing 9-
O-acyl- (4±6) and 9-O-benzoyl- (7) substituents was synthe-
sized with the expectation of increasing the antimicrobial activ-
ity. One of the berberrubine derivatives, 9-lauroylberberrubine
chloride was the most active against Gram-positive bacteria En-
terococcus faecalis, Staphylococcus aureus, Staphylococcus epider-
midis, Micrococcus luteus, Bacillus subtilis as well as the Gram-
negative bacterium Klebsiella pneumoniae in comparison to ber-
berine, the currently used antibiotic in clinic. This result sug-
gested that the presence of lipophilic substituents of certain
structures and sizes might be crucial for the optimal antimicro-
bial activity.
Table 1 NMR data for 9-valeryl- (5) and9-lauroylberberrubine chlor-
ides (6).
5
6
5
6
No.
1H
1H
13C
13C
1
7.81
±
7.81
±
105.39
130.66
149.36
149.76
108.28
120.19
26.32
55.27
145.11
137.89
147.49
170.38
32.88
26.32
21.54
13.69
±
105.41
130.66
149.38
149.75
108.28
120.20
26.19
1a
2
±
±
3
±
±
4
7.10
±
7.09
±
4a
Berberine, an alkaloid derived from the plant Berberis aristata
L. (Berberidaceae), has been shown to possess some antibac-
terial action, local anaesthetic action, antiheparin, anticoagu-
lant action and antitrachoma action (1±4). Berberrubine is 9-
demethylberberine, an alkaloid isolated from Berberis vulgaris
L., and is readily derived from berberine (5). Berberine had no
antitumor activity, but berberrubine and the ester derivatives
of berberrubine were reported to have a strong antitumor ac-
tivity (6). We reported previously that berberrubine repre-
sents a new class of antitumor agent which exhibits the topo-
isomerase II poison activity as well as catalytic inhibition ac-
tivity and may have a potential clinical value in cancer treat-
ment (7). Furthermore, as shown in Fig.1, several berberru-
bine derivatives and new acyl derivatives (5 and 6) were syn-
thesized to study the correlation between their structures and
biological activities. In this paper, we report the syntheses
and the antimicrobial activities of 9-O-acyl- and 9-O-benzoyl-
berberrubine derivatives.
5
3.22
4.95
9.93
±
3.22
4.93
9.98
±
6
55.20
8
144.24
137.88
147.49
170.39
33.22
8a
9
±
±
9-OCO
9-Valeryl
or
±
±
2.87
1.73
1.48
0.97
±
2.87
1.73
1.44
1.23±
0.84
±
24.29
9-Lauroyl
24.42
28.36
28.44
±
±
28.74
±
±
±
28.78
±
±
±
28.87
±
±
±
28.99
±
±
±
29.03
±
±
±
13.99
10
±
±
150.15
57.17
126.47
125.72
133.38
120.19
136.37
102.00
150.15
57.15
The structures of the new acyl derivatives (5 and 6) were de-
termined by NMR and MS experiments. Full signal assign-
ment of 1H and 13C was carried out with various NMR tech-
niques including DEPT, COSY, H,C-COSY, and long-range H,C-
10-OMe
11
4.03
8.20
8.28
±
4.02
8.21
8.28
±
126.49
125.70
133.40
120.42
136.37
102.00
12
1
COSY. The complete assignment of H and 13C chemical shifts
12a
for 9-valeryl- (5) and 9-lauroylberberrubine (6) chlorides are
described in Table 1.
13
9.04
±
9.06
±
13a
OCH2O
6.18
6.17
The biological activities of the compounds tested against a
panel of microorganisms are summarized in Tables 2 and 3.
The antibacterial activities of compounds 3±7 were compared
Planta Medica 66 (2000) 361±363
ꢀ Georg Thieme Verlag Stuttgart New York
ISSN: 0032-0943
·

362 Planta Med. 66 (2000)
Letters
with those of berberine (1) and berberrubine (2). The activi-
ties of the 9-O-acyl- and 9-O-benzoylberberrubine derivatives
against Gram-positive bacteria were increased as the length
of the chain at the C-9position increased. However, only 9-
lauroylberberrubine chloride showed inhibitory activity
against Gram-negative bacteria. Compounds 2, 3, and 4 have
no activity at 128mg/ml against any strains tested. Com-
pounds 5 and 7 exhibited weaker activity than kanamycin sul-
fate against Gram-positive bacteria and proved to be inactive
against Gram-negative bacteria (>128mg/ml). In particular,
compound 6, 9-lauroylberberrubine chloride, showed less in-
hibitory activity against Gram-positive bacteria by 1/2±1/8
than kanamycin sulfate, while it exerted stronger activity
against the Gram-negative bacterium K. pneumoniae (MIC 8)
than kanamycin sulfate (Table 2).
ed the highest activity against T. mentagrophytes, C. immitis,
and C. neoformans (Table 3).
Materials and Methods
Berberine is commercially available. 1H- and 13C-NMR spectra
were recorded on a Varian Mercury (300 MHz) using TMS as a
internal standard and DMSO-d6 as solvent. The mass spectra
were taken on a JEOL JMS HX 100±110A in the positive FAB
mode.
Berberrubine (2): Berberine (3 g) with urea (6 g) was stirred at
200 8C for 30 min. Boiling water was added to the mixture fol-
lowed by extraction with chloroform for 8~10 times. The or-
ganic layer was evaporated and chromatographed with
CHCl3 :MeOH (4:1) on a silica gel column to give berberru-
bine, yield: 2.3 g (76%).
The MICs of nystatin for some fungi and yeasts were deter-
mined and served as positive control for comparison with the
experimental compounds. Compounds 3 and 6 exhibited
weaker activity than nystatin against some fungi and yeasts,
while compounds 2 and 5 showed inhibitory activity against
some fungi only at high concentration (250mg/ml). Among
the compounds tested, berberrubine hydrochloride (3) exhibit-
Berberrubine hydrochloride (3): Berberrubine (0.56 g) in boil-
ing water (22.4 ml) was treated with 35% hydrochloride
(3 ml). After cooling of the mixture at 4 8C, the resulting crys-
tals were filtered. Drying of the filtrate gave berberrubine hy-
drochloride, yield: 0.43 g (70%).
Table 2 Antibacterial activities of berberine, berberrubine, andberberrubine derivatives.
Test organisms
MIC (mg/ml)
1
2
3
4
5
6
7
KM
Staphylococcus aureus subsp. aureus
ATCC 25923
128
>128
>128
>128
>128
4
>128
1
1
Staphylococcus epidermidis ATCC 0155
Enterococcus faecalis ATCC 29212
Micrococcus luteus ATCC 10240
Bacillus subtilis ATCC 6633
64
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
>128
32
4
32
8
128
>128
128
16
4
128
64
8
128
1
Escherichia coli ATCC 25922
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
>128
>128
>128
64
Escherichia coli ATCC 10536
8
Proteus mirabilis ATCC 21100
Pseudomonas aeruginosa ATCC 27853
128
>128
<128
Klebsiella pneumoniae subsp.
pneumoniae ATCC 10031
Determinedafter 24 hours of incubation at 37 8C for the bacteria.
All experiments were run in triplicate.
KM = Kanamycin sulfate.
Table 3 Antifungal activities of berberubine andberberrubine derivatives.
Test organisms
MIC (mg/ml)
2
3
5
6
NYS
1.56
Candida lusitaniae ATCC 42720
Candida krusei ATCC 6258
500
>500
>500
>500
>500
250
>500
>500
>500
>500
500
250
>500
>500
500
>500
500
500
250
6.25
Candida albicans ATCC 10231
3.125
3.125
1.56
Candida tropicalis ATCC 13803
Cryptococcus neoformans ATCC 36556
Coccidioides immitis ATCC 34020
Trichophyton mentagrophytes ATCC 9533
62.5
62.5
250
250
500
62.5
250
3.125
250
500
25
Determinedafter 24 ~48 hours of incubation at 28~30 8C for the yeast andfungi.
NYS = Nystatin.

Letters
Planta Med. 66 (2000) 363
9-O-Acyl- and 9-O-benzoylberberrubine chlorides (4±7): Dried
berberrubine (0.714 g) in acetonitrile (25 ml) was reacted
with acetyl (0.17 ml), valeryl (0.21 ml), lauroyl (0.51 ml) and
benzoyl (0.31 ml) chlorides at room temperature for 1 h (8).
The mixture was diluted with diethyl ether (50 ml). The re-
sulting crystals were filtered and washed with diethyl ether
to give 9-O-acetyl (0.575 g, 72%), 9-O-valeryl (0.626 g, 71%),
9-O-lauroyl (0.318 g, 73%) and 9-O-benzyol (0.575 g, 72%) ber-
berrubine chlorides. 5: FAB-MS 406, HRFAB-MS: C24H24NO5
calc 406.1654, found 406.1659. 6: FAB-MS 504, HRFAB-MS:
13 Schmeller T, Latz-Bruning B, Wink M. Biochemical activities of
berberine, palmatine and sanguinarine mediating chemical de-
fence against microorganisms and herbivores. Phytochemistry
1997; 44: 257 ± 66
14 Okunade AL, Hufford CD, Richardson MD, Peterson JR, Clark AM.
Antimicrobial properties of alkaloids from Xanthorhiza simplicis-
sima. J. Pharm. Sci. 1994; 83: 404±6
Dr. J. H. Kim
C
31H38NO5 calc 504.2750, found 504.2748.
Department of Biotechnology
College of Engineering
Yonsei University
134 Shinchon-dong
Seoul 120-749
The test compound was dissolved in H2O containing 2.5%
DMSO and its antibacterial activity was measured by the
broth dilution method in 96-well titer plates. After incubation
for 24 h, the microbial growth was examined by measuring
the optical density at 650 nm with a Model Emax Microplate
Reader (Molecular Devices) (9). The concentrations of com-
pound were examined in the range of 0.125±128mg/ml. The
MIC of the test compound was defined as the lowest concen-
tration at which there was no visible growth. Antifungal activ-
ities of the test compounds were examined by means of the
agar dilution method in Sabouraud medium for fungi and
yeasts (10). The concentrations of compound were examined
in the range of 0.975±500mg/ml.
Korea
E-mail: junghan@bubble.yonsei.ac.kr
Fax: +822-362-7265
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