Antimicrobial Activity of 9-O-Acyl- and 9-O-Alkylberberrubine Derivatives

Article abstract of DOI:10.1055/s-2002-23128

For the structure-activity relationship study on berberrubine derivatives, a series of compounds bearing 9-O-acyl- and 9-O-alkyl-substituents were synthesized and tested for antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. O

Full text of DOI:10.1055/s-2002-23128

Antimicrobial Activity of
9-O-Acyl- and 9-O-Alkylberberrubine
Derivatives
Sung Han Kim1, Sang Jun Lee2, Joo Hyoung Lee1, Won Suck Sun1,
Jung Han Kim1
Abstract
For the structure-activity relationship study on berberrubine de-
rivatives, a series of compounds bearing 9-O-acyl- and 9-O-alkyl-
substituents were synthesized and tested for antimicrobial activ-
ity against Gram-positive, Gram-negative bacteria and fungi. Oc-
tanoyl, decanoyl, lauroyl derivatives among the acyl analogs and
hexyl, heptyl, octyl, nonyl, decyl, undecyl derivatives among the
alkyl analogs showed strong antimicrobial activity against
Gram-positive bacteria and fungi. As a whole, alkyl analogs
were more active than acyl analogs for antimicrobial activity.
Synthesized derivatives had no activity on Gram-negative bac-
teria. Too short or too long substituents decreased activity. These
results suggest that the presence of lipophilic substituents with
moderate sizes might be crucial for the optimal antimicrobial ac-
tivity.
Berberine, isolated from a variety of plants [1], is widely used in
Asia as a drug due to its antimicrobial activity [2±7]. Berberru-
bine is an isoquinoline alkaloid isolated from the plant Berberis
vulgaris L. [8] and readily derived from berberine by pyrolysis [9].
277
In a previous paper, we reported that berberrubine showed a po-
tent activity as a DNA topoisomerase II inhibitor [10], however,
despite much resemblance in chemical structure, other proto-
berberine alkaloids such as berberine and palmatine [11] did
not act on topoisomerase II. These results forced us to study the
correlation between modified structure and biological activities.
Recently, we reported that 9-O-lauroylberberrubine derivatives
displayed active antibacterial potency against Gram-positive
bacteria [12].
In the present study, the effects of variations in the length of the
acyl side-chain at 9-O-position of berberrubine on the antimicro-
bial activity were examined. Also, antimicrobial activity of 9-O-
alkylberberrubine derivatives with C2-C18 alkyl chains were test-
ed against representative strains of Gram-positive, Gram-nega-
tive bacteria and fungal microorganisms by 2-fold dilution
methods. Acyl and alkyl derivatives were synthesized as present-
Affiliation: 1 Department of Biotechnology, Bioproducts Research Center, Yon-
sei University, Seoul, Korea ´ 2 Pharmaceutical & Health Research Institute, Pa-
cific R&D Center, Yongin-si, Kyounggi-do, Korea
Correspondence: Dr. J. H. Kim ´ Department of Biotechnology ´ College of
Engineering ´ Yonsei University ´ 134 Shinchon-dong ´ Seoul 120±749 ´ Korea ´
E-Mail: junghan@bubble.yonsei.ac.kr ´ Fax: +822±362±7265
Received: March 23, 2001 ´ Accepted: June 30, 2001
Bibliography: Planta Med 2002; 68: 277±281 ´ ꢀ Georg Thieme Verlag Stuttgart ´
New York ´ ISSN 0032-0943

Fig. 1 Synthesis of berberrubine (2), 9-O-acylberberrubines (3±14) and 9-O-alkylberberrubines (15±27).
Table 1 1H-NMRa data and mass spectra of 9-O-acyl-substituted derivatives of berberrubine (8±10)
CH3(CH3)nCH2-CO- at C9-O
ESIMSb
formula m/z
[M±Cl]+
CH3-
-CH2-
-CH2-CO- H-5
10-OMe H-6
OCH2O H-4
H-1
H-11
H-12
H-13
H-8
8
9
0.91
1.25~1.42 (m, 8H)
2.85
3.23
(t, 2H) (s, 3H)
3.22 4.02
(t, 2H) (s, 3H)
3.23 4.03
(t, 2H) (s, 3H)
4.03
4.94
6.18
7.10
7.81
8.21
8.28
9.05
9.93
C27H30NO5 448
C29H34NO5 476
C31H38NO5 504
(t, 3H) 1.76 (m, 2H)
(t, 2H)
(t, 2H) (s, 2H) (s, 1H) (2, H) (d, H) (d, 1H) (s, 1H) (s, 1H)
4.93 6.18 7.10 7.81 8.19 8.29 9.03 9.87
(t, 2H) (s, 2H) (s, 1H) (s, H) (d, H) (d, 1H) (s, 1H) (s, 1H)
4.94 6.18 7.10 7.82 8.22 8.29 9.03 9.92
(t, 2H) (s, 2H) (s, 1H) (s, H) (d, H) (d, 1H) (s, 1H) (s, 1H)
0.89
1.23~1.42 (m, 12H) 2.85
278
(t, 3H) 1.74 (m, 2H)
(t, 2H)
10 0.88
1.22~1.42 (m, 16H) 2.86
(t, 2H)
(t, 3H) 1.74 (m, 2H)
a
1H-NMR spectra were run in DMSO-d6 on a Varian Mercury-300 MHz (d from TMS).
b Mass spectra were determined on Fisons-VG platform in the positive ESI (electron spray ionization) mode.
Table 2 1H-NMRa data and mass spectra of 9-O-alkyl-substituted derivatives of berberrubine (19±24)
CH3(CH3)nCH2- at C9-O
ESIMSb
CH3-
-CH2-
-CH2-CO- H-5
10-OMe H-6
OCH2O H-4
H-1
H-11
H-12
H-13
H-8
formula
m/z
[M±Cl]+
19 0.99
1.29~1.45 (m, 6H)
1.24~1.37 (m, 8H)
4.28
3.23
(t, 2H) (s, 3H)
3.21 4.05
(t, 2H) (s, 3H)
3.21 4.07
(t, 2H) (s, 3H)
3.21 4.05
(t, 2H) (s, 3H)
3.21 4.05
(t, 2H) (s, 3H)
3.21 4.06
(t, 2H) (s, 3H9
4.05
4.94
6.18
7.09
7.80
8.01
8.18
8.93
9.74
C25H28NO4 406
C26H30NO4 420
C27H32NO4 434
C28N34NO4 448
C29N36NO4 462
CH38NO430 476
(t, 3H) 1.85 (m, 2H)
(t, 2H)
4.28
(t, 2H) (s, 2H) (s, 1H) (s, 1H) (d, 1H) (d, 1H) (s, 1H) (s, 1H)
4.94 6.17 7.10 7.80 7.98 8.19 8.92 9.76
(t, 2H) (s, 2H) (s, 1H) (s, 1H) (d, 1H) (d, 1H) (s, 1H) (s, 1H)
4.96 6.17 7.10 7.80 7.98 8.19 8.92 9.74
(t, 2H) (s, 2H) (s, 1H) (s, 1H) (d, 1H) (d, 1H) (s, 1H) (s, 1H)
4.96 6.17 7.09 7.80 7.98 8.19 8.92 9.74
(t, 2H) (s, 2H) (s, 1H) (s, 1H) (d, 1H) (d, 1H) (s, 1H) (s, 1H)
4.96 6.17 7.09 7.80 7.98 8.19 8.92 9.74
(t, 2H) (s, 2H) (s, 1H) (s, 1H) (d, 1H) (d, 1H) (s, 1H) (s, 1H)
4.96 6.17 7.02 7.80 7.98 8.19 8.92 9.72
(t, 2H) (s, 2H) (s, 1H) (s, 1H) (d, 1H) (d, 1H) (s, 1H) (s, 1H)
20 0.96
(t, 3H) 1.74 (m, 2H)
(t, 2H)
21 0.96
1.28~1.41 (m, 10H) 4.28
(t, 2H)
1.27~1.47 (m, 12H) 4.28
(t, 3H) 1.85 (m, 2H) (t, 2H)
23 0.89 1.24~1.47 (m, 14H) 4.28
(t, 3H) 1.86 (m, 2H) (t, 2H)
24 0.85 1.20~1.48 (m, 16H) 4.26
(t, 3H) 1.88 (m, 2H) (t, 2H)
(t, 3H) 1.84 (m, 2H)
22 0.96
a
1H-NMR spectra were run in DMSO-d6 on a Varian Mercury-300 MHz (d from TMS).
b Mass spectra were determined on Fisons-VG platform in the positive ESI (electron spray ionization) mode.

Table 3 Antibacterial activities of berberine (1), berberrubine (2), 9-O-acyl (3±14), and 9-O-alkyl (15±27) berberrubine derivatives
Test organisms
MIC (mg/ml)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Enterococcus faecalis
ATCC 29212
128
128
32
>128
>128
64
>128
>128
64
>128
>128
64
>128
>128
64
>128
>128
64
128
128
64
32
8
32
4
32
4
>128
>128
2
>128
>128
4
>128
>128
16
>128
>128
64
Staphylococcus aureus
ATCC 25923
Micrococcus luteus
ATCC 10240
16
64
4
1
Staphylococcus epidermidis >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
>128
>128
>128
>128
>128
>128
>128
>128
4
4
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
ATCC 0155
Bacillus subtilis
ATCC 6644
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
8
>128
>128
>128
>128
>128
Escherichia coli
ATCC 25922
>128
>128
>128
>128
>128
>128
>128
>128
Escherichia coli
ATCC 10536
Proteus mirabilis
ATCC 27853
Pseudomonas aeurginosa
ATCC 27853
15
16
17
18
19
20
21
22
23
24
25
26
27
km
Enterococcus faecalis
ATCC 29212
128
128
8
128
64
64
32
64
32
8
8
8
4
2
2
2
2
2
2
8
8
64
64
128
<128
4
>128
<128
32
4
8
2
Staphylococcus aureus
ATCC 25923
Micrococcus luteus
ATCC 10240
4
4
1
0.5
0.125
0.125
0.125
0.125
0.5
0.5
Staphylococcus epidermidis
ATCC 0155
64
64
8
8
2
8
2
4
1
2
0.5
2
0.5
2
1
8
1
64
64
128
0.5
279
Bacillus subtilis
ATCC 6644
128
>128
>128
>128
>128
64
32
32
<128
>128
>128
>128
>128
<128
>128
>128
>128
>128
2
64
Escherichia coli
ATCC 25922
>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
>128
>128
>128
>128
>128
Escherichia coli
ATCC 10536
>128
>128
>128
>128
>128
>128
8
Proteus mirabilis
ATCC 27853
128
>128
Pseudomonas aeruginosa
ATCC 27853
Determined after 24 hours of incubation at 37 8C forthe bacteria.
All experiments were run in triplicates.
km = kanamycin sulfate.
ed in Fig.1 for investigation of activity against Gram-positive tra for berberrubine derivative chlorides with antimicrobial ac-
(Enterococcus faecalis, Staphylococcus aureus, Micrococcus luteus, tivity are described in Table 1 and Table 2.
Staphylococcus epidermidis and Bacillus subtilis) and Gram-nega-
tive (Escherichia coli, Proteus mirabilis and Pseudomonas The biological activities of the compounds against a panel of mi-
aeruginosa) bacteria as well as the fungi (Candida krusei, Candida croorganisms are summarized in Tables 3 and 4. The antibacterial
lusitaniae, Candida albicans and Cryptococcus neoformans).
activities of synthesized compounds (3±27) were compared with
those of berberine (1), berberrubine (2) and kanamycin sulfate
The structures of the new acyl and alkyl derivatives (3±27) were (28) as a positive control. The activities of the acyl- and alkylber-
determined by NMR and MS experiments. Full signal assignment berrubine derivatives against Gram-positive bacteria were in-
1
of H and 13C was carried out with various NMR techniques in- creased as the length of the chain of the substituents increased.
cluding DEPT, COSY, H,C-COSY, and long-range H,C-COSY. The The change in lipophilicity of the protoberberinium salts caused
complete assignment of 1H-NMR chemical shifts and mass spec- by substituents influenced the antibacterial activity of derivatives.

Table 4 Antifungal activities of berberine (1), berberrubine (2), 9-O-acyl (3±14), and 9-O-alkyl (15±27) berberrubine derivatives.
Test organisms
MIC (mg/ml)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Candida krusei
IFO 1664
32
>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
128
32
16
32
32
16
8
2
2
4
2
64
>128
>128
>128
64
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
Candida lusitaniae
ATCC 42720
>128
128
16
>128
Candida albicans
ATCC 10231
4
4
2
Candida tropicalis
IFO 10241
Cryptococcus neoformans
ATCC 2344
8
4
15
16
17
18
19
20
21
22
23
24
25
26
27
AmpB
Candida krusei
IFO 1664
64
>128
>128
128
32
32
64
32
32
32
16
64
16
16
32
16
64
16
16
8
4
8
4
1
4
2
4
2
1
1
1
1
1
1
2
1
4
4
4
8
4
16
16
16
16
8
64
64
64
32
32
>128
>128
>128
>128
64
0.5
Candida lusitaniae
ATCC 42720
1
1
0.5
Candida albicans
ATCC 10231
2
1
0.125
0.25
0.125
Candida tropicalis
IFO 10241
1
1
Cryptococcus neoformans
ATCC 2344
16
0.5
0.5
Determined after 24±72 hours of incubation at 28±30 8C forthe fungi.
All experiments were run in triplicates.
AmpB = Amphotericin B.
280
However, longer chains than lauroyl in acyl analogs and dodecyl 9-O-Acylberberrubine chlorides (3±14): Dried berberrubine
in alkyl homologs decreased the antibacterial activity. Among (0.714 g) in acetonitrile (25 ml) was reacted with acetyl (0.17
the acyl derivatives, octanoyl (8), decanoyl (9) and lauroyl (10) ml), propionyl (0.18 m), butyryl (0.19 ml), valeryl (0.21 ml), hex-
berberrubine chlorides showed moderate activity. Among the al- anoyl (0.23 ml), octanoyl (0.31 ml), decanoyl (0.41 ml), lauroyl
kyl derivatives, hexyl (19), heptyl (20), octyl (21), nonyl (22), de- (0.51 ml), myristoyl (0.55 ml), palmitoyl (0.61 ml), benzoyl (0.31
cyl (23) and undecyl (24) exhibited strong antibacterial potency ml) and 3,4,5-trimethoxybenzoyl (0.52 g) chlorides at room tem-
against the tested organisms. As a whole, 9-O-alkylberberrubine perature for 1 h [13]. The mixture was diluted with diethyl ether
analogs were more active than 9-O-acylberberrubine analogs or (50 ml). The resulting crystals were filtered and washed with
the parent molecule, berberine and berberrubine, against Gram- ether. Chromatography with CHCl3: MeOH (4:1) on a silica gel
positive bacteria. For Gram-negative bacteria, the synthesized column gave the corresponding 9-O-acylberberrubine chlor-
compounds had little antibacterial activity. Additionally, antifun- ides.
gal activity of derivatives was compared with berberine (1) and
berberrubine (2), starting material, as well as amphotericin B as a 9-O-Alkylberberrubine chlorides (15 ± 27): Dried berberrubine
positive control. Similar results in antifungal activity were dis- (0.714 g) in DMF (100 ml) was reacted with ethyl (0.312 g), pro-
played as in antibacterial activity against Gram-positive bacteria.
pyl (0.340 g), butyl (0.368 g), pentyl (0.396 g), hexyl (0.424 g),
heptyl (0.452 g), octyl (0.480 g), nonyl (0.508 g), decyl (0.536 g),
As a result, 9-O-alkylberberrubine derivatives with C6 ± C11 alkyl undecyl (0.564 g), dodecyl (0.592 g), hexadecyl (0.704 g), octade-
chains (19 ± 24) had potent antimicrobial activity and potential cyl (0.816 g) iodide at 1208C for 5 h [9]. Evaporation and chroma-
as new antimicrobial agents.
tography with CHCl3: MeOH (4:1) on a silica gel column gave the
corresponding 9-O-alkylberberrubine iodides. These iodides
were converted into yellow-orange crystalline chlorides with
AgCl in hot MeOH [4].
Materials and Methods
Berberine (1) is commercially available. Berberrubine (2) was The test compound was dissolved in H2O containing 2.5% DMSO
synthesized from berberine [12]. 1H- and 13C-NMR spectra were as a negative control and its antibacterial activity was measured
recorded on a Varian Mercury (300 MHz) using TMS as a internal by the broth dilution method in 96-well titer plates. After incu-
standard and DMSO-d6 as a solvent. The mass spectra were taken bation for 24 h, the microbial growth was examined by measur-
by a Fisons-VG platform in the positive ESI mode.
ing the optical density at 650 nm with a Model Emax Microplate

Reader (Molecular Devices) [9]. The concentration of compound
were examined in the range of 0.125±128 mg/ml. The MIC of the
test compound was defined as the lowest concentration at which
there was no visible growth. Antifungal activities of the test com-
pounds were examined by means of the broth dilution method in
Sabouraud medium for fungi [14]. The concentration of com-
pound were examined in the range of 0.125±128 mg/ml.
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