Synthesis and antifungal activity of a novel series of 13-(4- isopropylbenzyl)berberine derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.09.045

By replacing the methyl group of 13-(4-isopropylbenzyl)berberine 2 with various acyl, alkyl, and benzyl groups via the demethylated intermediate, 13-(4-isopropylbenzyl)berberrubine 4, a novel series of 9-O-alkyl-13-(4- isopropylbenzyl)berberine derivatives was synthesized and examined for antifungal activities against various human pathogenic fungi. The introduction of various alkyl groups led to enhanced antifungal activity but that of acyl groups resulted in decrease of the activity. Among them, 9-O-butyl-13-(4- isopropylbenzyl)berberine 6d exhibited the most potent antifungal activities against Cryptococcus neoformans, Candida species (MIC = 0.25-1 μg/ml), and Aspergillus species (MIC = 2-4 μg/ml). The compound was found to be relatively safe up to 900 mg/kg in oral administration to mice.

Full text of DOI:10.1016/j.bmcl.2010.09.045

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6551–6554
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antifungal activity of a novel series
of 13-(4-isopropylbenzyl)berberine derivatives
Ki Duk Park , Sung Jin Cho à, Jae Sun Moon, Sung Uk Kim
⇑
Division of Biomaterials Science, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
By replacing the methyl group of 13-(4-isopropylbenzyl)berberine 2 with various acyl, alkyl, and benzyl
groups via the demethylated intermediate, 13-(4-isopropylbenzyl)berberrubine 4, a novel series of 9-O-
alkyl-13-(4-isopropylbenzyl)berberine derivatives was synthesized and examined for antifungal activi-
ties against various human pathogenic fungi. The introduction of various alkyl groups led to enhanced
antifungal activity but that of acyl groups resulted in decrease of the activity. Among them, 9-O-butyl-
13-(4-isopropylbenzyl)berberine 6d exhibited the most potent antifungal activities against Cryptococcus
Received 6 August 2010
Revised 7 September 2010
Accepted 8 September 2010
Available online 21 September 2010
Keywords:
neoformans, Candida species (MIC = 0.25–1
lg/ml), and Aspergillus species (MIC = 2–4 lg/ml). The com-
13-(4-Isopropylbenzyl)berberine
Berberine derivatives
Human pathogenic fungi
Antifungal activity
pound was found to be relatively safe up to 900 mg/kg in oral administration to mice.
Ó 2010 Elsevier Ltd. All rights reserved.
The incidences of infections by opportunistic fungi are increas-
ing especially in patients whose immune systems are compro-
mised by AIDS, cancer, diabetes, aging, and other causes.1,2
Although many antifungal compounds have been developed and
some drugs are under clinical trials, safe and effective antifungal
drugs have not yet been developed because of the similarity be-
tween fungi and mammalian cells.3,4 Therefore, amphotericin B
and the azole compounds remain the mainstream of fungal sys-
temic infection therapy despite their serious side effects and the
emergence of resistant strains.5,6 This has consequently resulted
in a strong demand for drugs that have low side effects and
broad-spectrum activity.
Medicinal plants that have been used for a long time may be
good sources of safe antifungal agents. Berberine 1, which is an
alkaloid isolated from Korean and Chinese medicinal plants, signif-
icantly inhibits growth of a wide range of Candida species.7,8 Re-
cently, berberine was combined with amphotericin B to treat
disseminated candidiasis in mice and investigated for the synergis-
tic interaction with fluconazole against fluconazole resistant clini-
cal isolates of Candida albicans.9–11 In addition, to develop the novel
antifungal agents, various berberine derivatives with substituents
at C-13 position were synthesized, which exhibited potent activity
against Candida species.12,13
Previously, we reported that the introduction of various aro-
matic groups at C-13 position of berberine remarkably improved
the antifungal activites.13 Among them, 13-(4-isopropylben-
zyl)berberine 2 including a lipophilic substituent at the para posi-
tion of benzyl group exhibited the most potent antifungal activities
(Fig. 1). The most significant structural feature leading to enhanced
activity was the presence of a methyl group on 9-hydroxyl. Thus,
13-(4-isopropylbenzyl)berberine 2 showed over fourfold stronger
activities than 13-(4-isopropylbenzyl)berberrubine 4 with a hydro-
xy group at 9-position instead against various the human patho-
genic fungi.
The purpose of the present study was to investigate whether
structural changes of the methyl group on 9-hydroxyl might en-
hance antifungal activities. A new series of 13-(4-isopropylben-
zyl)berberine analogs 6a–6o was synthesized by using other
basic moieties such as alkyl chains, olefins and aromatic groups
as the replacement of the methyl group in the 9-position, and
examined for antifungal activities against seven human pathogenic
fungi.
For the synthesis of 9-O-substituted-13-(isopropylben-
zyl)berberines, 13-(4-isopropylbenzyl)berberrubine 4 was synthe-
sized by the methods described in the previous study.13
Acetonylberberine 3 used as a key intermediate was obtained by
condensation of berberine with acetone. The treatment of 3 with
4-isopropylbenzyl bromide and sodium iodide (NaI) gave 13-(4-
isopropylbenzyl)berberine 2 by displacement of the acetonyl group
with the 4-isopropylbenzyl group. The pyrolysis of 2 was carried
out in DMF with high temperature to give 4. The treatment of 4
with various acyl chlorides, alkyl iodides and benzyl bromides gave
a salt form of 9-O-substituted derivatives 5. The iodide or bromide
⇑
Corresponding author. Tel.: +82 42 860 4554; fax: +82 42 861 2675.
E-mail address: kimsu@kribb.re.kr (S.U. Kim).
Present address: Division of Medicinal Chemistry and Natural Products, School of
Pharmacy, University of North Carolina at Chapel Hill, NC, USA.
à
Present address: Drug Discovery Program, Department of Medicinal Chemistry
and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, IL, USA.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.045

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K. D. Park et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6551–6554
O
O
O
N
O
N
9
OMe
OMe
13
OMe
OMe
1
2
Figure 1. Structural optimization of berberine 1.
salt forms of the target compounds were converted into yellow-or-
ange crystalline chlorides 6 using AgCl in hot MeOH (Scheme 1).
All of the synthesized compounds were tested for in vitro anti-
fungal activity against various Candida species, two Aspergillus spe-
cies, and Cryptococcus neoformans using the procedure described
by the broth microdilution method of the Clinical and Laboratory
Standards Institute (CLSI, formerly National Committee for Clinical
and Laboratory Standards NCCLS).14,15 The MIC was defined as the
lowest concentration that produced an optically clear well. Deter-
minations of MICs were performed in duplicate on at least three
separate experiments.
replacement of the methyl group with various aliphatic groups at
9-O position remarkably improved antifungal activity. The most
significant structural change leading to enhanced activity was the
introduction of alkyl chains as an alkyloxy group. Among them,
9-O-butyl-13-(4-isopropylbenzyl)berberine 6d exerted the most
potent antifungal activities with MIC values of 0.25–1 lg/ml
against several Candida species, and dramatically over eightfold
stronger antifungal activities than the corresponding acyloxy
substituted analog, 9-O-butyryl-13-(4-isopropylbenzyl)berberine
6b. These results indicate that the acyl groups at 9-O position
might be cleaved easily because the oxygen at C-9 position of berb-
errubine is present in the form of either ketone 4a or hydroxyl 4b
and reversibly transformed due to oxidative rearrangement in iso-
quinolium salts (Fig. 2).16,17 It can be seen that replacing the alkyl
chains with olefin groups as either the allyl 6e or crotyl 6f does not
The antifungal activities of the compounds against human path-
ogenic fungi are summarized in Table 1. The results were com-
pared with findings previously reported for 2, 4, and the proven
antifungal agent amphotericin B. These results indicate that the
O
O
N
O
O
O
O
O
Cl
b
a
N
N
OMe
OMe
OMe
OMe
OMe
OMe
2
1
3
O
O
O
O
X
N
N
d
c
OR
O
OMe
OMe
5
4
6a
6b
6c
6d
6e
6f
R = Ac
R = butyryl
R = propyl
R = butyl
R = allyl
O
O
Cl
N
R = crotyl
OR
6g
6h
R =
t-Butyl
e
R = 1,1-dimethylpropyl
6i R = 3-hydroxypropyl
6j R = benzyl
OMe
6k
6l R = 4-
R = 4-Isopropylbenzyl
-butylbenzyl
t
6m R = 4-CF3-benzyl
6n R = 4-CF3O-benzyl
6o R = 3,4,5-F-benzyl
6a-6o
Scheme 1. Synthesis of 9-O-alkyl-13-(4-isopropylbenzyl)berberine derivatives. Reagents and conditions: (a) acetone, 5 N NaOH, rt, 1 h; (b) 4-isopropylbenzylbromide, NaI,
CH3CN, 80 °C, 4 h; (c) DMF, reflux, 190 °C, 2 h; (d) various acyl chlorides, alkyl iodides and benzyl bromides, CH3CN, 120 °C, 4 h; (e) AgCl, MeOH, 60 °C, 2 h.

K. D. Park et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6551–6554
6553
Table 1
Structures and in vitro antifungal activities for 9-substituted-13-(4-isopropylbenzyl)berberine derivatives 6.
Compounds
R
MICa
(l
g/ml)
Aspergillus
Candida
Candida
tropicalis
Candida
lusitaniae
Candida
krusei
Aspergillus
terreus
Cryptococcus
neoformans
albicansb
fumigatus
2
4
CH3
H
C(O)CH3
C(O)CH2CH2CH3
CH2CH2CH3
CH2(CH2)2CH3
CH2CH@CH2
CH2CH@CHCH3
C(CH3)3
4
1
8
8
8
8
2
1
2
1
8
4
4
4
64
>128
>128
>128
4
2
8
2
32
>128
>128
>128
8
4
8
4
4
4
8
4
2
1
2
1
4
4
2
16
16
8
1
0.5
1
0.5
4
2
16
16
8
0.5
0.25
1
0.5
1
1
16
16
8
2
1
2
1
4
4
6a
6b
6c
6d
6e
6f
6g
6h
6i
32
32
8
32
16
8
C(CH3)2CH2CH3
CH2(CH2)2OH
2
1
2
6j
4
1
1
4
1
1
8
4
4
4
2
4
64
4
32
8
4
2
2
6k
6l
4
8
F
F
6m
6n
F
2
2
2
2
4
4
4
4
16
32
16
16
4
4
F
O
F
F
F
F
6o
F
4
4
8
1
4
32
1
32
16
4
AmpBc
0.5
0.5
0.5
0.25
a
The MIC value was determined by methods of CLSI. The final concentration of antifungal agents was between 0.125 and 128
experiments.
lg/ml. The values are given as means of three
b
Test organisms: Candida albicans ATCC 10231, Candida tropicalis ATCC 13803, Candida lusitaniae ATCC 42720, Candida krusei ATCC 6258, Aspergillus fumigatus ATCC 16424,
Aspergillus terreus ATCC 46941, and Cryptococcus neoformans ATCC 36556.
c
AmpB: amphotericin B.
result in improved activity. Hence, the presence of unsaturation in
the alkyl group did not enhance the activity as seen in compounds
6c, 6d, 6e and 6f. Furthermore, comparison of 6c and 6d with 6g
and 6h showed that the substitution of methyl groups on the C-1
position of the alkyl chains led to decreased activity. As the polarity
is further increased in the cases of the hydroxy-propyl 6i, there is a
decrease in antifungal activity compared to the compound 6c. In
addition, the introduction of various benzyl groups at 9-O position
showed diverse antifungal activities depending on the functional
groups of the substituents on the aromatic ring. Compound 6k
and 6l exhibited stronger activities than 6j and 6o, consistent with
the previous requirement for potency, a lipophilic alkyl group at
the benzyl moiety. The activity of these compounds with a benzyl
group at 9-O position showed similar tendency to that of the pre-
viously synthesized compounds with a benzyl group at C-13
position.
To examine the toxicity of compound 6d, the acute toxicity test
of the compound was performed. Compound 6d was orally admin-
istered to ICR mice with various doses (33–900 mg/kg), and the
animals were closely monitored. No animal died in the 7-day treat-
ment, indicating that LD50 of 6d was over 900 mg/kg. No changes in
clinical signs, body weights, and the gross necropsy of mice admin-
istrated with these dosages were observed. This result suggests
that compound 6d is considerably safe in vivo up to 900 mg/kg
in oral administration.
On the basis of our previous13 and current studies, our results
suggest two structural requirements for enhancing the antifungal
activity: 1) the introduction of a benzyl group including lipophilic
substituents at C-13 position of berberine and 2) the modification
of the methyl group at 9-O position with an alkyl group. Especially,
the introduction of an alky group at 9-O position led to much bet-
ter antifungal activity than that of an acyl group. Among novel ser-
O
O
N
N
O
O
OH
O
OMe
OMe
4b
4a
Figure 2. Rearrangement in compound 4.

6554
K. D. Park et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6551–6554
ies of 9-O-alkyl-13-(isopropylbenzyl)berberine derivatives, com-
pound 6d with a butyl group exhibited 4–32-fold stronger anti-
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Supplementary data
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Supplementary data (materials and methods, and ESI-MS, NMR,
and elementary analysis data) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2010.09.045.