Synthesis and antimicrobial activities of 3-O-alkyl analogues of (+)-catechin: Improvement of stability and proposed action mechanism

Article abstract of DOI:10.1016/j.ejmech.2009.11.045

We report here the synthesis and biological properties of 3-O-alkyl analogues of (+)-catechin (5), which itself is one of the major natural polyphenols found in green tea and has several physiological activities. Starting from 5, a series of 3-O-alkyl-(+)-catechin derivatives were investigated as potent antimicrobial agents. The presence of an alkyl chain rather than acyl on 3-O- showed an increase in antimicrobial activity which may be due to stability in standard culture condition. The most promising compound is 8e, 3-O-decyl analogue, with the MIC of 0.5-2, 32-128 and 2-4?μg/mL against Gram-positive bacteria, Gram-negative bacteria and human pathogenic fungi, respectively. Regarding action mechanism, the antimicrobial activity is possibly due to the lipophilicity and disrupting ability of the analogues to the liposome membrane.

Full text of DOI:10.1016/j.ejmech.2009.11.045

European Journal of Medicinal Chemistry 45 (2010) 1028–1033
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activities of 3-O-alkyl analogues of (þ)-catechin:
Improvement of stability and proposed action mechanism
,
1,
2
,1,3
*
*
Ki Duk Park
, Sung Jin Cho
Laboratory of Cellular Function Modulator, Korea Research Institute of Bioscience and Biotechnology, Yuseong, 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:
We report here the synthesis and biological properties of 3-O-alkyl analogues of (þ)-catechin (5), which itself
is one of the major natural polyphenols found in green tea and has several physiological activities. Starting
from 5, a series of 3-O-alkyl-(þ)-catechin derivatives were investigated as potent antimicrobial agents. The
presence of an alkyl chain rather than acyl on 3-O- showed an increase in antimicrobial activity which may be
due to stability in standard culture condition. The most promising compound is 8e, 3-O-decyl analogue, with
Received 3 August 2009
Received in revised form
17 November 2009
Accepted 20 November 2009
Available online 26 November 2009
the MIC of 0.5–2, 32–128 and 2–4 mg/mL against Gram-positive bacteria, Gram-negative bacteria and human
pathogenic fungi, respectively. Regarding action mechanism, the antimicrobial activity is possibly due to the
lipophilicity and disrupting ability of the analogues to the liposome membrane.
Published by Elsevier Masson SAS.
Keywords:
3-O-acyl-(þ)-catechins
3-O-Alkyl-(þ)-catechins
Antimicrobial activity
1. Introduction
catechingallatessuchas(ꢀ)-ECG(3)and(ꢀ)-EGCG(1)aresusceptible
to hydrolysis by bacterial and possibly host esterases. To prevent the
Greentea, madesolely withtheleavesofCamelliasinensis, isoneof
the most popular and commonly consumed beverages next to water
in the world [1]. It has beenpaidmuch attention over last decadewith
respect to the beneficial biological activities of its components, cate-
chins, which have been reported to have antimutagenic [2], antioxi-
dant [3], antibacterial [4,5], antitumor and cancer preventive [6]
properties. Green tea contains primarily catechins such as (ꢀ)-epi-
gallocatechin-3-gallate (EGCG) (1), (ꢀ)-epigallocatechin (EGC) (2),
(ꢀ)-epicatechin-3-gallate (ECG) (3), (ꢀ)-epicatechin (EC) (4) and
(þ)-catechin(C)(5)(Fig.1)[7]. (ꢀ)-EGCG(1)isthemostabundantand
biologically promising polyphenol in green tea, making up more than
40% of the total polyphenolic mixture [8], and there has been
extensive investigation into the ways by which (ꢀ)-EGCG (1) might
act [9–11]. Several studies have shown that the presence of the
3-galloyl moiety in catechins led to higher biological activities
[12–14]. For example, (ꢀ)-EGCG (1) and (ꢀ)-ECG (3) showed stronger
anticanceractivitiesthan(ꢀ)-EGC (2) and (ꢀ)-EC (4) [2,6]. However, it
causes the major limitations of low bioavailability which could be
related to their low stability in neutral or slightly alkaline solutions
and their inability to cross cellular membranes [15]. In addition,
esterase-mediated removal of the galloyl moiety from catechin
gallates, Anderson and coworkers have synthesized (ꢀ)-ECG (3)
derivatives through the replacement of hydrolytically susceptible
ester bond with a more stable amide bond [16]. Landis-Piwowar and
coworkers developed acetyl-protected (ꢀ)-EGCG (1) analogues as
putative pro-drugs which exhibited more potent proteasome inhib-
itory activity than (ꢀ)-EGCG (1) in cultured tumor cell [17]. However,
stability problems still remain after deacetylation in the cell.
It is of great importance to understand the use of individual
catechins because this can provide the information of intrinsic
metabolism and bioavailability in the absence of the drug–drug
interactions from other catechins and impurities. Another
promising direction is to generate more chemically stable
analogues of green tea catechins.
In our previous study, we thus reported that the synthesized 3-
O-acyl analogues of (ꢀ)-EC (4) and (þ)-C (5) with the lipophilic
substituents enhanced antimicrobial activities [18]. Furthermore,
we found that 3-O-alkyl and acyl analogues of (ꢀ)-EC (4) showed
potent anticancer activities against various cancer cell line and alkyl
analogues exerted better activities than acyl analogues [19].
In this study, we synthesized a series of 3-O-alkyl-(þ)-C deriv-
atives (8a–8l), evaluated their antimicrobial activities, and exam-
ined stability of 3-O-acyl and alkyl analogues in culture media (pH
7.4) using HPLC analysis. Furthermore, the interaction between
those analogues and the lipid membranes was investigated using
liposome as a model membrane to propose the possible mecha-
nism of the activity.
* Corresponding authors. Tel.: þ82 42 860 4564; fax: þ82 42 861 2675.
E-mail addresses: kdpark@email.unc.edu (K.D. Park), sjcho@uic.edu (S.J. Cho).
1
These authors contributed equally to this work.
Present address: Division of Medicinal Chemistry and Natural Products, School
2
of Pharmacy, University of North Carolina at Chapel Hill, NC, USA.
3
Present address: Drug Discovery Program, Department of Medicinal Chemistry
and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, IL, USA.
0223-5234/$ – see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.11.045

K.D. Park, S.J. Cho / European Journal of Medicinal Chemistry 45 (2010) 1028–1033
OH
1029
3. Results
OH
3'
OH
OH
OH
4'
5'
3.1. Antimicrobial activities of 3-O-alkyl-(þ)-catechin derivatives
B
HO
O
7
2
HO
O
C
OH
A
The antimicrobial activities of the compounds 8a–8l against
Gram-positive bacteria, Gram-negative bacteria and human path-
ogenic fungi are summarized in Tables 1 and 2. The antimicrobial
activities of synthesized compounds 8a–8l were compared with
those of major catechins, 3-O-decanoyl-(þ)-catechin (9) [18] and
the positive controls, kanamycin sulfate and ciprofloxacin for
antibacterial and amphotericin B and itraconazole for antifungal.
In the previous study, the introduction of various aromatic ring
and aliphatic chain as an acyl group at the 3-O position of (ꢀ)-EC (4)
or (þ)-C (5) exhibited mild antibacterial activities against Gram-
positive bacteria and antifungal activities [18]. In this study, we
tested whether introducing an alkyl group at the 3-O position
instead of an acyl group to (þ)-C (5) enhances the antimicrobial
activity from preventing of enzymatic or non-enzymatic cleavage of
the acyl group in assay [20]. We found that 3-O-alkyl-(þ)-C deriv-
atives showed significantly enhanced antimicrobial activities, 2 to
4-folds more active than 3-O-acyl-(þ)-C derivatives in general.
Among them, (þ)-C derivatives bearing an alkyl chain of carbon
atoms in the close vicinity of C8 to C10 showed strong antimicrobial
activities. Especially, the 3-O-decyl-(þ)-C (8e) exerted the strongest
OH
3
O
5
OH
OH
OH
OH
O
D
(-)-Epigallocatechin (EGC) (2)
OH
OH
OH
(-)-Epigallocatechin-3-gallate(EGCG) (1)
HO
O
OH
OH
OH
OH
HO
O
(-)-Epicatechin (EC) (4)
O
OH
OH
OH
OH
O
OH
HO
O
OH
OH
(-)-Epicatechin-3-gallate (ECG) (3)
OH
(+)-Catechin (C) (5)
Fig. 1. Structures of major tea catechins.
antimicrobial activities (MIC ¼ 0.5–2
mg/mL and 2–4 mg/mL against
Gram-positive bacteria and fungi, respectively) and showed 4–8
times the activities than the previous acyloxy analogue, 3-O-dec-
anoyl-(þ)-C (9). For Gram-negative bacteria, the synthesized
compounds have shown less antibacterial activities compared to
2. Chemistry
kanamycin sulfate (MIC
¼
32–128 mg/mL) and ciprofloxacin
(MIC ¼ 0–0.125 g/mL). It has been observed that the introduction
m
The synthesis of the 3-O-alkyl-(þ)-C derivatives is outlined in
Scheme 1. For the introduction of an alkyl group at 3-hydroxy
group, the phenolic groups of (þ)-C (5) were benzylated by
treatment with benzyl bromide and K2CO3 to give 5,7,30,40-tetra-O-
benzyl-(þ)-catechin (6) in 74% yield. The treatment of the
benzylated intermediate (6) with cesium hydroxide (CsOH), tetra-
butylammonium iodide (TBAI) and various substituted benzyl
bromide or various alkyl iodide gave 5,7,30,40-tetra-O-benzyl-3-O-
alkyl-(þ)-catechins (7a–7l) in 46–72% yields. The debenzylation of
the alkylated compounds (7a–7l) was carried out with Pd/C in the
presence of H2 to give the corresponding target compounds 8a–8l
in 78–89% yields.
of a lipophilic substituent with appropriate sizes (8d–8f) on the 3-O
maximized the antimicrobial activities in the series of aliphatic
chain analogues. Also, structural change in the linker of an ester to
ether on the 3-O position significantly enhanced antimicrobial
activities.
3.2. Stability of compounds
We investigated the stability of 3-O-decyl-(þ)-C (8e) compared
with 3-O-decanoyl-(þ)-C (9) and (ꢀ)-EGCG (1) in aqueous PBS
buffer (pH 7.4). We observed that both compounds 8e and 9 were
considerably much more stable than (ꢀ)-EGCG (1) (Fig. 2). The data
OBn
OBn
OH
OH
OBn
OBn
BnO
O
BnO
O
HO
O
b
a
O
R
OH
OH
OBn
OBn
OH
5
6
7a-7l
OH
F
8a R = propyl
8b R = butyl
8i R =
OH
8k R =
8l R =
8c R = hexyl
8d R = octyl
HO
O
F
F
c
8e R = decyl
8f R = dodecyl
8g R = tetradecyl
OMe
OMe
F
F
O
R
8j R =
OH
8h R = hexadecyl
OMe
8a-8l
Scheme 1. Synthesis of 3-O-alkyl-(þ)-C analogues (8a–8l). Reagents and conditions: (a) benzyl bromide, K2CO3, DMF, rt, 20 h; (b) R–X, cesium hydroxide, tetrabutylammonium
iodide, DMF, ꢀ20 ꢁC, 20 h; (c) Pd/C, H2, MeOH, rt, 5 h.

1030
K.D. Park, S.J. Cho / European Journal of Medicinal Chemistry 45 (2010) 1028–1033
Table 1
Antibacterial activities of major catechins (1–5) and 3-O-alkyl-(þ)-C derivatives (8a–8l).
Test organisms
MIC (
mg/mL)
1
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
9a
Km Cpfx
Enterococcus faecalis ATCC 29212
Staphylococcus aureus ATCC 25923
Micrococcus luteus ATCC 10240
Staphylococcus epidermidis ATCC 0155
Bacillus Subtilis ATCC 6633
Escherichia coli ATCC 25922
Escherichia coli ATCC 10536
Proteus mirabilis ATCC 27853
Klebsiella pneumonia ATCC 10031
>128
>128
32
128
>128
64
64
32
64
128
32
16
8
16
64
8
4
2
8
16
4
2
1
2
4
2
1
0.5
1
2
2
1
1
2
2
16
8
4
8
8
32
16
16
16
32
32
64
32
64
64
64
32
16
32
32
8
16
8
16
32
8
16
8
16
32
16 32
0.5
0.25
<0.06
0.25
<0.06
0.125
0.06
0.125
<0.06
8
2
16
8
4
4
32
2
>128 >128 >128 >128
>128 >128 >128 >128 >128 128
>128 >128
>128 >128
64
64
64 >128 >128 >128 >128 128 128 >128
>128 >128 >128 >128
128 >128 >128
128 >128 >128
8
8
64
128
64
16
64 >128
16
128
128
128
128
64
64
32
32
128
128
32 32
32 64
128 128 128
Compounds 2–5 (MIC > 128) were not shown. Determined after 24 h of incubation at 37 ꢁC for the bacteria. All experiments were run in triplicates. Km ¼ Kanamycin sulfate.
Cpfx ¼ Ciprofloxacin.
a
3-O-decanoyl-(þ)-C [18].
showed that more than 90% and 75% of the initial concentration of
compounds 8e and 9, respectively, remained in the culture medium
after an incubation period of 16 h while only 15% of the initial
concentration of (ꢀ)-EGCG (1) remained (Fig. 2). Certain studies
have indicated the most active tea polyphenols are unstable in
neutral or alkaline medium [21]. Several possible mechanisms have
been reported for explaining their instability such as ester cleavage
of the gallate moiety and oxidation at the phenolic groups on B and
D rings [22]. As such, introduction of an aliphatic chain instead of
the reactive phenolic groups on D ring and absence of the pyro-
galloyl B-ring led to enhanced stability. Consistent with antimi-
crobial activities of both compounds, we observed that
introduction of alkyl group facilitated stability compared with acyl
group and this modification is likely to prevent the compounds
from not only enzymatic but also non-enzymatic cleavage in
culture medium.
supplemented in Fig. 4 that only compounds 8d–8f including
a suitable length of alkyl chain caused more calcein leakage than
compounds including shorter (8c) or longer chain (8g and 8h).
4. Discussion
Although (ꢀ)-EGCG (1) and (ꢀ)-ECG (3), major catechins of
green tea, have been shown various biological activities including
antioxidant, antimicrobial and anticancer, their instability due to
enzymatic or non-enzymatic cleavage of the 3-galloyl group has
hampered their clinical use. In this study, we synthesized a series of
catechin analogues to enhance not only antimicrobial activities but
also stability at pH 7.4. The synthesized compounds showed
increased levels of antimicrobial activities. Among them, 3-O-
decyl-(þ)-C (8e) was examined stability at pH 7.4 compared with 3-
O-decanoyl-(þ)-C (9) and EGCG (1). We found that the replacement
of the 3-galloyl group led to significant increased stability and the
introduction of an ether bond led to higher stability (more than
10%) than an ester bond at pH 7.4. These results indicate that ether
bond prevent from non-enzymatic cleavage more than ester bond.
Accordingly, 3-O-alkyl-(þ)-catechins are likely to minimize both
enzymatic and non-enzymatic cleavage in vivo assay.
3.3. Proposed action mechanism
The antimicrobial activities of (þ)-C derivatives increased with
elongation of an alkyl chain of suitable length (Table 1). Thus, we
presumed that the antibacterial activities of the (þ)-C derivatives
and their interaction with the bacterial membrane are closely
correlated.
Indeed, the percent incorporation of the (þ)-C derivatives (8a–
8l) into liposomes increased with elongation of the alkyl chain
lengths of the derivatives (Fig. 3). While c Log P value was signifi-
cantly increased (3.5–9) according to the increment of alkyl chain
length or the introduction of lipophilic substituents, their incor-
porative abilities were shown over 90% as long as c Log P value of
compounds is more than 4 in the series. Besides, (ꢀ)-EGCG (1) and
(þ)-C (5) were not incorporated well into the liposome membrane
out of proportion to the corresponding c Log P values possibly due
to many polar hydroxy groups on the molecule.
To propose action mechanism of the synthesized compounds,
we prepared model membrane using liposome. The synthesized
compounds showed significant ability of incorporation into lipo-
somes upon length of aliphatic chain. Despite almost 100% incor-
poration ability, compounds 8g and 8h including much longer
aliphatic chain (C14–C16
)
exhibited weaker activities than
compounds 8d–8f (C8–C12) (Fig. 3 and Tables 1 and 2). Consistent
with these findings, compounds including a suitable length of alkyl
chain (8d–8f) caused more calcein leakage than compounds
including shorter (8c) or longer chain (8g and 8h) (Fig. 4). Thus, it is
likely that the activity disrupting membrane structure in addition
to the amount incorporated into the lipid bilayers should affect the
antibacterial activities of (þ)-C derivatives such as compounds 8e
and 8f because the presence of the alkyl chain accelerated the
adsorption on the surface of the membrane and the disruption of
the membrane by increasing the lipophilicity.
The percent incorporation of compounds 8c–8h was almost
100%, but the antimicrobial activities of compounds 8g and 8h
including much longer aliphatic chain (C14–C16) were generally
weaker than those of compounds 8d–8f (C8–C12). It was
Table 2
Antifungal activities of major catechins (1–5) and 3-O-alkyl-(þ)-C derivatives (8a–8l).
Test organisms
MIC (
m
g/ml)
8c
8b
8d
8e
8f
8g
8h
8j
8k
8l
9a
AmpB
Itrc
Candida krusei IFO 1664
128
32
32
32
16
8
8
8
2
4
4
2
4
8
4
4
8
16
16
8
64
32
32
32
128
64
64
64
32
32
64
64
32
16
64
>128
64
32
0.5
0.5
0.25
0.5
0.25
0.125
0.06
0.06
Candida lusitaniae ATCC 42720
Candida albicans ATCC 10231
Candida tropicalis IFO 10241
64
32
16
32
128
16
Compounds 1–5, 8a and 8i (MIC > 128) were not shown. Determined after 24–72 h of incubation at 28–30 ꢁC for the fungi. All experiments were run in triplicates.
AmpB ¼ Amphotericin B. Itrc ¼ Itraconazole.
a
3-O-decanoyl-(þ)-C [18].

K.D. Park, S.J. Cho / European Journal of Medicinal Chemistry 45 (2010) 1028–1033
1031
100
100
80
60
40
20
0
OH
80
OH
8e
HO
O
60
40
20
0
9
O
EGCG (1)
OH
O
(CH2)8CH3
9
0
4
8
Time (hours)
12
16
8c
8d
8e
8f
8g
8h
Fig. 4. Effect of 3-O-alkyl analogues of (þ)-C (8c–8h) on calcein leakage.
Fig. 2. Stability of (ꢀ)-EGCG (1), 8e and 9 in standard culture condition in the absence
of cells.
extracted with Et2O (ꢂ2). The combined organic layers were dried
over Na2SO4, and concentrated in vacuo. The residue was purified by
silica gel chromatography (CH2Cl2) and recrystallized from ether/
MeOH to afford the title compound as an off-white solid (3.32 g, 74%
5. Conclusion
It has been paid much attention over last decade with respect to
the beneficial biological activities of its components, catechins. The
present work reports the chemical synthesis of 12 new 3-O-alkyl
analogues of (þ)-C (5), the antimicrobial activities, the stability in
pH 7.4, and possible action mechanism of activities using model
membrane. We showed that introducing of alkyl group instead of
acyl group enhanced activities and stability in pH 7.4. Using model
membrane, we suggested that the synthesized compounds showed
potent activities may effectively adsorb and disrupt the surface of
the membrane in microsome.
yield):mp125–126.5ꢁC;1HNMR (300 MHz, CDCl3)
d7.44–7.23 (m, 20
H), 7.03 (s, 1 H), 6.94 (s, 2 H), 6.27 (m, 1 H), 6.20 (m, 1 H), 5.17 (s, 2 H),
5.16 (m, 2 H), 5.03(s, 2 H), 4.98(s, 2 H), 4.63(d, J ¼ 8.5 Hz,1 H), 4.00 (m,
1 H), 3.10 (m, 1 H), 2.65 (m, 1 H), 1.58 (d, J ¼ 3.5 Hz, 1 H).
6.1.1.2. 5,7,30,40-Tetra-O-benzyl-3-O-alkyl-(þ)-catechins (7a–7l). Com-
pound 6 (2 g, 3.08 mmol) was reacted with tetrabutylammonium
iodide (TBAI) (1.5 equiv), cesium hydroxide (CsOH) (1.5 equiv), and
various substituted benzyl bromides or aliphatic alkyl iodides
(1.5 equiv) for 24 h at room temperature. The reaction mixture
was diluted with ethyl ether and washed successively with water.
The organic layer was dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica gel chromatography
(MeOH/CH2Cl2 1:90) to afford the title compound as off-white
solids in 46–72% yields.
6. Experimental
6.1. Chemistry
1H and 13C NMR spectra were obtained with a Varian Mercury
spectrometer at 300 and 75 MHz, respectively. 1H chemical shifts
(d) were reported in ppm downfield from internal Me4Si. Mass
spectra were measured by Fisons-VG platform in positive mode
electrospray ionization (ESI).
6.1.1.3. 3-O-Alkyl-(þ)-catechins (8a–8l). The debenzylation of
compounds 7a–7l was carried out with Pd/C in presence of H2 to
afford the title compounds 8a–8l (78–89% yields) as white sticky
foams.
8a: 1H NMR (DMSO-d6, 300 MHz)
d 9.30–8.84 (m, 4H, OH (A,B-
6.1.1. General procedure for the preparation of 3-O-alkyl-
(þ)-catechins (8a–8l)
ring)), 6.77–6.60 (m, 3H, aromatic proton (B-ring)), 5.91, 5.75 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.63 (d, J ¼ 7.5 Hz, 1H,
C2-H), 3.65 (m,1H, C3-H), 3.34, 3.13 (2m, 2H, CH2– at C3-O), 2.71 (dd,
J ¼ 16.4, 5.0 Hz, 1H, C4-Hequatorial), 2.40 (dd, J ¼ 15.5, 7.5 Hz, 1H, C4-
6.1.1.1. 5,7,30,40-Tetra-O-benzyl-(þ)-catechin (6). (þ)-Catechinhydrate
(2 g, 6.89 mmol) dissolved in N,N-dimethylformamide (DMF) was
reacted with K2CO3 (5.7 g, 41.34 mmol) and benzyl bromide (3.8 ml,
27.56 mmol) for 20 h at rt. To the resulting mixture were added water
and Et2O. The organic layer was separated, and the water layer was
Haxial), 1.28 (m, 2H, C3–O–CH2CH2CH3), 0.74 (t, J ¼ 7.3 Hz, 3H, C3–O–
27
CH2CH2CH3); [
z: 332.3.
a]
þ5.1ꢁ (c 0.70; DMSO); MS (positive ESI mode) m/
D
8b: 1H NMR (DMSO-d6, 300 MHz)
d 9.24–8.86 (m, 4H, OH (A,B-
100
80
60
40
20
0
10
8
ring)), 6.73–6.57 (m, 3H, aromatic proton (B-ring)), 5.89, 5.70 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.65 (d, J ¼ 7.2 Hz, 1H,
C2-H), 3.65 (m, 1H, C3-H), 3.39, 3.18 (2m, 2H, CH2- at C3-O), 2.67 (dd,
J ¼ 16.5, 5.1 Hz, 1H, C4-Hequatorial), 2.38 (dd, J ¼ 15.6, 7.5 Hz, 1H, C4-
Haxial), 1.36–1.09 (m, 4H, C3–O–CH2(CH2)2CH3), 0.77 (t, J ¼ 7.2 Hz,
6
27
3H, C3–O–CH2(CH2)2CH3); [
tive ESI mode) m/z: 346.4.
a
]
ꢀ62.5ꢁ (c 1.04; DMSO); MS (posi-
D
4
8c: 1H NMR (DMSO-d6, 300 MHz)
d 9.25–8.84 (m, 4H, OH (A,B-
ring)), 6.74–6.56 (m, 3H, aromatic proton (B-ring)), 5.89, 5.71 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.64 (d, J ¼ 7.1 Hz, 1H,
C2-H), 3.65 (m, 1H, C3-H), 3.39, 3.17 (2m, 2H, CH2- at C3-O), 2.66 (dd,
J ¼ 16.6, 5.3 Hz, 1H, C4-Hequatorial), 2.38 (dd, J ¼ 15.7, 7.5 Hz, 1H, C4-
2
cLogP
Incorporation
0
1 5 8a 8b 8c 8d 8e 8f 8g 8h 8i 8j 8k 8l
Haxial), 1.36–1.10 (m, 8H, C3–O–CH2(CH2)4CH3), 0.80 (t, J ¼ 7.0 Hz,
27
3H, C3–O–CH2(CH2)4CH3); [
tive ESI mode) m/z: 374.1.
a
]
þ12.4ꢁ (c 0.87; DMSO); MS (posi-
D
Fig. 3. Percent incorporation of (ꢀ)-EGCG (1), (þ)-C (5) and 3-O-alkyl analogues of
(þ)-C (8a–8l).

1032
K.D. Park, S.J. Cho / European Journal of Medicinal Chemistry 45 (2010) 1028–1033
8d: 1H NMR (DMSO-d6, 300 MHz)
d
9.24–8.83 (m, 4H, OH (A,B-
2H, aromatic proton (A-ring)), 4.81 (d, J ¼ 6.6 Hz, 1H, C2-H), 4.54–
4.33 (m, 2H, Ar-CH2– at C3-O), 3.75 (m, 1H, C3-H), 2.83 (dd, J ¼ 17.3,
ring)), 6.74–6.55 (m, 3H, aromatic proton (B-ring)), 5.89, 5.70 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.65 (d, J ¼ 6.9 Hz, 1H,
C2-H), 3.64 (m,1H, C3-H), 3.38, 3.17 (2m, 2H, CH2– at C3-O), 2.65 (dd,
J ¼ 16.7, 5.5 Hz, 1H, C4-Hequatorial), 2.38 (dd, J ¼ 15.8, 7.5 Hz, 1H, C4-
5.3 Hz, 1H, C4-Hequatorial), 2.64 (dd, J ¼ 16.4, 7.3 Hz, 1H, C4-Haxial);
27
[a
]
þ60.0ꢁ (c 0.6; DMSO); MS (positive ESI mode) m/z: 434.3.
D
Haxial), 1.38–1.08 (m, 12H, C3–O–CH2(CH2)6CH3), 0.80 (t, J ¼ 7.0 Hz,
6.2. Antimicrobial activity
27
3H, C3–O–CH2(CH2)6CH3); [
ESI mode) m/z: 402.4.
a]
þ6.5ꢁ (c 1.15; DMSO); MS (positive
D
The MIC is the lowest concentration of the antimicrobial agent
that prevents the development of viable growth after overnight
incubation [23]. MIC values of the synthesized compounds against
Gram-positive and Gram-negative test bacteria were determined
by method of NCCLS [24]. Muller Hinton agar (MHA) was used for
MIC determination. All the test cultures were streaked on the
soybean casein digest agar (SCDA) and incubated overnight at
37 ꢁC. Turbidity of all the bacterial cultures was adjusted to 0.5
McFarland standard by preparing bacterial suspension of four to six
well isolated colonies. The cultures were further diluted 10-fold to
get inoculum size of 1.2 ꢂ 107 CFU/mL. Stock solution of 4 mg/mL
was prepared in DMSO and was diluted to get final concentration of
8e: 1H NMR (DMSO-d6, 300 MHz)
d 9.23–8.83 (m, 4H, OH (A,B-
ring)), 6.73–6.55 (m, 3H, aromatic proton (B-ring)), 5.90, 5.70 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.65 (d, J ¼ 6.8 Hz, 1H,
C2-H), 3.64 (m, 1H, C3-H), 3.37, 3.16 (2m, 2H, CH2- at C3-O), 2.65 (dd,
J ¼ 16.7, 5.6 Hz, 1H, C4-Hequatorial), 2.37 (dd, J ¼ 15.8, 7.5 Hz, 1H, C4-
Haxial), 1.37–1.05 (m, 16H, C3–O–CH2(CH2)8CH3), 0.85 (t, J ¼ 6.6 Hz,
27
3H, C3–O–CH2(CH2)8CH3); [
tive ESI mode) m/z: 430.3.
a]
þ14.5ꢁ (c 1.00; DMSO); MS (posi-
D
8f: 1H NMR (DMSO-d6, 300 MHz)
d 9.23–8.84 (m, 4H, OH (A,B-
ring)), 6.72–6.56 (m, 3H, aromatic proton (B-ring)), 5.89, 5.70 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.65 (d, J ¼ 6.6 Hz, 1H,
C2-H), 3.64 (m, 1H, C3-H), 3.36, 3.15 (2m, 2H, CH2- at C3-O), 2.65 (dd,
J ¼ 16.8, 5.7 Hz, 1H, C4-Hequatorial), 2.37 (dd, J ¼ 15.9, 7.5 Hz, 1H, C4-
128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 mg/mL 320 mL of each
dilution was added to 20 mL cooled 45 ꢁC molten MHA (separate
flask was taken for each dilution). After thorough mixing, the
medium was poured in sterilized petri plates. The test bacterial
cultures were spotted in a predefined pattern by ascetically trans-
Haxial), 1.38–1.10 (m, 20H, C3–O–CH2(CH2)10CH3), 0.85 (t, J ¼ 6.3 Hz,
27
3H, C3–O–CH2(CH2)10CH3); [
tive ESI mode) m/z: 458.2.
a
]
þ48.2ꢁ (c 0.74; DMSO); MS (posi-
D
8g: 1H NMR (DMSO-d6, 300 MHz)
d
9.22–8.84 (m, 4H, OH (A,B-
ferring 10 mL of each culture on the surface of presolidified agar
ring)), 6.71–6.56 (m, 3H, aromatic proton (B-ring)), 5.89, 5.69 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.64 (d, J ¼ 6.6 Hz, 1H,
C2-H), 3.64 (m, 1H, C3-H), 3.36, 3.15 (2m, 2H, CH2– at C3-O), 2.65
(dd, J ¼ 16.8, 5.7 Hz, 1H, C4-Hequatorial), 2.36 (dd, J ¼ 15.9, 7.5 Hz, 1H,
plates. The spotted plates were incubated at 35 ꢁC for 24 h.
MIC values of the synthesized compounds against fungi were
determined by method of NCCLS [25]. RPMI-1640 broth was used
for MIC determination. All the test cultures were streaked of the
Sabouraud dextrose agar (SDA) and incubated overnight at 35 ꢁC.
Cell density adjusted with a spectrophotometer by adding suffi-
cient sterile saline to increase the transmittance to that produced
by a 0.5 McFarland standard. This procedure yielded a yeast stock
suspension of 1.0 ꢂ 106 to 5.0 ꢂ 106 cell/mL. A working suspension
is made by a 1:100 dilution followed by a 1:20 dilution of the stock
suspension with RPMI-1640 broth medium, which results in
5.0 ꢂ 102 to 2.5 ꢂ 103 cell/mL. Tubes are incubated at 35 ꢁC for 48 h
in ambient air.
C4-Haxial), 1.39–1.08 (m, 24H, C3–O–CH2(CH2)12CH3), 0.86 (t, J ¼ 6.3
27
Hz, 3H, C3–O–CH2(CH2)12CH3); [
(positive ESI mode) m/z: 486.4.
a]
þ47.1ꢁ (c 1.02; DMSO); MS
D
8h: 1H NMR (DMSO-d6, 300 MHz)
d 9.22–8.85 (m, 4H, OH (A,B-
ring)), 6.72–6.55 (m, 3H, aromatic proton (B-ring)), 5.90, 5.69 (2d,
J ¼ 2.4, 2.1 Hz, 2H, aromatic proton (A-ring)), 4.64 (d, J ¼ 6.5 Hz, 1H,
C2-H), 3.65 (m, 1H, C3-H), 3.36, 3.14 (2m, 2H, CH2– at C3-O), 2.64
(dd, J ¼ 16.9, 5.7 Hz, 1H, C4-Hequatorial), 2.35 (dd, J ¼ 15.9, 7.5 Hz, 1H,
C4-Haxial), 1.40–1.10 (m, 28H, C3–O–CH2(CH2)14CH3), 0.86 (t, J ¼ 6.2
27
Hz, 3H, C3–O–CH2(CH2)14CH3); [
(positive ESI mode) m/z: 514.4.
a
]
D
þ4.9ꢁ (c 1.04; DMSO); MS
6.3. Stability of 3-O-decyl- and decanoyl-(þ)-catechins (8e and 9)
8i: 1H NMR (DMSO-d6, 300 MHz)
d
9.30–8.79 (m, 4H, OH (A,B-
at pH 7.4
ring)), 7.46–6.84 (m, 5H, aromatic proton (Ar-CH2– at C3-O)), 6.74–
6.60 (m, 3H, aromatic proton (B-ring)), 5.90, 5.73 (2d, J ¼ 2.4, 2.1 Hz,
2H, aromatic proton (A-ring)), 4.68 (d, J ¼ 6.6 Hz, 1H, C2-H), 4.53–
4.41 (m, 2H, Ar-CH2– at C3-O), 3.85 (m, 1H, C3-H), 2.94 (dd, J ¼ 17.0,
To examine the stability of catechin derivatives in culture
medium, a 1% DMSO/aqueous 50 mM PBS buffer (pH 7.4, 10 mL)
containing either compound 8e (0.86 mg, 2
mmol, 200 mM) or 9
5.7 Hz, 1H, C4-Hequatorial), 2.50 (dd, J ¼ 16.2, 7.5 Hz, 1H, C4-Haxial);
(0.89 mg, 2 mol, 200
m
m
M) was incubated at 37 ꢁC. The pH of the
27
[
a]
þ9.8ꢁ (c 1.12; DMSO); MS (positive ESI mode) m/z: 380.2.
solution was measured both before and after each reaction and was
found to be within 0.1 pH unit of the listed pH value. The solutions
were analyzed by HPLC using a photodiode array detector (210–
D
8j: 1H NMR (DMSO-d6, 300 MHz)
d 9.42–8.87 (m, 4H, OH (A,B-
ring)), 7.43–6.92 (m, 2H, aromatic proton (Ar-CH2– at C3-O)), 6.77–
6.60 (m, 3H, aromatic proton (B-ring)), 5.88, 5.63 (2d, J ¼ 2.4, 2.1 Hz,
2H, aromatic proton (A-ring)), 4.74 (d, J ¼ 6.6 Hz, 1H, C2-H), 4.55–
4.34 (m, 2H, Ar-CH2– at C3-O), 3.85 (m, 1H, C3-H), 3.73 (m, 9H, CH3–
340 nm). Samples (50 mL) were injected onto a mBondapak C-18
column (3.9 ꢂ 300 mm, Waters Corp. Cat. No. WAT027324). A
mobile phase (35/65 CH3CN/H2O) was employed for 30 min using
a flow rate of 1 mL/min. The column was maintained at 37 ꢁC. The
relative percentage of catechin derivatives remaining was deter-
mined by integration of the HPLC peak compared with the internal
standard at 254 nm.
O–), 2.83 (dd, J ¼ 17.2, 5.5 Hz, 1H, C4-Hequatorial), 2.48 (dd, J ¼ 16.3,
27
7.4 Hz, 1H, C4-Haxial); [
mode) m/z: 470.4.
a
]
D
þ87.0ꢁ (c 1.00; DMSO); MS (positive ESI
8k: 1H NMR (DMSO-d6, 300 MHz)
d 9.34–8.85 (m, 4H, OH (A,B-
ring)), 7.41–6.91 (m, 3H, aromatic proton (Ar-CH2– at C3-O)), 6.63–
6.56 (m, 3H, aromatic proton (B-ring)), 5.93, 5.69 (2d, J ¼ 2.4, 2.1 Hz,
2H, aromatic proton (A-ring)), 4.74 (d, J ¼ 6.7 Hz, 1H, C2-H), 4.45–
4.25 (m, 2H, Ar-CH2- at C3-O), 3.75 (m, 1H, C3-H), 2.83 (dd, J ¼ 17.2,
6.4. Incorporation into liposome
The affinities of the synthesized compounds for lipid bilayers
were measured as previously reported with slight modification
[26]. Briefly, egg PC was dissolved in a small amount of chloroform
and solvent evaporated off with a rotary evaporator. The thin film of
egg PC was dried with a vacuum pump. An aqueous glucose solu-
tion (300 mM) was then poured into the flask, and the mixture was
5.4 Hz, 1H, C4-Hequatorial), 2.64 (dd, J ¼ 16.2, 7.4 Hz, 1H, C4-Haxial);
27
[
a]
þ35.8ꢁ (c 1.37; DMSO); MS (positive ESI mode) m/z: 416.3.
D
8l: 1H NMR (DMSO-d6, 300 MHz)
d 9.37–8.97 (m, 4H, OH (A,B-
ring)), 7.31–6.98 (m, 2H, aromatic proton (Ar-CH2– at C3-O)), 6.73–
6.54 (m, 3H, aromatic proton (B-ring)), 5.93, 5.72(2d, J ¼ 2.4, 2.1 Hz,

K.D. Park, S.J. Cho / European Journal of Medicinal Chemistry 45 (2010) 1028–1033
1033
sonicated using an ultrasonicator. The resulting solution of multi-
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5 min at 20 ꢁC. The final concentration of egg PC in the liposomal
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Acknowledgment
We thank Sulgi Lee and A Reum Han for helpful supporting and
discussion.