Cytotoxic N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-dipeptides: Structure-activity relationships and synergistic studies

Article abstract of DOI:10.1016/j.ejmech.2010.02.035

The synthesis and cytotoxic evaluation of a series of Fmoc-based dipeptides are described. Among the thirty compounds, 4a, 8a, 12a, 2b, 4b, 10b, 3c, 4c and 6c showed potent activity against HepG2, Hep3B, MCF-7, MDA-MB-231, A549 and Ca9-22 human cancer cell lines. The most active compounds (10a and 10c) showed relatively good sensitivity toward HepG2 and Ca9-22 cell lines with IC₅₀ values of 1.0 and 0.4 μM, respectively. Additionally, compound 10c was threefold more potent than doxorubicin, the positive control, against the Ca9-22 cell line. Furthermore, 10c showed a synergistic effect and increased the cytotoxicity of doxorubicin against the MDA-MB-231 cancer cell line. Therefore, 10c could be used as a new lead compound for therapeutic development.

Full text of DOI:10.1016/j.ejmech.2010.02.035

European Journal of Medicinal Chemistry 45 (2010) 2494e2502
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cytotoxic N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-dipeptides:
Structureeactivity relationships and synergistic studies
Chiao-Ting Yen a, Chin-Chung Wu a, Jin-Ching Lee d, Shu-Li Chen a, Susan L. Morris-Natschke c,
,
a,e,f,
**
Pei-Wen Hsieh b , Yang-Chang Wu
*
a Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b Graduate Institute of Natural Products, Chang Gung University, Tao-Yuan 333, Taiwan
c Natural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
d Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan
e Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 804, Taiwan
f National Sun Yat-Sen UniversityeKaohsiung Medical University Joint Research Center, Kaohsiung 804, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and cytotoxic evaluation of a series of Fmoc-based dipeptides are described. Among the
thirty compounds, 4a, 8a, 12a, 2b, 4b, 10b, 3c, 4c and 6c showed potent activity against HepG2, Hep3B,
MCF-7, MDA-MB-231, A549 and Ca9-22 human cancer cell lines. The most active compounds (10a and
10c) showed relatively good sensitivity toward HepG2 and Ca9-22 cell lines with IC50 values of 1.0 and
Received 5 September 2009
Received in revised form
12 February 2010
Accepted 15 February 2010
Available online 1 March 2010
0.4 mM, respectively. Additionally, compound 10c was threefold more potent than doxorubicin, the
positive control, against the Ca9-22 cell line. Furthermore, 10c showed a synergistic effect and increased
the cytotoxicity of doxorubicin against the MDA-MB-231 cancer cell line. Therefore, 10c could be used as
a new lead compound for therapeutic development.
Keywords:
Fmoc-based dipeptide
Cytotoxicity
Ó 2010 Elsevier Masson SAS. All rights reserved.
Human cancer cell line
HepG2
Ca9-22
1. Introduction
and antithrombotic activities [6]. Furthermore, the dipeptide
derivatives, linking with long chain fatty acid [7e9], triterpenoids
“Cancer” is, in fact, a general term that refers to over 100 distinct
diseases affecting many different tissues and cell types. However, all
forms of cancer are characterized by abnormal cell growth resulting
from a relatively small number of inherited or environmentally-
induced genetic mutations [1]. In the United States, cancer is the
leading cause of death for individuals less than 85 years of age [2].
Therefore, research and development of new generation anticancer
agents continue.
Certain peptides containing phenylalanine, serine, or alanine
residues have shown antitumor activity byenhancing nucleic acid or
protein metabolism [3]. Of these, dipeptide derivatives have been
recognized for decades and showed diverse biological activities,
including anti-inflammatory [4], antinoceptive [4], antimalarial [5]
[10e12], steroids [13,14], cerebrosides [15], glycolipids [16e20],
flavonoids [21], and vitamin A (such as retinal) [22], also exhibited
inhibitory effect on eukaryotic DNA polymerase
a [23]. Conversely,
dipeptide derivatives may be used as carriers for targeting cytotoxic
agents to tumor for chemotherapy [24,25].
More recently, a cyclodipeptide, cordycedipeptide A, showed
better effect on L-929, A375, and HeLa cancer cell lines [26]. In
addition, Matijing-Su (MTS, N-(N-benzoyl-L-phenylalanyl)-O-
acetyl-L-phenylalanol), a dipeptide derivative, was isolated from
a Chinese ethnic drug Matijing (Dichondra repens Forst.) which has
been widely used in the treatment of chronic liver diseases in China
[27]. Furthermore, the cytotoxicities and anti-hepatitis B virus (anti-
HBV) activities of MTS-derivatives were evaluated. A series of new
MTS-derivatives showed that both hydroxymethyl and methyl
carboxylate substituted on the C-terminal might be the pharmaco-
phores from the evaluation of their cytotoxicities and anti-HBV
activities [27]. Thus, in our research on anticancer agents, we eval-
uated of the Fmoc-acetate dipeptides [28] against HepG2 and Hep3B
(liver), MCF-7 and MDA-MB-231 (breast), Ca9-22 (gingival) and
A549 (lung) human cancer cell lines. The results showed that 2b, 6b
* Corresponding author. Tel.: þ886 3 211 8800x3105; fax: þ886 3 211 8643.
** Corresponding author at: Graduate Institute of Natural Products, Kaohsiung
Medical University, Kaohsiung 807, Taiwan. Tel.: þ886 7 312 1101x2197; fax: þ886
7 311 4773.
E-mail addresses: pewehs@mail.cgu.edu.tw (P.-W. Hsieh), yachwu@kmu.edu.tw
(Y.-C. Wu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.035

C.-T. Yen et al. / European Journal of Medicinal Chemistry 45 (2010) 2494e2502
2495
and 10b were equipotent against the tested cancer cell lines
(Table 1). Therefore, additional analogs were prepared to investigate
this new class of anticancer Fmoc-dipeptides. Additionally, because
combination therapy is frequently used clinically for treating cancer,
we also combined selected bioactive synthetic dipeptides with
doxorubicin in a fixed ratio concentration to evaluate possible
synergistic effects.
1be10b, 1ae10a and 1ce10c, respectively, as well as doxorubicin,
included as a positive control.
Regarding the cytotoxic activity of 1be10b (Table 1), examina-
tion of paired analogs confirmed that stereochemistry did affect
potency. S,R-Configured Fmoc-acetate dipeptides (2b, 4b, 6b and
8b) were more potent than S,S-configured analogs (1b, 3b, 5b and
7b) against most cell lines. Data comparison between analogs with
aromatic amino acids (1be8b) and analogs with aliphatic amino
acids (9b and 10b) showed no significant preference for either class.
However, for compounds with a single phenyl ring (1b, 2b, 7b and
8b) at N-terminal ones, analogs 7b and 8b (tyrosine) were less
active than 1b, 2b (phenylalanine). This observation suggested that
addition of an OH (7b and 8b) at the phenyl para-position
decreased cytotoxic activity.
Since Xu and his co-workers proposed hydroxyl substituted on
the C-terminal might increase the cytotoxicities [27]. Therefore, we
evaluated the cytotoxicity of Fmoc dipeptide analogs with
a hydroxyl group. Among ten Fmoc-alcohol dipeptides (1ae10a),
only compounds 6a (naphthalene), 8a (tyrosine), and 10a (valine)
were more potent than acetyl ones against HepG2 cancer cell line.
Additionally, 8a and 10a showed respectively the highest potency
against Ca9-22 and HepG2 human cancer cell lines, with IC50 values
2. Chemistry
Synthetic routes were similar to previously described pathway
[28]. The desired Fmoc-alcohol dipeptides (1ae10a) were inter-
mediates in this methodology. In addition, benzoates (1ce10c)
were prepared from 1ae10a, respectively, by esterification with
benzoic anhydride in pyridine to obtain the desired products. All
compounds (1a,b,ce10a,b,c, Tables 1e3) were >96% pure and were
analyzed by HPLC as tools and characterized by spectroscopic data,
as shown in the Experimental Section.
3. Results and discussion
The previously and newly synthesized compounds were
examined for in vitro cytotoxic activity against several human
cancer cell lines, including HepG2, Hep3B, MDA-MB-231, MCF-7,
A549 and Ca9-22. Tables 1e3 list the IC50 values obtained with
of 2.1 and 1.0 mM, respectively Compound 8a was significantly more
potent than 2a against six test cancer cell lines, indicating the
importance of an OH at the para-position of phenyl moiety in these
Table 1
Cytotoxic effects of Fmoc-acetate dipeptides.
O
H
R
B
N
A
HN
O
O
O
O
Compd.
Cancer cell line (IC50 value,a
m
M)
R
Config.
HepG2
Hep3B
MCF-7
A549
Ca9-22
MDA-MB-231
A
B
1b
2b
>55
>55
>55
>55
12.3 ꢀ 5.3 ꢁ 10ꢂ4
>55
>55
S
S
S
R
7.2 ꢀ 8.9 ꢁ 10ꢂ4
15.1 ꢀ 10.0 ꢁ 10ꢂ4
5.7 ꢀ 2.0 ꢁ 10ꢂ4
20.1 ꢀ 2.5 ꢁ 10ꢂ5
11.7 ꢀ 1.0 ꢁ 10ꢂ3
3b
4b
17.5 ꢀ 6.1 ꢁ 10ꢂ4
14.1 ꢀ 5.0 ꢁ 10ꢂ4
>55
>55
30.6 ꢀ 1.5 ꢁ 10ꢂ4
10.7 ꢀ 1.7 ꢁ 10ꢂ4
27.5 ꢀ 7.0 ꢁ 10ꢂ4
11.4 ꢀ 3.3 ꢁ 10ꢂ5
31.4 ꢀ 3.7 ꢁ 10ꢂ4
19.5 ꢀ 2.5 ꢁ 10ꢂ4
S
S
S
R
11.2 ꢀ 3.5 ꢁ 10ꢂ4
13.7 ꢀ 3.8 ꢁ 10ꢂ4
HN
5b
6b
28.3 ꢀ 1.6 ꢁ 10ꢂ5
26.9 ꢀ 10.0 ꢁ 10ꢂ4
>55
30.7 ꢀ 1.4 ꢁ 10ꢂ3
31.2 ꢀ 3.3 ꢁ 10ꢂ4
11.9 ꢀ 2.0 ꢁ 10ꢂ4
14.5 ꢀ 1.1 ꢁ 10ꢂ4
22.6 ꢀ 5.9 ꢁ 10ꢂ4
8.9 ꢀ 3.9 ꢁ 10ꢂ4
20.9 ꢀ 4.5 ꢁ 10ꢂ5
>55
S
S
S
R
26.6 ꢀ 2.6 ꢁ 10ꢂ5
7b
8b
14.9 ꢀ 1.62 ꢁ 10ꢂ3
18.8 ꢀ 3.6 ꢁ 10ꢂ4
>55
>55
>55
>55
>55
>55
>55
>55
S
S
S
R
HO
30.4 ꢀ 1.80 ꢁ 10ꢂ4
12.8 ꢀ 59 ꢁ 10ꢂ4
9b
10b
14.6 ꢀ 1.4 ꢁ 10ꢂ3
20.3 ꢀ 1.4 ꢁ 10ꢂ3
>55
>55
>55
>55
>55
Isopropyl
S
S
S
R
30.3 ꢀ 2.4 ꢁ 10ꢂ4
30.1 ꢀ 3.9 ꢁ 10ꢂ5
7.4 ꢀ 3.7 ꢁ 10ꢂ4
6.4 ꢀ 3.9 ꢁ 10ꢂ4
6.6 ꢀ 1.2 ꢁ 10ꢂ4
Doxo.b
0.5
0.6
0.7
0.4
1.3
0.1
a
IC50 represents the 50% inhibitory concentration. Results are presented as mean ꢀ S.E.M. (n ¼ 2).
b
Doxorubicin: positive control.

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C.-T. Yen et al. / European Journal of Medicinal Chemistry 45 (2010) 2494e2502
Table 2
Cytotoxic effects of Fmoc-alcohol dipeptides.
R
A
H
N
B
HN
O
OH
O
O
Compd.
Cancer cell line (IC50 value,a
m
M)
R
Config.
HepG2
Hep3B
MCF-7
A549
Ca9-22
MDA-MB-231
A
B
1a
2a
>55
>55
>55
>55
>55
>55
>55
>55
>55
>55
>55
>55
S
S
S
R
3a
4a
19.0 ꢀ 2.0 ꢁ 10ꢂ4
16.5 ꢀ 2.0 ꢁ 10ꢂ4
>55
>55
30.8 ꢀ 1.5 ꢁ 10ꢂ3
16.7 ꢀ 7.1 ꢁ 10ꢂ5
>55
>55
S
S
S
R
9.7 ꢀ 4.5 ꢁ 10ꢂ4
14.5 ꢀ 3.0 ꢁ 10ꢂ4
13.2 ꢀ 2.0 ꢁ 10ꢂ4
25.9 ꢀ 2.0 ꢁ 10ꢂ4
HN
5a
6a
25.8 ꢀ 3.3 ꢁ 10ꢂ4
5.8 ꢀ 1.7 ꢁ 10ꢂ4
>55
>55
>55
29.7 ꢀ 5.8 ꢁ 10ꢂ4
>55
13.6 ꢀ 4.5 ꢁ 10ꢂ5
12.1 ꢀ 1.6 ꢁ 10ꢂ5
>55
>55
S
S
S
R
15.6 ꢀ 2.5 ꢁ 10ꢂ4
7a
8a
17.4 ꢀ 7.5 ꢁ 10ꢂ5
2.5 ꢀ 2.6 ꢁ 10ꢂ4
15.5 ꢀ 1.3 ꢁ 10ꢂ4
3.7 ꢀ 2.8 ꢁ 10ꢂ4
>55
>55
24.4 ꢀ 2.3 ꢁ 10ꢂ4
2.1 ꢀ 1.9 ꢁ 10ꢂ4
>55
S
S
S
R
HO
21.3 ꢀ 8.8 ꢁ 10ꢂ4
18.8 ꢀ 4.5 ꢁ 10ꢂ4
31.5 ꢀ 1.3 ꢁ 10ꢂ3
9a
10a
9.7 ꢀ 2.5 ꢁ 10ꢂ4
1.0 ꢀ 4.5 ꢁ 10ꢂ5
>55
>55
>55
>55
>55
14.8 ꢀ 1.7 ꢁ 10ꢂ4
10.1 ꢀ 3.5 ꢁ 10ꢂ4
>55
>55
Isopropyl
S
S
S
R
35.2 ꢀ 2.5 ꢁ 10ꢂ3
Doxo.b
0.5
0.6
0.7
0.4
1.3
0.1
a
IC50 represents the 50% inhibitory concentration. Results are presented as mean ꢀ S.E.M. (n ¼ 2).
b
Doxorubicin: positive control.
analogs, but these results reversed the conclusion of C-terminal with
acetyl derivatives. Moreover, two compounds 4a and 8a showed
activity against all tested cancer cell lines (IC50 values of 2e30 M).
4b were more active than 3a, 3b, respectively). In contrast, with
tyrosine-phenylalaninol benzoate compounds, 7c (S,S) was more
potent than 8c (S,R) and 4c (S,R).
m
Interestingly, the same stereochemical preference (S,R more potent
than S,S) was also found.
On the other hand, all synthetics were also evaluated on the
anti-hepatitis C virus (anti-HCV) activities in Huh-7 cell model
[29,30]. Only compounds 4c and 8c showed inhibitory effects on
Certain Fmoc-benzoate dipeptides (3c, 4c and 6ce10c), which
have a conjugated aromatic ester attached to the phenylalaninol
hydroxyl, showed better activity compared with corresponding
Fmoc-acetate (b series) and Fmoc-alcohol (a series) dipeptides,
particularly against the Ca9-22 human gingival cancer cell line.
the replication of HCV at 10
mM (the inhibition percentages were
72% and 87%, respectively), but they were no active at 5
mM. The
results also found the stereochemical preference for S,R-forms. In
addition, compounds 4c and 8c could be as lead compounds for
anti-HCV agents.
With an impressive IC50 value of 0.4 mM, 10c [O(CO)C6H5] was more
potent than doxorubicin, the positive control, as well as 16-fold
more active than 10b [O(CO)CH3] and 40-fold more active than 10a
(OH) against this cell line. These findings suggest that inhibition of
Ca9-22 cell growth is increased by introduction of an aromatic ring
ester at the phenylalaninol. Compound 9c, a stereochemical isomer
of 10c, was less potent but selectively active against Ca9-22 (IC50
4. Conclusion
Several previously synthesized novel anti-inflammatory Fmoc-
acetate dipeptide analogs also showed in vitro cytotoxic activity,
which led to the synthesis of additional compounds, including
Fmoc-hydroxy and -benzoate analogs as highly potent cytotoxic
agents. The cytotoxicity results showed that Fmoc-alcohol dipep-
tides 4a and 8a presented cytotoxicity against all cell lines.
However, 10a emerged as a noteworthy candidate for future
development in terms of potency and selective cytotoxicity, espe-
cially toward the HepG2 cell line. Additionally, when the hydroxyl
in the phenylalaninol of 10a was esterified as a benzoate group, the
data revealed that the resulting 10c was the most potent compound
against the Ca9-22 cell line. It was active at the nanomolar level and
2.9
(Hep3B).
The data also demonstrated that Fmoc tryptophan-
phenylalaninol benzoate dipeptides were active but relatively non-
selective; compounds 3c and 4c had IC50 values of 4.3e23.2
mM), while 10c showed activity against only one other cell line
mM
against the tested cell lines. With these two compounds, the S,R-
configured isomer (4c) was generally equipotent or more potent
than the S,S-configured isomer (3c), consistent with the trend found
for the corresponding paired acetate and hydroxyl compounds (4a,

C.-T. Yen et al. / European Journal of Medicinal Chemistry 45 (2010) 2494e2502
2497
Table 3
Cytotoxic effects of Fmoc-benzoate dipeptides.
O
R
H
N
B
A
HN
O
O
O
O
Compd.
Cancer cell line (IC50 value,a
m
M)
R
Config.
HepG2
Hep3B
MCF-7
A549
Ca9-22
MDA-MB-231
A
B
1c
2c
>55
>55
>55
>55
>55
29.8 ꢀ 8.9 ꢁ 10ꢂ4
>55
>55
>55
>55
S
S
S
R
18.9 ꢀ 5. ꢁ 10ꢂ4
1.9 ꢀ 1.9 ꢁ 10ꢂ4
3c
4c
14.2 ꢀ 3. ꢁ 10ꢂ4
23.2 ꢀ 3. ꢁ 10ꢂ5
7.3 ꢀ 4.5 ꢁ 10ꢂ5
4.3 ꢀ 1.4 ꢁ 10ꢂ4
12.2 ꢀ 2. ꢁ 10ꢂ4
11.0 ꢀ 2. ꢁ 10ꢂ4
5.5 ꢀ 4.5 ꢁ 10ꢂ5
4.7 ꢀ 1.2 ꢁ 10ꢂ4
10.8 ꢀ 8. ꢁ 10ꢂ4
3.9 ꢀ 2.1 ꢁ 10ꢂ4
19.6 ꢀ 1.0 ꢁ 10ꢂ3
10.2 ꢀ 5.3 ꢁ 10ꢂ4
S
S
S
R
HN
5c
6c
>55
>55
>55
>55
>55
>55
>55
S
S
S
R
26.9 ꢀ 4. ꢁ 10ꢂ5
26.6 ꢀ 4 ꢁ 10ꢂ5
31.2 ꢀ 2. ꢁ 10ꢂ4
14.5 ꢀ 4.5 ꢁ 10ꢂ5
8.9 ꢀ 4.5 ꢁ 10ꢂ5
7c
8c
>55
>55
7.0 ꢀ 4.7 ꢁ 10ꢂ5
>55
9.7 ꢀ 1.8 ꢁ 10ꢂ3
>55
5.8 ꢀ 2.8 ꢁ 10ꢂ5
23.8 ꢀ 3.7 ꢁ 10ꢂ4
6.4 ꢀ 4.8 ꢁ 10ꢂ4
10.4 ꢀ 5.6 ꢁ 10ꢂ4
24.2 ꢀ 4.5 ꢁ 10ꢂ4
>55
S
S
S
R
HO
9c
10c
>55
>55
>55
>55
>55
>55
>55
2.9 ꢀ 3.5 ꢁ 10ꢂ5
0.4 ꢀ 1.7 ꢁ 10ꢂ5
>55
>55
Isopropyl
S
S
S
R
8.3 ꢀ 3.5 ꢁ 10ꢂ4
Doxo.b
0.5
0.6
0.7
0.4
1.3
0.1
a
IC50 represents the 50% inhibitory concentration. Results are presented as mean ꢀ S.E.M. (n ¼ 2).
b
Doxorubicin: positive control.
threefold more potent than doxorubicin, the positive control. The
were measured on a JASCO J-810 spectrometer in CH3CN. NMR
spectra, which contained 1D (1H, 13C, DEPT) and 2D (COSY, TOCSY,
HSQC, HMBC and NOESY), were performed in 400 and 100 MHz for
possible synergistic effects of compounds 10a and 10c with doxo-
rubicin were also investigated (Table 4). A 1:1 combination of 14c
with doxorubicin significantly increased cytotoxicity against MDA-
MB-231, compared to the activity of doxorubicin alone.
Overall, in this paper we report the cytotoxicity of novel Fmoc-
based dipeptides and the discovery of a synergistic combination of
10c and doxorubicin. The investigation should help to identify
small dipeptide molecules as lead compounds for therapeutic
development.
1H and 13C, respectively. 1H NMR: C5D5N as solvent,
d
¼ 7.21, 7.59,
8.72 ppm and 13C NMR: C5D5N as solvent,
d
¼ 123.5, 135.3,
149.8 ppm. Low resolution ESI-MS spectra were obtained on an API
3000Ô (Applied Biosystems) in positive or negative mode (solvent:
CH3OH), high resolution ESI-MS spectra on a Bruker Daltonics APEX
II 30e spectrometer in positive or negative mode (solvent: CH3OH).
5.1.1. General procedure for synthesis of compounds 1ae6a and
9ae10a
5. Experimental
Fmoc-L-amino acid (1.0 mmol) was introduced to L- or D-phe-
nylalaninol (1.0 mmol) in CH2Cl2 (10 ml) by using with HBTU
(1.5 mmol) and DIEA (1.5 mmol), and the mixture was stirred at rt
for 6 h. The residue obtained after evaporation of the solvent was
purified by column chromatography over silica gel using CHCl3 as
eluent to afford the products.
5.1. Materials and methods
Unless stated otherwise, the chemicals were acquired from
commercial sources and used without further purification. All
amino acids were purchased from ACROS, Sigma and Aldrich.
Compound purity was checked by high performance liquid chro-
matography (HPLC, Supleco 5
m
m, 4.6 mm ꢁ 250 mm C-18) and
5.1.1.1. (9H-Fluoren-9-yl)methyl (S)-1-((S)-1-hydroxy-3-phenylpropan-
measured by two diverse conditions, one is MeCN and the other is
90% MeOH as isocratic system. Melting points were determined on
a Melt-Temp II apparatus, optical rotations were measured with
JASCO P-1020 digital polarimeter, UV spectra were obtained on
a Hitachi 200-20 spectrophotometer in CH3CN, and CD spectra
2-ylamino)-1-oxo-3-phenylpropan-2-ylcarbamate (1a). Yield: 55%,
[
a
]
¼ ꢂ9.0ꢃ (c 0.1, CH3OH). Mp: 202e204 ꢃC. 1H NMR (C5D5N,
D
400 MHz):
d
¼ 3.12 (1H, dd, J ¼ 7.0, 13.4 Hz, H-9a), 3.24e3.31 (2H,
m, H-8a, 9b), 3.49 (1H, dd, J ¼ 6.4, 13.6 Hz, H-8b), 3.89 (2H, t,
J ¼ 5.2 Hz, H-1a, b), 4.26 (1H, t, J ¼ 7.2 Hz, Fmoc CH), 4.40 (1H, dd,

2498
C.-T. Yen et al. / European Journal of Medicinal Chemistry 45 (2010) 2494e2502
J ¼ 7.0, 10.6 Hz, Fmoc CH2), 4.58 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2),
4.69e4.77 (1H, m, H-2), 5.09e5.14 (1H, m, H-5), 7.13e7.19 (2H, m,
Ar CH), 7.22e7.28 (6H, m, Ar CH, Fmoc Ar CH), 7.28e7.35 (2H, m,
Fmoc Ar CH), 7.39e7.42 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.61 (2H, d,
J ¼ 7.6 Hz, Fmoc Ar CH), 7.84 (2H, d, J ¼ 8.0 Hz, Fmoc Ar CH), 8.95
(1H, d, J ¼ 8.4 Hz, NH-3), 9.05 (1H, d, J ¼ 8.8 Hz, NH-6). 13C NMR
(C-5), 63.0 (C-2), 66.7 (Fmoc CH2), 111.5, 111.9, 119.2, 121.7, 124.3,
128.6, 137.5 (Trp Ar CH), 120.4, 125.7, 125.8, 127.5, 128.0 (Fmoc Ar
CH), 126.4, 128.6, 129.9, 139.6 (Ar CH), 141.6, 144.6 (Fmoc Ar quat C),
157.1 (Fmoc CO), 172.7 (C-4). HRESI-MS m/z 582.2525 [M þ Na]þ
(calcd for C35H33N3O4Na 582.6596).
(C5D5N, 100 MHz):
d
¼ 37.5 (C-9), 39.2 (C-8), 47.7 (Fmoc CH), 53.6
5.1.1.5. (9H-Fluoren-9-yl)methyl(S)-1-((S)-1-hydroxy-3-phenylpropan-
(C-1), 57.3 (C-5), 62.9 (C-2), 66.7 (Fmoc CH2), 120.4, 125.6, 125.8,
127.5, 128.0 (Fmoc Ar CH), 126.4, 126.8, 128.6, 129.9, 130.0, 138.5,
139.5 (Ar CH), 141.6, 144.6 (Fmoc Ar quat C), 157.0 (Fmoc CO), 172.1
(C-4). HRESI-MS m/z 543.2416 [M þ Na]þ (calcd for C35H34N2O5Na
543.6236).
2-ylamino)-3-(naphthalen-2-yl)-1-oxopropan-2-ylcarbamate
(5a).
Yield: 50%, [
a
]D ¼ ꢂ16.3ꢃ (c 0.1, CH3OH). Mp: 202e204 ꢃC. 1H NMR
(C5D5N, 400 MHz):
d
¼ 3.12 (1H, dd, J ¼ 6.8, 13.6 Hz, H-9a), 3.26 (1H,
dd, J ¼ 6.8, 13.6 Hz, H-9b), 3.44 (1H, dd, J ¼ 8.4, 13.6 Hz, H-8a), 3.66
(1H, dd, J ¼ 6.2, 13.4 Hz, H-8b), 3.90 (2H, dd, J ¼ 5.0, 9.0 Hz, H-1a, b),
4.21 (1H, t, J ¼ 7.2 Hz, Fmoc CH), 4.36 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc
CH2), 4.51 (1H, dd, J ¼ 7.0, 10.6 Hz, Fmoc CH2), 4.71e4.77 (2H, m, H-
2, 5), 7.15 (2H, q, J ¼ 15.4 Hz, Ar CH), 7.11e7.25 (7H, m, Ar CH, Fmoc
Ar CH), 7.31e7.44 (8H, m, Ar CH, Fmoc Ar CH), 7.46e7.51 (2H, m, Ar
CH), 7.72e7.79 (4H, m, Ar CH, Fmoc Ar CH), 7.82 (2H, d, J ¼ 7.6 Hz,
Fmoc Ar CH), 9.12 (2H, t, J ¼ 7.8 Hz, NH-3, 6). 13C NMR (C5D5N,
5.1.1.2. (9H-Fluoren-9-yl)methyl(S)-1-((R)-1-hydroxy-3-phenylpropan-
2-ylamino)-1-oxo-3-phenylpropan-2-ylcarbamate (2a). Yield: 59%,
[
a
]
¼ þ5.7ꢃ (c 0.1, CH3OH). Mp: 204e207 ꢃC. 1H NMR (C5D5N,
D
400 MHz):
d
¼ 3.09 (1H, dd, J ¼ 7.2, 13.6 Hz, H-9a), 3.20e3.26 (2H,
m, H-8a, 9b), 3.43 (1H, dd, J ¼ 6.9, 12.4 Hz, H-8b), 3.94 (2H, d,
J ¼ 4.8 Hz, H-1a, b), 4.24 (1H, t, J ¼ 7.2 Hz, Fmoc CH), 4.36 (1H, dd,
J ¼ 7.0, 10.6 Hz, Fmoc CH2), 4.56 (1H, dd, J ¼ 6.8, 10.4 Hz, Fmoc CH2),
4.71e4.79 (1H, m, H-2), 5.07e5.12 (1H, m, H-5), 7.18e7.37 (10H, m,
Ar CH, Fmoc Ar CH), 7.40 (4H, d, J ¼ 8.0 Hz, Ar CH, Fmoc Ar CH), 7.59
(2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.83 (2H, d, J ¼ 8.0 Hz, Fmoc Ar CH),
8.93 (1H, d, J ¼ 8.4 Hz, NH-3), 9.01 (1H, d, J ¼ 8.8 Hz, NH-6). 13C NMR
100 MHz):
d
¼ 37.4 (C-9), 39.3 (C-8), 47.5 (Fmoc CH), 53.6 (C-2), 57.3
(C-5), 62.9 (C-1), 66.7 (Fmoc CH2), 120.3, 125.5, 125.6, 125.7, 127.1,
128.1 (Fmoc Ar CH), 110.0, 119.9, 124.4, 126.4, 127.4, 128.0, 128.3,
128.4, 128.6, 129.8, 132.7, 133.9, 136.1, 139.5 (Ar CH), 141.5, 144.4
(Fmoc Ar quat C), 157.1 (Fmoc CO), 172.1 (C-4). HRESI-MS m/z
593.2573 [M þ Na]þ (calcd for C37H34N2O4Na 593.6823).
(C5D5N, 100 MHz):
d
¼ 37.6 (C-9), 39.4 (C-8), 47.6 (Fmoc CH), 53.5
(C-1), 57.3 (C-5), 63.0 (C-2), 66.6 (Fmoc CH2), 120.4, 125.6, 125.7,
127.5, 128.0 (Fmoc Ar CH), 126.5, 126.8, 128.7, 129.9, 130.0, 138.4,
139.6 (Ar CH), 141.6, 144.6 (Fmoc Ar quat C), 157.0 (Fmoc CO), 172.0
(C-4). HRESI-MS m/z 543.2416 [M þ Na]þ (calcd for C35H34N2O5Na
543.6236).
5.1.1.6. (9H-Fluoren-9-yl)methyl (S)-1-((S)-1-hydroxy-3-phenylpropan-
2-ylamino)-3-(naphthalen-2-yl)-1-oxopropan-2-ylcarbamate
(6a).
Yield: 52%, [
a
]D ¼ þ5.2ꢃ (c 0.1, CH3OH). Mp: 202e204 ꢃC. 1H NMR
(C5D5N, 400 MHz):
d
¼ 3.09 (1H, dd, J ¼ 7.6, 13.6 Hz, H-9a), 3.20 (1H,
dd, J ¼ 6.4, 13.6 Hz, H-8a), 3.39 (1H, dd, J ¼ 7.8, 13.4 Hz, H-9b), 3.58
(1H, dd, J ¼ 6.2, 13.8 Hz, H-8b), 3.94 (2H, m, H-1a, b), 4.20 (1H, t,
J ¼ 7.0 Hz, Fmoc CH), 4.33 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2), 4.50
(1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2), 4.72e4.81 (1H, m, H-2), 5.20
(1H, q, J ¼ 15.8 Hz, H-5), 7.15 (2H, q, J ¼ 15.4 Hz, Ar CH), 7.16e7.26
(6H, m, Ar CH), 7.33e7.40 (4H, m, Fmoc Ar CH), 7.41e7.48 (4H, m, Ar
CH), 7.55 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.74e7.78 (2H, m, Fmoc Ar
CH), 7.81 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 9.05 (1H, d, J ¼ 8.4 Hz, NH-
6), 9.11 (1H, d, J ¼ 8.4 Hz, NH-3). 13C NMR (C5D5N, 100 MHz):
5.1.1.3. (9H-Fluoren-9-yl)methyl (S)-1-((S)-1-hydroxy-3-phenylpropan-
2-ylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-ylcarbamate (3a). Yield:
53%, [
400 MHz):
a
]
¼ ꢂ10.5ꢃ (c 0.1, CH3OH). Mp: 162e165 ꢃC. 1H NMR (C5D5N,
D
d
¼ 3.13 (1H, dd, J ¼ 6.8, 13.6 Hz, H-9a), 3.25 (1H, dd,
J ¼ 6.8, 13.6 Hz, H-9b), 3.64 (1H, dd, J ¼ 7.6, 14.4 Hz, H-8a), 3.80 (1H,
dd, J ¼ 6.2,14.6 Hz, H-8b), 3.88 (2H, t, J ¼ 5.4 Hz, H-1a, b), 4.27 (1H, t,
J ¼ 7.2 Hz, Fmoc CH), 4.43 (1H, dd, J ¼ 7.6, 10.4 Hz, Fmoc CH2), 4.57
(1H, dd, J ¼ 7.6, 10.4 Hz, Fmoc CH2), 4.71e4.77 (1H, m, H-2),
5.23e5.29 (1H, m, H-5), 7.11e7.16 (2H, m, Trp Ar CH), 7.22e7.26 (5H,
m, Ar CH, Fmoc Ar CH), 7.36e7.42 (4H, m, Trp Ar CH), 7.49e7.52 (2H,
m, Fmoc Ar CH), 7.62 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.83 (2H, d,
J ¼ 7.6 Hz, Fmoc Ar CH), 7.87 (1H, d, J ¼ 8.0 Hz, Fmoc Ar CH), 8.93
(2H, m, NH-3, 6), 11.9 (1H, s, Trp NH). 13C NMR (C5D5N, 100 MHz):
d
¼ 37.5 (C-9), 39.5 (C-8), 47.5 (Fmoc CH), 53.5 (C-2), 57.3 (C-5), 63.0
(C-1), 66.7 (Fmoc CH2), 120.3, 125.5, 125.6, 125.8, 127.1, 128.2 (Fmoc
Ar CH), 110.0, 119.9, 124.4, 126.3, 127.4, 128.0, 128.3, 128.4, 128.6,
129.9, 132.87,133.9, 136.01, 139.5 (Ar CH), 141.5, 144.4 (Fmoc Ar quat
C), 157.0 (Fmoc CO), 172.1 (C-4). HRESI-MS m/z 593.2573 [M þ Na]þ
(calcd for C37H34N2O4Na 593.6823).
d
¼ 29.3 (C-9), 37.5 (C-8), 47.7 (Fmoc CH), 53.6 (C-1), 57.0 (C-5), 62.9
(C-2), 66.7 (Fmoc CH2), 111.5, 111.9, 119.2, 121.7, 124.3, 128.6, 137.5
(Trp Ar CH), 120.4, 125.7, 125.8, 127.5, 128.0 (Fmoc Ar CH), 126.4,
128.6, 129.9, 139.6 (Ar CH), 141.6, 144.6 (Fmoc Ar quat C), 157.1
(Fmoc CO), 172.7 (C-4). HRESI-MS m/z 582.2525 [M þ Na]þ (calcd
for C35H33N3O4Na 582.6596).
5.1.1.7. (9H-Fluoren-9-yl)methyl (S)-1-((S)-1-hydroxy-3-phenylpropan-
2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate (9a). Yield: 57%,
[
a
]D ¼ ꢂ23.7ꢃ (c 0.1, CH3OH). Mp: 206e208 ꢃC. 1H NMR (C5D5N,
400 MHz):
d
¼ 1.08 (6H, t, J ¼ 7.2 Hz, H-11a, b, c, 12a, b, c), 2.37e2.46
(1H, m, H-10), 3.13 (1H, dd, J ¼ 6.8, 13.6 Hz, H-9a), 3.25 (1H, dd,
J ¼ 6.8, 13.6 Hz, H-9b), 4.00 (2H, t, J ¼ 6.0 Hz, H-1), 4.35 (1H, t,
J ¼ 7.2 Hz, Fmoc CH), 4.54 (1H, dd, J ¼ 6.8, 10.4 Hz, H-8a), 4.64e4.73
(2H, m, H-2, 8b), 4.74e4.82 (1H, m, H-5), 7.14 (1H, t, J ¼ 7.4 Hz, Ar
CH), 7.23e7.29 (4H, m, Ar CH, Fmoc Ar CH), 7.38e7.42 (4H, m, Ar CH,
Fmoc Ar CH), 7.70 (2H, dd, J ¼ 7.6 Hz, Fmoc Ar CH), 7.85 (2H, d,
J ¼ 7.2 Hz, Fmoc Ar CH), 8.76 (1H, d, J ¼ 8.4 Hz, NH-6), 8.99 (1H, d,
5.1.1.4. (9H-Fluoren-9-yl)methyl (S)-1-((S)-1-hydroxy-3-phenylpropan-
2-ylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-ylcarbamate (4a). Yield:
52%, [
a
]D ¼ þ18.2ꢃ (c 0.1, CH3OH). 1H NMR (C5D5N, 400 MHz):
d
¼ 3.05 (1H, dd, J ¼ 7.0, 13.4 Hz, H-9a), 3.18 (1H, dd, J ¼ 6.8, 14.2 Hz,
H-9b), 3.59 (1H, dd, J ¼ 7.4, 14.2 Hz, H-8a), 3.73 (1H, dd, J ¼ 6.6,
14.2 Hz, H-8b), 3.93 (2H, d, J ¼ 4.8 Hz, H-1a, b), 4.25 (1H, t,
J ¼ 7.2 Hz, Fmoc CH), 4.38 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2), 4.55
(1H, dd, J ¼ 7.6, 10.4 Hz, Fmoc CH2), 4.77e4.81 (1H, m, H-2),
5.20e5.26 (1H, m, H-5), 7.16e7.20 (2H, m, Trp Ar CH), 7.24e7.29
(3H, m, Trp Ar CH, Ar CH), 7.34e7.39 (4H, m, Ar CH), 7.55 (2H, d,
J ¼ 8.0 Hz, Fmoc Ar CH), 7.59e7.62 (4H, m, Fmoc Ar CH), 7.82 (2H, d,
J ¼ 7.6 Hz, Fmoc Ar CH), 7.92 (1H, d, J ¼ 8.4 Hz, Trp Ar CH), 8.94 (2H,
dd, J ¼ 8.0, 12.4 Hz, NH-3, 6), 11.9 (1H, s, Trp NH). 13C NMR (C5D5N,
J ¼ 8.4 Hz, NH-3). 13C NMR (C5D5N, 100 MHz):
¼ 18.7 (C-11), 19.8
d
(C-12), 31.6 (C-10), 37.6 (C-9), 47.8 (Fmoc CH), 53.5 (C-1), 61.7 (C-5),
63.1 (C-2), 66.6 (Fmoc CH2), 120.4, 125.7, 127.5, 128.0 (Fmoc Ar CH),
126.4, 128.6, 129.9, 139.6 (Ar CH), 141.7, 144.6, 144.8 (Fmoc Ar quat
C), 157.4 (Fmoc CO), 172.2 (C-7). HRESI-MS m/z 495.2416 [M þ Na]þ
(calcd for C29H32N2O4Na 495.5808).
5.1.1.8. (9H-Fluoren-9-yl)methyl(S)-1-((R)-1-hydroxy-3-phenylpropan-
2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate (10a). Yield: 56%,
100 MHz):
d
¼ 29.6 (C-9), 37.5 (C-8), 47.7 (Fmoc CH), 53.5 (C-1), 57.1

C.-T. Yen et al. / European Journal of Medicinal Chemistry 45 (2010) 2494e2502
2499
[
a
]
¼ þ10.4ꢃ (c 0.1, CH3OH). Mp: 206e208 ꢃC. 1H NMR (C5D5N,
(log
400 MHz): d
3
): 264 (3.79), 218 (4.31), 210 (4.37). 1H NMR (C5D5N,
¼ 3.12 (2H, t, J ¼ 8.2 Hz, H-10a, b), 3.60 (1H, dd, J ¼ 7.6,
D
400 MHz):
d
¼ 1.00 (6H, t, J ¼ 7.0 Hz, H-11a, b, c,12a, b, c), 2.28e2.37
(1H, m, H-10), 3.16 (1H, dd, J ¼ 8.2, 13.8 Hz, H-9a), 3.34 (1H, dd,
J ¼ 6.2, 13.8 Hz, H-9b), 3.98 (2H, t, J ¼ 4.8 Hz, H-1), 4.32 (1H, t,
J ¼ 7.0 Hz, Fmoc CH), 4.47 (1H, dd, J ¼ 7.0, 10.6 Hz, H-8a), 4.59e4.69
(2H, m, H-2, 8b), 4.79e4.87 (1H, m, H-5), 7.23e7.28 (3H, m, Ar CH),
7.32 (2H, t, J ¼ 7.6 Hz, Fmoc Ar CH), 7.36e7.41 (2H, m, Ar CH), 7.46
(2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.67 (2H, dd, J ¼ 6.4 Hz, Fmoc Ar CH),
7.84 (2H, d, J ¼ 7.8 Hz, Fmoc Ar CH), 8.74 (1H, d, J ¼ 8.4 Hz, NH-6),
14.4 Hz, H-9a), 3.77 (1H, dd, J ¼ 7.7, 14.2 Hz, H-9b), 4.26 (1H, t,
J ¼ 7.2 Hz, Fmoc CH), 4.43 (1H, dd, J ¼ 7.0, 10.6 Hz, Fmoc CH2),
4.46e4.53 (2H, m, H-2a, Fmoc CH2), 4.55e4.59 (1H, m, H-2b),
4.97e5.01 (1H, m, H-6), 5.22 (1H, q, J ¼ 15.2 Hz, H-3), 7.11 (1H, t,
J ¼ 7.2 Hz, Trp Ar CH), 7.15 (1H, t, J ¼ 7.2 Hz, Trp Ar CH), 7.22e7.26
(8H, m, Ar CH), 7.34e7.42 (3H, t, J ¼ 7.0 Hz, Trp Ar CH), 7.45e7.52
(4H, m, Fmoc Ar CH), 7.61 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.82 (3H, t,
J ¼ 6.4 Hz, Fmoc Ar CH, Ar CH), 8.13 (2H, d, J ¼ 7.2 Hz, Ar CH), 9.11
(1H, d, J ¼ 8.0 Hz, NH-4), 9.39 (1H, d, J ¼ 8.4 Hz, NH-7), 11.8 (1H, s,
8.85 (1H, d, J ¼ 8.4 Hz, NH-3). 13C NMR (C5D5N, 100 MHz):
¼ 18.3
d
(C-11), 19.7 (C-12), 31.8 (C-10), 37.8 (C-9), 47.8 (Fmoc CH), 53.5 (C-
1), 61.5 (C-5), 63.6 (C-2), 66.6 (Fmoc CH2), 120.4, 125.6, 125.7, 127.5,
128.0 (Fmoc Ar CH), 126.5, 128.7, 129.9, 139.7 (Ar CH), 141.7, 144.5,
144.7 (Fmoc Ar quat C), 157.4 (Fmoc CO), 172.1 (C-7). HRESI-MS m/z
495.2416 [M þ Na]þ (calcd for C29H32N2O4Na 495.5808).
Trp NH). 13C NMR (C5D5N, 100 MHz):
d
¼ 29.4 (C-10), 37.6 (C-11),
47.7 (Fmoc CH), 50.3 (C-2), 57.0 (C-6), 65.8 (C-3), 66.8 (Fmoc CH2),
111.3, 112.0, 119.1, 121.7, 124.2, 128.5, 137.4 (Trp Ar CH), 120.4, 125.7,
125.8, 127.5, 128.0 (Fmoc Ar CH), 126.8, 128.7, 129.7, 130.0, 138.5 (Ar
CH), 141.6, 144.6 (Fmoc Ar quat C), 157.1 (Fmoc CO), 166.4 (C-1),
173.0 (C-8). HRESI-MS m/z 686.2787 [M þ Na]þ (calcd for
C42H37N3O5Na 686.7657).
5.1.2. General procedure for synthesis of compounds 1ce6c and
9ce10c
A solution of compounds 1ae6a and 9ae10a with benzoic
anhydride (340 mg/1 mmol), respectively, in pyridine (1 ml) was
stirred at rt for 6 h. The reaction mixture was evaporated under
vacuum and purified by column chromatography over silica gel
using CHCl3 as eluent to afford the products.
5.1.2.4. (S)-2-((R)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
(1H-indol-3-yl)propanamido)-3-phenylpropyl benzoate (4c). Yield:
MeOH
68%, [
(log
400 MHz):
a
]D ¼ ꢂ12.1ꢃ (c 0.1, CH3OH). Mp: 223e224 ꢃC. UV
l
nm
max
3
): 264 (4.21), 220 (4.72), 209 (4.82). 1H NMR (C5D5N,
d
¼ 2.99 (2H, dt, J ¼ 6.8 Hz, H-10a, b), 3.56 (1H, dd,
5.1.2.1. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
J ¼ 7.4, 14.2 Hz, H-9a), 3.74 (1H, dd, J ¼ 7.4, 14.2 Hz, H-9b), 4.23 (1H,
t, J ¼ 7.2 Hz, Fmoc CH), 4.32 (1H, dd, J ¼ 7.4, 10.6 Hz, Fmoc CH2), 4.45
(1H, m, Fmoc CH2), 4.52e4.60 (2H, m, H-2a, b), 4.99e5.02 (1H, m,
H-6), 5.21 (1H, q, J ¼ 15.2 Hz, H-3), 7.15e7.32 (12H, m, Ar CH, Trp Ar
CH), 7.37e7.44 (4H, m, Fmoc Ar CH), 7.55e7.61 (2H, d, J ¼ 7.6 Hz,
Fmoc Ar CH), 7.83 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.88 (1H, d,
J ¼ 8.0 Hz, Ar CH), 8.13 (2H, d, J ¼ 7.2 Hz, Ar CH), 9.09 (1H, d,
J ¼ 8.4 Hz, NH-4), 9.35 (1H, d, J ¼ 8.0 Hz, NH-7), 11.9 (1H, s, Trp NH).
phenyl-propanamido)-3-phenylpropyl benzoate (1c). Yield: 64%,
[
a
]
¼ ꢂ21.6ꢃ (c 0.1, CH3OH). Mp: 126e128 ꢃC. 1H NMR (C5D5N,
D
400 MHz):
d
¼ 3.12 (2H, t, J ¼ 6.8 Hz, H-10a, b), 3.23 (1H, dd, J ¼ 7.8,
13.4 Hz, H-9a), 3.47 (1H, dd, J ¼ 7.0, 13.4 Hz, H-9b), 4.25 (1H, t,
J ¼ 7.0 Hz, Fmoc CH), 4.41 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2), 4.47
(1H, dd, J ¼ 6.0, 11.2 Hz, Fmoc CH2), 4.51e4.69 (2H, m, H-2),
4.94e4.98 (1H, m, H-3), 5.00e5.08 (1H, m, H-5), 7.10e7.29 (8H, m,
Ar CH), 7.35 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.41 (4H, t, J ¼ 7.8 Hz, Ar
CH), 7.43e7.56 (2H, m, Fmoc Ar CH), 7.60 (2H, d, J ¼ 7.6 Hz, Fmoc Ar
CH), 7.85 (2H, d, J ¼ 8.0 Hz, Fmoc Ar CH), 8.17 (2H, d, J ¼ 7.6 Hz. Ar
CH), 8.45 (1H, m, Ar CH), 9.16 (1H, d, J ¼ 8.4 Hz, NH-4), 9.36 (1H, d,
13C NMR (C5D5N, 100 MHz):
d
¼ 29.5 (C-10), 37.7 (C-11), 47.6 (Fmoc
CH), 50.1 (C-2), 57.1 (C-6), 65.9 (C-3), 66.6 (Fmoc CH2), 111.4, 112.0,
119.3, 121.7, 124.4, 128.7, 137.5 (Trp Ar CH), 120.4, 125.7, 125.8, 127.5,
128.0 (Fmoc Ar CH), 126.8, 128.7, 129.7, 130.0, 138.5 (Ar CH), 141.6,
144.6 (Fmoc Ar quat C), 157.1 (Fmoc CO), 166.4 (C-1), 173.0 (C-8).
HRESI-MS m/z 686.2787 [M þ Na]þ (calcd for C42H37N3O5Na
686.7657).
J ¼ 8.0 Hz, NH-7). 13C NMR (C5D5N, 100 MHz):
¼ 37.6 (C-10), 39.2
d
(C-9), 47.7 (Fmoc CH), 50.3 (C-2), 57.3 (C-6), 65.9 (C-3), 66.6 (Fmoc
CH2), 120.4, 125.6, 125.7, 127.5, 128.0 (Fmoc Ar CH), 126.8, 128.7,
128.8,129.7,130.0, 138.3 (Ar CH),141.6,144.6 (Fmoc Ar quat C),157.0
(Fmoc CO), 166.4 (C-1), 172.1 (C-5). HRESI-MS m/z 647.2678
[M þ Na]þ (calcd for C40H36N2O5Na 647.7296).
5.1.2.5. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
(naphthalen-2-yl)propanamido)-3-phenylpropyl benzoate (5c).
MeOH
Yield: 66%, [
nm (log
400 MHz):
a
]D ¼ ꢂ11.2ꢃ (c 0.1, CH3OH). Mp: 193e195 ꢃC. UV
l
max
5.1.2.2. (S)-2-((R)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
3
): 264 (4.27), 224 (5.00), 211 (4.91). 1H NMR (C5D5N,
phenyl-propanamido)-3-phenylpropyl benzoate (2c). Yield: 62%,
d
¼ 3.12 (2H, m, H-10a, b), 3.40 (1H, dd, J ¼ 8.0, 13.6 Hz,
[
a
]
¼ þ19.2ꢃ (c 0.1, CH3OH). Mp: 178e180 ꢃC. 1H NMR (C5D5N,
H-9a), 3.64 (1H, dd, J ¼ 6.8, 13.6 Hz, H-9b), 4.23 (1H, t, J ¼ 7.0 Hz,
Fmoc CH), 4.40 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2), 4.49e4.58 (3H,
m, H-2a, b, Fmoc CH2), 4.97e5.00 (1H, m, H-6), 5.16 (1H, q,
J ¼ 15.8 Hz, H-3), 7.14e7.27 (4H, m, Ar CH), 7.34e7.46 (8H, m, Ar CH,
Fmoc Ar CH), 7.50e7.56 (3H, m, Ar CH, Fmoc Ar CH), 7.71e7.76 (4H,
m, Ar CH, Fmoc Ar CH), 7.83 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.13 (2H,
d, J ¼ 7.2 Hz, Ar CH), 9.27 (1H, d, J ¼ 8.8 Hz, NH-4), 9.46 (1H, d,
D
400 MHz):
d
¼ 3.05 (2H, d, J ¼ 7.2 Hz, H-10a, b), 3.22 (1H, dd, J ¼ 8.0,
13.6 Hz, H-9a), 3.41 (1H, dd, J ¼ 6.6, 13.4 Hz, H-9b), 4.21 (1H, t,
J ¼ 7.0 Hz, Fmoc CH), 4.28 (1H, dd, J ¼ 7.2, 10.4 Hz, H-2 a), 4.47 (1H,
dd, J ¼ 6.4, 11.2 Hz, Fmoc CH2), 4.54 (1H, dd, J ¼ 7.0, 10.2 Hz, H-2 b),
4.59 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2), 4.96e5.07 (2H, m, H-3, 5),
7.19e7.35 (5H, m, Ar CH), 7.35e7.43 (2H, m, Fmoc Ar CH), 7.40e7.50
(2H, m, Ar CH, Fmoc Ar CH), 7.50e7.59 (5H, d, J ¼ 7.6 Hz, Ar CH,
Fmoc Ar CH), 7.84 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.13 (2H, d,
J ¼ 7.6 Hz. Ar CH), 8.45 (1H, m, Ar CH), 9.10 (1H, d, J ¼ 8.4 Hz, NH-4),
J ¼ 8.0 Hz, NH-7). 13C NMR (C5D5N, 100 MHz):
¼ 37.5 (C-10), 39.4
d
(C-9), 47.6 (Fmoc CH), 50.4 (C-2), 57.4 (C-6), 65.9 (C-2), 66.6 (Fmoc
CH2), 120.4, 125.6, 125.7, 125.8, 127.4, 128.0 (Fmoc Ar CH), 126.3,
126.8, 127.9, 128.1, 128.3, 128.8, 129.7, 130.0, 132.7, 133.4, 133.9,
136.1, 138.4 (Ar CH), 141.6, 144.5 144.6 (Fmoc Ar quat C), 157.1 (Fmoc
CO), 166.3 (C-1), 172.5 (C-5). HRESI-MS m/z 697.2835 [M þ Na]þ
(calcd for C44H38N2O5Na 697.7883).
9.31 (1H, d, J ¼ 8.4 Hz, NH-7). 13C NMR (C5D5N, 100 MHz):
¼ 37.8
d
(C-10), 39.2 (C-9), 47.6 (Fmoc CH), 50.2 (C-2), 57.3 (C-6), 66.0 (C-3),
66.6 (Fmoc CH2), 120.4, 125.6, 125.7, 127.4, 128.0 (Fmoc Ar CH),
126.9, 128.7, 128.8, 129.7, 130.2, 138.4 (Ar CH), 141.6, 144.5 (Fmoc Ar
quat C), 157.0 (Fmoc CO), 166.4 (C-1), 172.3 (C-5). HRESI-MS m/z
647.2678 [M þ Na]þ (calcd for C40H36N2O5Na 647.7296).
5.1.2.6. (S)-2-((R)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
(naphthalen-2-yl)propanamido)-3-phenylpropyl benzoate (6c).
MeOH
5.1.2.3. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
Yield: 60%, [
nm (log
400 MHz):
a
]
¼ þ9.6ꢃ (c 0.1, CH3OH). Mp: 238e240 ꢃC. UV
l
D
max
(1H-indol-3-yl)propanamido)-3-phenylpropyl benzoate (3c). Yield:
3
): 264 (4.12), 224 (4.86), 211 (4.72). 1H NMR (C5D5N,
MeOH
64%, [
a
]D ¼ ꢂ30.2ꢃ (c 0.1, CH3OH). Mp: 204e206 ꢃC. UV
l
nm
d
¼ 3.00 (2H, m, H-10a, b), 3.36 (1H, dd, J ¼ 8.0, 13.6 Hz,
max

2500
C.-T. Yen et al. / European Journal of Medicinal Chemistry 45 (2010) 2494e2502
H-9a), 3.63 (1H, dd, J ¼ 6.8, 13.6 Hz, H-9b), 4.28 (1H, t, J ¼ 7.0 Hz,
Fmoc CH), 4.42 (1H, dd, J ¼ 7.2, 10.4 Hz, Fmoc CH2), 4.51e4.59 (3H,
m, H-2a, b, Fmoc CH2), 5.00e5.08 (1H, m, H-6), 5.23 (1H, q,
J ¼ 15.8 Hz, H-3), 7.15e7.29 (4H, m, Ar CH), 7.34e7.46 (8H, m, Ar CH,
Fmoc Ar CH), 7.50e7.56 (3H, m, Ar CH, Fmoc Ar CH), 7.71e7.76 (4H,
m, Ar CH, Fmoc Ar CH), 7.84 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.12 (2H,
d, J ¼ 7.2 Hz, Ar CH), 9.31 (1H, d, J ¼ 8.8 Hz, NH-4), 9.43 (1H, d,
with benzoic anhydride (340 mg/1 mmol), respectively, in pyridine
(1 ml) was stirred at rt for 6 h. The reaction mixture was evaporated
under vacuum and purified by column chromatography over silica
gel using CHCl3 as eluent to afford the products.
5.1.3.1. (9H-Fluoren-9-yl)methyl(S)-1-((S)-1-hydroxy-3-phenylpropan-
2-ylamino)-3-(4-hydroxyphenyl)-1-oxopropan-2-ylcarbamate (7a).
J ¼ 8.0 Hz, NH-7). 13C NMR (C5D5N, 100 MHz):
d
¼ 37.5 (C-10), 39.4
Yield: 89%, [
(C5D5N, 400 MHz):
a
]D ¼ ꢂ13.6ꢃ (c 0.1, CH3OH). Mp: 217e219 ꢃC. 1H NMR
(C-9), 47.6 (Fmoc CH), 50.4 (C-2), 57.4 (C-6), 65.9 (C-2), 66.6 (Fmoc
CH2), 120.4, 125.6, 125.6, 125.9, 127.4, 128.0 (Fmoc Ar CH), 126.3,
126.8, 127.9, 128.3, 128.4, 128.8, 129.7, 130.0, 132.7, 133.4, 133.9,
136.1, 138.4 (Ar CH), 141.6, 144.5 144.6 (Fmoc Ar quat C), 157.1 (Fmoc
CO), 166.2 (C-1), 173.0 (C-5). HRESI-MS m/z 697.2835 [M þ Na]þ
(calcd for C44H38N2O5Na 697.7883).
d
¼ 3.14 (1H, dd, J ¼ 7.0, 13.4 Hz, H-9a), 3.27 (2H,
dd, J ¼ 7.2, 13.6 Hz, H-8a, 9b), 3.48 (1H, dd, J ¼ 6.0, 14.0 Hz, H-8b),
3.93 (2H, dd, J ¼ 3.0, 5.0 Hz, H-1), 4.27 (1H, t, J ¼ 7.2 Hz, Fmoc CH),
4.39 (1H, dd, J ¼ 7.2,10.4 Hz, Fmoc CH2), 4.58 (1H, dd, J ¼ 7.2,10.4 Hz,
Fmoc CH2), 4.71e4.76 (1H, m, H-2), 5.07e5.13 (1H, m, H-5), 7.08 (2H,
d, J ¼ 8.4 Hz, Ar CH), 7.14 (1H, t, J ¼ 7.4 Hz, Ar CH), 7.22e7.31 (6H, m,
Ar CH, Fmoc Ar CH), 7.36e7.43 (4H, m, Ar CH, Fmoc Ar CH), 7.62 (2H,
d, J ¼ 7.4 Hz, Fmoc Ar CH), 7.83 (2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 8.94
5.1.2.7. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
methylbutanamido)-3-phenylpropyl benzoate (9c). Yield: 62%,
(2H, d, J ¼ 8.4 Hz, NH-3, 6). 13C NMR (C5D5N, 100 MHz):
¼ 37.5 (C-
d
[
a]
¼ ꢂ2.6ꢃ (c 0.1, CH3OH). Mp: 214e216 ꢃC. UV lMmaexOH nm (log
3
):
9), 38.5 (C-8), 47.7 (Fmoc CH), 53.6 (C-1), 57.6 (C-2), 63.0 (C-5), 66.7
(Fmoc CH2), 116.1, 126.4, 128.6, 129.9, 131.0, 139.5 (Ar CH), 120.3,
125.6, 125.8, 127.5, 128.0, 157.6 (Fmoc Ar CH), 141.6, 144.6 (Fmoc Ar
quat C), 157.1 (Fmoc CO), 172.3 (C-4). HRESI-MS m/z 559.2365
[M þ Na]þ (calcd for C33H32N2O5Na 559.6230).
D
263 (3.98), 208 (4.63). 1H NMR (C5D5N, 400 MHz):
d
¼ 1.02 (6H, dd,
J ¼ 6.8 Hz, H-12a, b, c, 13a, b, c), 2.31e2.40 (1H, m, H-11), 3.13 (2H, t,
J ¼ 5.2 Hz, H-10a, b), 4.33 (1H, t, J ¼ 6.8 Hz, Fmoc CH), 4.50e4.58
(3H, m, H-2a, b, Fmoc CH2), 4.67e4.73 (2H, m, H-3, 6), 7.16 (1H, t,
J ¼ 7.2 Hz, Ar CH), 7.26 (4H, t, J ¼ 7.6 Hz, Ar CH), 7.36e7.45 (6H, m, Ar
CH, Fmoc Ar CH), 7.53 (2H, d, J ¼ 7.2 Hz, Ar CH), 7.68 (2H, dd,
J ¼ 7.6 Hz, Fmoc Ar CH), 7.85 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.22
(2H, d, J ¼ 7.2 Hz, Ar CH), 8.90 (1H, d, J ¼ 9.2 Hz, NH-4), 9.34 (1H, d,
5.1.3.2. (9H-Fluoren-9-yl)methyl (S)-1-((S)-1-hydroxy-3-phenylpropan-
2-ylamino)-3-(4-hydroxyphenyl)-1-oxopropan-2-ylcarbamate
(8a).
Yield: 87%, [
a
]D ¼ þ9.6ꢃ (c 0.1, CH3OH). Mp: 221e223 ꢃC. 1H NMR
J ¼ 8.0 Hz, NH-7). 13C NMR (C5D5N, 100 MHz):
d
¼ 18.8 (C-12), 19.7
(C5D5N, 400 MHz):
d
¼ 3.14 (1H, dd, J ¼ 7.6, 13.6 Hz, H-9a),
(C-13), 31.5 (C-11), 37.9 (C-10), 47.8 (Fmoc CH), 50.2 (C-2), 61.7 (C-
6), 65.9 (C-3), 66.6 (Fmoc CH2), 120.4, 125.7, 127.5, 128.0 (Fmoc Ar
CH), 126.8, 128.8, 129.7, 130.0, 133.4, 138.3 (Ar CH), 141.7, 144.5,
144.7 (Fmoc Ar quat C), 157.4 (Fmoc CO), 166.5 (C-1), 172.5 (C-5).
HRESI-MS m/z 599.2678 [M þ Na]þ (calcd for C29H32N2O4Na
599.6868).
3.21e3.28 (2H, m, H-8a, 9b), 3.42 (1H, dd, J ¼ 6.2, 13.8 Hz, H-8b),
3.95 (2H, d, J ¼ 5.2 Hz, H-1), 4.26 (1H, t, J ¼ 7.2 Hz, Fmoc CH), 4.36
(1H, dd, J ¼ 7.26, 10.4 Hz, H-1a), 4.58 (1H, dd, J ¼ 7.2, 10.2 Hz, H-1b),
4.74e4.80 (1H, m, H-2), 5.06-5.11 (1H, m, H-5), 7.10 (2H, d,
J ¼ 8.4 Hz, Ar CH), 7.27 (5H, t, J ¼ 9.0 Hz, Ar CH), 7.31 (2H, t,
J ¼ 7.6 Hz, Fmoc Ar CH), 7.37 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.41
(2H, t, J ¼ 6.8 Hz, Ar CH), 7.62 (2H, t, J ¼ 5.6 Hz, Fmoc Ar CH), 7.82
(2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 8.91 (2H, d, J ¼ 8.4 Hz, NH-3, 6). 13C
5.1.2.8. (S)-2-((R)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
methylbutanamido)-3-phenylpropyl benzoate (10c). Yield: 64%,
NMR (C5D5N, 100 MHz):
d
¼ 37.6 (C-9), 38.7 (C-8), 47.7 (Fmoc CH),
[
a]
¼ þ21.6ꢃ (c 0.1, CH3OH). Mp: 233e235 ꢃC. UV lMmaexOH nm (log
3
):
53.5 (C-1), 57.7 (C-2), 63.1 (C-5), 66.7 (Fmoc CH2),116.2,126.5,128.7,
130.0, 131.1, 139.7 (Ar CH), 120.4, 125.6, 125.8, 127.5, 128.0, 157.7
(Fmoc Ar CH), 141.6, 144.6 (Fmoc Ar quat C), 157.1 (Fmoc CO), 172.3
(C-4). HRESI-MS m/z 559.2365 [M þ Na]þ (calcd for C33H32N2O5Na
559.6230).
D
263 (4.05), 208 (4.65). 1H NMR (C5D5N, 400 MHz):
d
¼ 0.95 (6H, dd,
J ¼ 6.6, 15.8 Hz, H-12a, b, c, 13a, b, c), 2.28e2.33 (1H, m, H-11), 3.15
(2H, d, J ¼ 8.0 Hz, H-10a, b), 4.28 (1H, t, J ¼ 7.0 Hz, Fmoc CH), 4.37
(1H, dd, J ¼ 6.8, 10.4 Hz, H-2a), 4.51e4.58 (2H, m, H-2b, Fmoc CH2),
4.61e4.68 (2H, m, Fmoc CH2, H-3), 5.11 (1H, q, J ¼ 15.0 Hz, H-6),
7.22e7.28 (3H, m, Ar CH), 7.31e7.35 (3H, m, Ar CH), 7.37e7.44 (6H,
m, Ar CH, Fmoc Ar CH), 7.65 (2H, dd, J ¼ 7.4 Hz, Fmoc Ar CH), 7.84
(2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.15 (2H, d, J ¼ 6.8 Hz, Ar CH), 8.84
(1H, d, J ¼ 8.8 Hz, NH-4), 9.26 (1H, d, J ¼ 8.4 Hz, NH-7). 13C NMR
5.1.3.3. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
(4-hydroxyphenyl)propanamido)-3-phenylpropyl benzoate (7c).
MeOH
Yield: 88%, [
a
]D ¼ ꢂ15.1ꢃ (c 0.1, CH3OH). Mp: 161e162 ꢃC. UV
l
max
nm (log
3
): 265 (3.98), 208 (4.55). 1H NMR (C5D5N, 400 MHz):
(C5D5N, 100 MHz):
d
¼ 18.6 (C-12), 19.7 (C-13), 31.5 (C-11), 37.8 (C-
d
¼ 3.13 (2H, m, H-10a, 9a), 3.23 (1H, dd, J ¼ 8.3, 13.7 Hz, H-10b),
10), 47.8 (Fmoc CH), 50.1 (C-2), 61.7 (C-6), 66.5 (C-3, Fmoc CH2),
120.4, 125.6, 125.7, 127.4, 128.0 (Fmoc Ar CH), 126.9, 128.7, 128.8,
129.7, 130.0, 133.2, 138.6 (Ar CH), 141.7, 144.5, 144.7 (Fmoc Ar quat
C), 157.4 (Fmoc CO), 166.4 (C-1), 172.5 (C-5). HRESI-MS m/z
599.2678 [M þ Na]þ (calcd for C29H32N2O4Na 599.6868).
3.46 (1H, dd, J ¼ 6.6, 13.4 Hz, H-9b), 4.25 (1H, t, J ¼ 7.2 Hz, Fmoc CH),
4.37 (1H, dd, J ¼ 7.2, 10.6 Hz, H-2a), 4.50e4.59 (3H, m, H-2b, Fmoc
CH2), 4.96e4.99 (1H, m, H-3), 5.05 (1H, q, J ¼ 15.0 Hz, H-6), 7.06
(2H, d, J ¼ 8.4 Hz, Ar CH), 7.14 (2H, dd, J ¼ 8.4, 12.4 Hz, Ar CH),
7.23e7.37 (3H, m, Ar CH), 7.34e7.41 (8H, m, Ar CH, Fmoc Ar CH),
7.50 (1H, t, J ¼ 6.4 Hz, Ar CH), 7.61 (2H, d, J ¼ 7.4 Hz, Fmoc Ar CH),
7.83 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.18 (2H, d, J ¼ 7.2 Hz, Ar CH),
9.05 (1H, d, J ¼ 8.0 Hz, NH-3), 9.39 (1H, d, J ¼ 8.2 Hz, NH-6). 13C NMR
5.1.3. General procedure for synthesis of compounds 7a, 7c, 8a
and 8c
Fmoc-L-O-t-butyl-tyrosine (1.0 mmol) was introduced to L- or
D-phenylalaninol (1.0 mmol) in CH2Cl2 (10 ml) by using with HBTU
(1.5 mmol) and DIEA (1.5 mmol), and the mixture was stirred at rt
for 6 h. The reaction mixture was evaporated under vacuum and
purified by column chromatography over silica gel using CHCl3 as
eluent to afford the intermediates. A solution of above compounds
(C5D5N, 100 MHz):
d
¼ 37.6 (C-10), 38.4 (C-9), 47.6 (Fmoc CH), 50.3
(C-2), 57.6 (C-3), 66.6 (C-6), 66.7 (Fmoc CH2), 116.1, 126.7, 128.8,
130.0, 130.9, 138.4 (Ar CH), 120.3, 125.6, 125.7, 127.4, 128.0, 157.6
(Fmoc Ar CH), 141.6, 144.5 (Fmoc Ar quat C), 157.1 (Fmoc CO), 166.4
(C-1), 172.3 (C-5). HRESI-MS m/z 663.2627 [M þ Na]þ (calcd for
C40H36N2O6Na 663.7290).
in TFA/TIS/H2O ¼ 95/2.5/2.5 (100
ml/mg), respectively, were stirred
at rt for 1 h. The solvent was evaporated under vacuum and the
residue was purified by suction (MeOH as solvent) to give the
desired products 7a and 8a. A solution of compounds 7a and 8a
5.1.3.4. (S)-2-((R)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
(4-hydroxyphenyl)propanamido)-3-phenylpropyl benzoate (8c).
MeOH
Yield: 88%, [
a
]D ¼ þ5.6ꢃ (c 0.1, CH3OH). Mp: 204e206 ꢃC. UV
l
max

C.-T. Yen et al. / European Journal of Medicinal Chemistry 45 (2010) 2494e2502
2501
Table 4
(PALL, Pensacola, USA). The membrane was blocked with 5% skim
milk in PBS-T 4 h at room temperature and stained with goat
polyclonal anti-NS3 antibody (Virostat, Maine, USA). As a loading
control, membrane was also probed with rabbit polyclonal anti-
GAPDH antibody (GeneTex, CA, USA). Subsequently, the bound
antibody was further probed with horseradish peroxidase-
conjugated with either anti-goat or anti-rabbit IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA, USA). Finally, the
signals were revealed with an ECL western blotting system (Amer-
sham Biosciences). Densitometric quantification of relative signal in
the western blot was determined by the Quantity OneÒ analysis
software (Bio-Rad, CA, USA). Cell viability was determined by the
MTS assay essentially as described [30].
The cytotoxicity of combination of 10a and 10c with doxorubicin, respectively.
Compound Cancer cell line (IC50 value,a
m
g/ml)
A549
HepG2
Hep3B
MCF-7
Ca9-22
MDA-MB-
231
10a:Doxo
(1:1)
10a:Doxo
(1:5)
0.50
0.68
0.60
0.42
0.64
0.99 ꢀ 0.52
1.87 ꢀ 1.13
ꢀ 0.23
ꢀ 0.01
ꢀ 0.07
ꢀ 0.74
ꢀ 0.54
0.78
0.91
0.99
0.61
0.25
ꢀ 1.29
ꢀ 0.76
ꢀ 1.23
ꢀ 1.54
ꢀ 1.23
10c:Doxo
(1:1)
10c:Doxo
(1:5)
0.13
0.37
0.19
0.30
0.16
0.03 ꢀ 0.06
0.23 ꢀ 0.74
ꢀ 0.46
ꢀ 1.12
ꢀ 0.05
ꢀ 2.77
ꢀ 1.73
0.13
0.34
0.15
0.23
0.12
ꢀ 0.04
ꢀ 1.65
ꢀ 1.22
ꢀ 0.87
ꢀ 0.09
Doxorubicinb 0.18
0.47
0.33
0.26
0.14
0.43
a
Acknowledgements
IC50 represents the 50% inhibitory concentration. Results are presented as
mean ꢀ S.E.M. (n ¼ 2).
b
Positive control.
The investigation was supported by research grants to P. W.
Hsieh from the Chang Gung Medical Research Foundation and
National Science Council of the Republic of China in Taiwan, as well
as by research grants to Y. C. Wu from the National Sun Yat-Sen
UniversityeKaohsiung Medical University Joint Research Center,
and Center of Excellence for Environmental Medicine (KMU-EM-
97-2.1a).
nm (log
¼ 3.10 (2H, m, H-10a, 9a), 3.22 (1H, dd, J ¼ 8.0, 13.2 Hz, H-10b),
3
): 264 (4.30), 208 (4.86). 1H NMR (C5D5N, 400 MHz):
d
3.41 (1H, dd, J ¼ 6.6, 13.8 Hz, H-9b), 4.21e4.31 (2H, m, Fmoc CH,
H-2a), 4.48 (1H, dd, J ¼ 6.4, 11.2 Hz, H-2b), 4.53e4.62 (2H, m, Fmoc
CH2), 4.99e5.03 (2H, m, H-3, 6), 7.11 (2H, d, J ¼ 8.0 Hz, Ar CH),
7.23e7.26 (6H, m, Ar CH), 7.30e7.34 (4H, m, Ar CH), 7.35e7.45 (4H,
m, Ar CH, Fmoc Ar CH), 7.60 (2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 7.83 (2H,
d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.15 (2H, d, J ¼ 7.2 Hz, Ar CH), 9.06 (1H, d,
J ¼ 8.4 Hz, NH-3), 9.34 (1H, d, J ¼ 8.4 Hz, NH-6). 13C NMR (C5D5N,
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.02.035.
100 MHz):
d
¼ 37.8 (C-10), 38.5 (C-9), 47.6 (Fmoc CH), 50.2 (C-2),
57.7 (C-3), 66.0 (C-6), 66.6 (Fmoc CH2), 116.2, 126.9, 128.7, 130.0,
131.1, 138.5 (Ar CH), 120.3, 125.6, 125.8, 127.4, 128.0, 157.7 (Fmoc Ar
CH), 141.6, 144.6 (Fmoc Ar quat C), 157.0 (Fmoc CO), 166.4 (C-1),
172.6 (C-5). HRESI-MS m/z 663.2627 [M þ Na]þ (calcd for
C40H36N2O6Na 663.7290).
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5.2.1. Cell viability assays
Compounds were tested against six cancer cell lines: HepG2,
Hep3B (human hepatoma cell); A549 (human lung cancer cell);
MCF-7, MDA-MB-231 (human breast cancer cell); and Ca9-22
(human oral epithelial cancer cell) using the MTT method [7].
Freshly trypsinized cell suspensions were seeded in 96 well
microtiter plates at densities of 5000e10,000 cells per well with
tested compounds added from DMSO-diluted stock. After 72 h in
culture, attached cells were incubated with MTT (0.5 mg/ml, 1 h)
and subsequently solubilized in DMSO for determining of IC50. For
combination experiments, cells were exposed to the combined
treatments for 72 h with the same ranges of doses. A fixed drug ratio
was used: 10a/doxorubicin 1:1, 10a/doxorubicin 1:5, 10c/doxoru-
bicin 1:1 and 10c/doxorubicin 1:5 (Table 4). The absorbency at
550 nm was then measured using a microplate reader. The cytotoxic
activity of compounds was expressed as the concentration inhibit-
ing cell growth by 50% (IC50) calculated from the survival curves.
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subgenomic-replicon, were seeded in 24-well plate at density of
5 ꢁ104 cells per well and treated with compound at indicated
concentration for 4 days. The inhibitory assay was performed with
western blot according to the manufacturer's instruction. In brief,
the protein concentration of cell extracts was measured by the
protein assay buffer (Bio-Rad, CA, USA). 5 mg of protein for each
sample was resolved by 8% SDS-polyacrylamide gel. Proteins in gel
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