Design and synthesis of new N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-dipeptides as anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2008.11.008

Twenty-four new dipeptide analogs (1-24) of aurantiamide acetate were designed, synthesized, and assayed for effects on superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB. Among them, seven N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-dipeptides (6, 9, 12, 14, 17, 18 and 20) showed potent inhibitory effects. Compounds 9 and 18 showed the most selective effects against human neutrophil elastase release, with IC₅₀ values of 0.8 ± 0.1 and 1.7 ± 0.6 μM, respectively, and were 130-fold more potent than phenylmethylsulfonyl fluoride (PMSF), the positive control, in this anti-inflammatory assay. These two compounds could be developed as new lead anti-inflammatory agents.

Full text of DOI:10.1016/j.ejmech.2008.11.008

European Journal of Medicinal Chemistry 44 (2009) 1933–1940
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of new N-(fluorenyl-9-methoxycarbonyl)
(Fmoc)-dipeptides as anti-inflammatory agents
,
b,
**
Chiao-Ting Yen a, Tsong-Long Hwang b, Yang-Chang Wu a , Pei-Wen Hsieh
*
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
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-four new dipeptide analogs (1–24) of aurantiamide acetate were designed, synthesized, and
assayed for effects on superoxide anion generation and elastase release by human neutrophils in
response to fMLP/CB. Among them, seven N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-dipeptides (6, 9, 12,
14, 17, 18 and 20) showed potent inhibitory effects. Compounds 9 and 18 showed the most selective
Received 28 August 2008
Received in revised form
3 November 2008
Accepted 14 November 2008
Available online 27 November 2008
effects against human neutrophil elastase release, with IC50 values of 0.8 ꢀ 0.1 and 1.7 ꢀ 0.6
mM,
respectively, and were 130-fold more potent than phenylmethylsulfonyl fluoride (PMSF), the positive
control, in this anti-inflammatory assay. These two compounds could be developed as new lead anti-
inflammatory agents.
Keywords:
Fmoc-based dipeptides
Aurantiamide derivatives
Anti-inflammatory agents
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
aurantiamide (Fig. 1), a major component of Zanthoxylum dissitum
and Aspergillus penicilloides [9,10], exhibited anti-bacterial [11,12],
Human neutrophils are active phagocytes that act as a crucial
component of immunity [1]. They play important roles in the host
defense against microorganisms and in the pathogenesis of various
diseases, such as rheumatoid arthritis [1–3], ischemia-reperfusion
injury, chronic obstructive pulmonary disease (COPD), and asthma
[4–6]. In response to diverse stimuli, activated neutrophils secrete
a series of cytotoxins, including superoxide anion (Oꢁ2 ), which is
a precursor of other reactive oxygen species (ROS), as well as granule
protease and bioactive lipids [1,2,7]. Therefore, it is essential to
control respiratory burst and degranulation in physiological condi-
tions, while potentiating these functions in infected tissues and
organs [1]. Only a few agents are now used to modulate pro-
inflammatory responses of neutrophils; therefore, research and
development of newgeneration anti-inflammatoryagents continue.
In previous reports, aurantiamide acetate (Fig. 1), a dipeptide
composed of N-benzoylphenylalanine and phenylalaninol acetate,
was isolated from Polygonum chinensis, and showed inhibitory effects
anti-inflammatory [10], antioxidant [13], and anti-HIV effects [9].
Other research has shown that several N-Fmoc amino acids
exert anti-inflammatory activity by recruitment of neutrophils into
the inflammatory site [14,15]. In addition, these compounds also
increased intracellular Ca2þ concentrations in Madin Darby canine
kidney (MDCK) renal tubular cells [16].
Therefore, we evaluated eight L-Fmoc amino acids for inhibitory
effects on superoxide anion generation and elastase release by
human neutrophils in response to fMLP/CB. The results revealed
that most of the tested L-configured N-Fmoc aromatic amino acids
were equipotent in both assays and exhibited non-selective
inhibitory activities (Table 1).
Accordingly, we designed and synthesized a series of aur-
antiamide acetate analogs in an attempt to obtain new anti-
inflammatory agents. As shown in Fig. 2, the points of synthetic
modification included (1) the N-terminal substitution, (2) the amino
acid residue (part A); and (3) the configuration (S- or R-) of phe-
nylalaninol. All newly synthesized analogs were assayed for effects
on superoxide anion generation and elastase release by fMLP-
activated human neutrophils. Herein the synthesis, bioactivity, and
anti-inflammatory structure–activity relationships are described.
on Oꢁ2 generation induced by formyl-
L-methionyl-L-leucyl-L-phenyl-
alanine (fMLP) in human neutrophils [8]. Additionally, its analog
* Corresponding author. Graduate Institute of Natural Products, Kaohsiung
Medical University, 100, SHIH CHUAN 1st Rd, Kaohsiung 807, Taiwan. Tel.: þ886
(0)7 312 1101x2197; fax: þ886 (0)7 311 4773.
2. Chemistry
** Corresponding author. Graduate Institute of Natural Products, Chang Gung
University, 259 Wen-Hwa First Road, Kwei-Shan Tao-Yuan 333, Taiwan. Tel.: þ886
(0)3 211 8800x3105; fax: þ886 (0)3 211 8506.
The synthetic routes to the compounds are shown in Schemes
1–3. The compounds were prepared by liquid-phase peptide
synthesis via a previously described pathway (Scheme 1). In this
E-mail addresses: yachwu@kmu.edu.tw (Y.-C. Wu), pewehs@mail.cgu.edu.tw
(P.-W. Hsieh).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.11.008

1934
C.-T. Yen et al. / European Journal of Medicinal Chemistry 44 (2009) 1933–1940
O
H
N
H
N
HN
O
CH3
HN
OH
O
O
O
O
aurantiamide
aurantiamide acetate
Fig. 1. Structures of aurantiamide and aurantiamide acetate.
Table 1
Inhibitory effects of Fmoc amino acids on superoxide anion generation and elastase
release by human neutrophils in response to fMLP/CB.
Fig. 2. Design of aurantiamide acetate analogs.
Compound
Superoxide anion
IC50
M)a or (Inh %)
Elastase
IC50
M)a or (Inh %)
(Fmoc) group of 5, 6, 11 and 12 was removed by treatment with 20%
piperidine in CH2Cl2 to afford 7, 8, 13 and 14, respectively (Scheme
3). All compounds (1–24, Table 2) were >99% pure and were
characterized by spectroscopic data, as shown in Section 5.
(
m
(m
Fmoc-
Fmoc-
Fmoc-
Fmoc-
Fmoc-
Fmoc-
Fmoc-
Fmoc-
DPIb
L
L
L
L
L
L
L
L
-phenylalanine
-O-t-butyltyrosine
-tyrosine
-trityl-histidine
-tryptophan
-naphthalanine
-valine
14.1 ꢀ 3.3
5.5 ꢀ 0.7
14.2 ꢀ 1.3
5.6 ꢀ 0.9
2.4 ꢀ 0.3***
0.5 ꢀ 0.0
2.9 ꢀ 0.1***
0.6 ꢀ 0.0
3.3 ꢀ 0.3
(45.4 ꢀ 1.7)**
2.1 ꢀ 0.2
1.2 ꢀ 1.4
3. Results and discussion
(29.4 ꢀ 2.8)***
12.8 ꢀ 0.8
0.7 ꢀ 0.4
(14.5 ꢀ 1.7)**
(39.8 ꢀ 5.4)**
-isoleucine
Compounds 1–24 were evaluated for anti-inflammatory activity
based on effects against superoxide anion generation and elastase
release by human neutrophils in response to fMLP/CB. These assay
systems were performed using established protocols [14], and are
widely used to identify potential anti-inflammatory compounds.
Table 3 lists the results for the test compounds, as well as diphe-
nyleneiodonium (DPI) and phenylmethylsulfonyl fluoride (PMSF),
included as positive controls for superoxide anion generation and
elastase release, respectively.
PMSFb
130.9 ꢀ 29.1
Percentage of inhibition (Inh %) at 10
m
g/mL. Results are presented as mean ꢀ S.E.M.
(n ¼ 3–4). **P < 0.01, ***P < 0.001 compared with the control value.
a
Concentration necessary for 50% inhibition (IC50).
DPI and PMSF were used as positive controls.
b
method, S- or R-phenylalaninol reacted with an N-protected amino
acid in the presence of coupling agents O-benzotriazole-N,N,N0,N0-
tetramethyl-uronium-hexafluoro-phosphate (HBTU) and diisopro-
pylethylamine (DIEA) in CH2Cl2 to give an intermediate protected
dipeptide alcohol. The synthetic acetate analogs were prepared
from this intermediate by esterification, followed by condensation
with acetic anhydride in pyridine to obtain the desired products as
shown in Scheme 1. For 9 and 10, the tert-butyl protecting group
was removed by treatment with TFA/TIS/H2O (95/2.5/2.5) to give 11
and 12 (Scheme 2). Moreover, the 9-fluorenylmethoxycarbonyl
Among phenylalanine–phenylalaninol analogs 1–8, compounds
1, 2, 3 and 6 showed 39.3%, 40.9%, 42.1% and 31.3% inhibition,
respectively, in the superoxide anion assay at a concentration of
10
mg/mL, while only compound 6 inhibited fMLP-induced elastase
release (IC50 12.9 ꢀ 3.7
m
M). The results indicated that replacement
of benzyloxycarbonyl (Cbz, 1 and 2) by Fmoc (5 and 6) on the N-
terminus increased inhibitory effects on elastase release, but
slightly decreased inhibition of superoxide anion generation.
Furthermore, when the N-terminal substitution was t-Boc (3 and 4)
OH
H2N
R
R
O
H
N
H
N
R
HN
X
OH
HN
X
O
CH3
OH
HN
X
O
O
a
b
O
1,2: X = Cbz; 3,4: X = Boc; 5,6, 9-12,15-24: X = Fmoc
R =amino acid residues
Scheme 1. Reagents: (a) HBTU: O-benzotriazole-N,N,N0,N00-tetramethyl-uronium-hexafluoro-phosphate, DIEA: diisopropylethylamine, CH2Cl2, 6 h, room temperature. (b) Acetic
anhydride/pyridine, 1 h, room temperature.
O
HO
O
O
H
N
H
N
HN
O
CH3
a
HN
O
CH3
O
O
O
O
O
O
9, 10
11,12
Scheme 2. Reagents: (a) TFA/TIS/H20 ¼ 95/2.5/2.5, 0.5 h, room temperature.

C.-T. Yen et al. / European Journal of Medicinal Chemistry 44 (2009) 1933–1940
1935
R1
O
R1
H
N
O
H
N
HN
CH3
O
H2N
O
CH3
a
O
O
O
O
5, 6: R1=C6H5
11, 12: R1=C6H4OH
7, 8: R1=C6H5
13, 14: R1=C6H4OH
Scheme 3. Reagents: (a) 20% piperidine/CH2Cl2, 1 h, room temperature.
or hydrogen (7 and 8), the effects on neutrophil elastase release also
decreased compared to 6, while effects on superoxide anion
generation were not significant, except for 3. Therefore, an Fmoc
group at the N-terminus appears important for inhibiting neutro-
phil elastase release induced by fMLP.
In a continuing search for more potent analogs, we evaluated
the anti-inflammatory effects of N-Fmoc synthetic dipeptides
containing various amino acids other than phenylalanine.
Among the sixteen Fmoc dipeptides (5, 6, 9–12, and 15–24),
none showed significant activity against superoxide anion gener-
ation, except for 6, which showed a slight inhibitory effect (31.3%
inhibition at 10 mg/mL). However, dipeptides 6, 9, 12, 17, 18 and 20
did show selective inhibition of human neutrophil elastase release.
Analogs 9 (Fmoc O-t-butyltyrosine) and 18 (Fmoc tryptophan)
demonstrated the highest potency, with IC50 values of 0.8 and
1.7
m
M, respectively. Compounds 12 (Fmoc tyrosine) and 14 (tyro-
M, respectively) than 9, but
sine) were less potent (IC50 6.5 and 3.7
m
Table 2
slightly more potent than 6 (Fmoc phenylalanine) against elastase
release. These findings suggest that anti-inflammatory activity in
the analogs is increased by addition of an OH or O-t-butyl substit-
uent at the para-position of the phenyl ring.
Furthermore, comparison of the anti-inflammatory data of N-
Fmoc amino acids and N-Fmoc dipeptides (5, 6, 9–12, and 15–24)
Aurantiamide acetate derivatives.
O
R
H
N
B
A
HN
X
O
CH3
O
(Tables 1 and 3, respectively) indicated that introduction of an
L- or
D-phenylalaninol acetate residue in the latter compounds abolished
inhibition of superoxide anion generation. Therefore, even though
the N-Fmoc amino acids were also generally more potent against
human neutrophil elastase release than the corresponding N-Fmoc
Compound
R
X
Configuration
A
B
1
2
3
4
5
6
7
8
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
Cbz
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
R
S
R
S
R
S
R
S
R
S
R
S
R
t-Boc
Fmoc
H
Table 3
Inhibitory effects of dipeptides on superoxide anion generation and elastase release
by human neutrophils in response to fMLP/CB.
Compound
Superoxide anion
IC50
M)a or (Inh %)
Elastase
IC50
M)a or (Inh %)
9
CH2C6H4OC(CH3)3
CH2C6H4OH
Fmoc
Fmoc
H
(
m
(m
10
11
12
13
14
15
16
1
2
3
4
5
6
7
(39.3 ꢀ 4.1)***
(40.9 ꢀ 2.9)***
(42.1 ꢀ 7.9)**
(4.7 ꢀ 1.5)*
(2.8 ꢀ 5.2)
(27.2 ꢀ 6.4)*
(35.4 ꢀ 4.1)***
(18.9 ꢀ 5.5)*
(11.0 ꢀ 1.8)**
(39.5 ꢀ 6.2)**
12.9 ꢀ 3.7***
>20.0c***
CH2C6H4OH
trityl
3''
(31.3 ꢀ 6.4)**
>20.0b*
N
4''
N
8
>20.0
>20.0
2''
9
(25.9 ꢀ 5.5)*
(22.3 ꢀ 2.5)*
(-0.5 ꢀ 6.3)
(8.0 ꢀ 2.0)*
(-0.5 ꢀ 6.3)
(8.0 ꢀ 2.0)*
(9.5 ꢀ 2.0)**
(6.6 ꢀ 1.3)**
(24.5 ꢀ 0.6)***
(18.6 ꢀ 6.6)*
(5.0 ꢀ 2.4)
0.8 ꢀ 0.1***
(31.9 ꢀ 5.6)**
(22.8 ꢀ 5.8)**
6.5 ꢀ 2.0**
Fmoc
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
DPId
PMSFd
1''
5''
(22.8 ꢀ 5.8)**
3.7 ꢀ 1.2**
17
18
S
S
S
R
6''
(32.0 ꢀ 2.7)***
(-2.9 ꢀ 3.2)
5.5 ꢀ 0.6**
7''
8''
5''
4''
1''
HN
1.7 ꢀ 0.6*
Fmoc
9''
3''
(37.8 ꢀ 6.1)**
14.5 ꢀ 0.6***
(27.7 ꢀ 6.5)
(34.6 ꢀ 1.8)***
(25.8 ꢀ 5.1)**
(22.5 ꢀ 1.1)***
(2.1 ꢀ 2.8)
2''
(-6.3 ꢀ 0.9)**
(-2.3 ꢀ 1.3)
(2.2 ꢀ 2.3)
19
20
S
S
S
R
3''
4''
7''
(6.0 ꢀ 4.0)
9''
0.7 ꢀ 0.4
5''
6''
2''
1''
130.9 ꢀ 29.1
Fmoc
10''
Percentage of inhibition (Inh %) at 10
m
g/mL. Results are presented as mean ꢀ S.E.M.
8''
(n ¼ 3–4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control value.
a
Concentration necessary for 50% inhibition (IC50).
These compounds elicited superoxide anion generation by human neutrophils
b
21
22
23
24
isopropyl
Fmoc
Fmoc
S
S
S
S
S
R
S
R
in the absence of CB/fMLP.
c
These compounds elicited superoxide anion generation by human neutrophils
sec-butyl
in the presence of CB.
d
DPI and PMSF were used as positive controls.

1936
C.-T. Yen et al. / European Journal of Medicinal Chemistry 44 (2009) 1933–1940
dipeptides (except for 9, 17, and 18), the inhibitory selectivity of the
dipeptides for elastase release increased dramatically.
H-11b), 4.25 (2H, dd, J ¼ 5.6, 4.8 Hz, H-4a, b), 4.78–4.86 (1H, m, H-8),
5.49 (1H, q, J ¼ 15.6 Hz, H-5), 7.11 (1H, t, J ¼ 3.8 Hz, Ar CH), 7.18–7.31
(7H, m, Ar CH), 7.33–7.44 (7H, m, Ar CH), 9.28 (1H, d, J ¼ 8.4 Hz, NH-6),
Dipeptides with both S- or R-configured phenylalaninol residues
were synthesized and evaluated in the anti-inflammatory assays.
Comparison of data for paired dipeptides (Table 3) showed that
configuration had no general effect on superoxide anion genera-
tion. The inhibitory effects on elastase release were in the order 9
(S) j 17 (S) j 18 (R) > 12 (R) > 6 (R) > 20 (R), suggesting that there
was no preference for either the S- or R-configuration on the anti-
inflammatory activity.
9.35 (1H, d, J ¼ 8.8 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
¼ 20.7 (C-
d
1), 37.5 (C-10), 38.8 (C-11), 50.2 (C-5), 55.7 (C-8), 65.4 (C-4), 126.7 (C-
40), 126.9(C-400), 128.1, 128.6, 131.5 (Cbz Ar CH), 128.8 (C-20, 60, 200, 600),
129.8 (C-30, 50, 300, 500), 138.3 (C-100), 138.4 (C-10), 167.8 (Cbz CO), 170.7
(C-2), 172.1 (C-7).
5.1.1.2. (R)-2-((S)-2-Benzamido-3-phenylpropanamido)-3-phenyl-
propyl acetate (2). Yield: 89%, [
NMR (C5D5N, 400 MHz):
a
]
¼ þ30.3ꢂ (c 0.1, CH3OH). 1H
D
4. Conclusion
d
¼ 1.90 (3H, s, H-1a, b, c), 2.97 (2H, dd,
J ¼ 2.8, 2.8 Hz, H-10a, b), 3.28 (1H, dd, J ¼ 7.6, 7.6 Hz, H-11a), 3.44
(1H, dd, J ¼ 6.8, 6.4 Hz, H-11b), 4.26 (2H, dd, J ¼ 6.0, 6.0 Hz, H-4a,
b), 4.33 (1H, dd, J ¼ 4.8, 4.4 Hz, H-8), 4.80–4.88 (1H, m, H-5), 5.48
(1H, q, J ¼ 15.6 Hz, H-5), 7.12 (1H, t, J ¼ 3.6 Hz, Ar CH), 7.18–7.27
(7H, m, Ar CH), 7.30–7.44 (7H, m, Ar CH), 9.26 (1H, d, J ¼ 8.4 Hz,
NH-6), 9.35 (1H, d, J ¼ 8.8 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
Several novel aurantiamide acetate analogs of Fmoc and t-Boc
substituted dipeptides were synthesized, and several representa-
tives of this series were identified as highly potent anti-inflamma-
tory agents. Our studies revealed that Fmoc substitution is important
for strong inhibition of elastase release in human neutrophils. Our
results also showed that synthetic dipeptides with aromatic amino
acids (9–12 and 17–20) were more effective than those with
d
¼ 20.5 (C-1), 37.7 (C-10), 38.8 (C-11), 50.1 (C-5), 55.7 (C-8), 65.3
(C-4), 126.9 (C-40), 126.9(C-400), 128.1, 128.8, 131.5 (Cbz Ar CH),
128.9 (C-20, 60, 200, 600), 129.8 (C-30, 50, 300, 500), 138.3 (C-100), 138.5 (C-
10), 167.8 (Cbz CO), 170.6 (C-2), 172.1 (C-7).
aliphatic amino acids (21–24). In addition, Fmoc-L-tryptophan-
phenylalaninol dipeptides 17 and 18 possessed enhanced and
selective inhibitory effects against elastase release compared with
the original Fmoc-
L
-tryptophan amino acid. Notably, dipeptide 9 was
5.1.1.3. (S)-2-((S)-2-(Tert-butoxycarbonylamino)-3-phenylpropan-
efficacious in the elastase assay at the nanomolar level. Furthermore,
9 and 18 were 130-fold more potent than PMSF as a positive control
for elastase release. Thus, these two compounds could be developed
as new lead anti-inflammatory agents.
amido)-3-phenylpropyl acetate (3). Yield: 92%, [
a
]
¼ ꢁ9.3ꢂ (c 0.1,
D
CH3OH). 1H NMR (C5D5N, 400 MHz):
d
¼ 1.44 (9H, s, t-Boc CH3), 1.98
(3H, s, H-1), 2.97 (2H, t, J ¼ 8.2 Hz, H-10a, b), 3.19 (1H, dd, J ¼ 7.6,
7.6 Hz, H-11a), 3.44 (1H, dd, J ¼ 7.2, 7.2 Hz, H-11b), 4.20 (2H, t,
J ¼ 6.0 Hz, H-4a, b), 4.74–4.79 (1H, m, H-8), 4.95 (1H, q, J ¼ 15.8 Hz,
H-5), 7.18–7.31 (10 H, m, Ar CH), 8.17 (1H, d, J ¼ 8.4 Hz, NH-6), 9.07
5. Experimental
(1H, d, J ¼ 8.8 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
¼ 20.7 (C-1),
d
5.1. Materials and methods
28.4 (t-Boc CH3), 37.5 (C-10), 39.5 (C-11), 50.0 (C-5), 56.7 (C-8), 65.2
(C-4), 78.6 (t-Boc quat C), 126.8 (C-40, 400), 128.6 (C-20, 60), 128.8 (C-
200, 600), 129.7 (C-30, 50), 129.9 (C-300, 500), 138.4 (C-10, 100), 156.3 (Cbz
CO), 170.6 (C-2), 172.3 (C-7).
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 thin layer chromatography (TLC)
on precoated silica gel plates (Merck, Kieselgel 60 F254, layer
thickness 0.25 mm). 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 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 1H and
5.1.1.4. (R)-2-((S)-2-(Tert-butoxycarbonylamino)-3-phenylpropan-
amido)-3-phenylpropyl acetate (4). Yield: 90%, [
a
]
¼ þ28.0ꢂ (c 0.1,
D
CH3OH). 1H NMR (C5D5N, 400 MHz):
d
¼ 1.40 (9H, s, t-Boc CH3), 1.92
(3H, s, H-1), 2.93 (2H, d, J ¼ 6.6 Hz, H-10a, b), 3.16 (1H, dd, J ¼ 7.6,
7.6 Hz, H-11a), 3.36 (1H, dd, J ¼ 6.8, 6.8 Hz, H-11b), 4.25 (2H, dddd,
J ¼ 6.0, 6.0, 4.8, 4.8 Hz, H-4a, b), 4.79–4.84 (1H, m, H-8), 4.95 (1H, q,
J ¼ 15.0 Hz, H-5), 7.18–7.33 (10 H, m, Ar CH), 8.10 (1H, d, J ¼ 8.4 Hz,
NH-6), 9.03 (1H, d, J ¼ 8.0 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.5 (C-1), 28.4 (t-Boc CH3), 37.7 (C-10), 39.4 (C-11), 49.9 (C-5),
13C, respectively. 1H NMR: C5D5N as solvent,
and 13C NMR: C5D5N as solvent,
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 spec-
trometer in positive or negative mode (solvent: CH3OH).
d
¼ 7.21, 7.59, 8.72 ppm
56.6 (C-8), 65.3 (C-4), 78.6 (t-Boc quat C), 126.8 (C-40, 400), 128.6 (C-
20, 60), 128.9 (C-200, 600), 129.7 (C-30, 50), 129.9 (C-300, 500), 138.4 (C-10),
138.5 (C-100), 156.3 (Cbz CO), 170.6 (C-2), 172.3 (C-7).
d
¼ 123.5, 135.3, 149.8 ppm. Low-
5.1.1.5. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
phenylpropan-amido)-3-phenylpropyl acetate (5). Yield: 84%,
MeOH
[a]
¼ ꢁ9.0ꢂ (c 0.1, CH3OH). UV
l
max
nm (log
3
): 300 (2.83), 288
¼ 1.99
D
5.1.1. General procedure for synthesis of compounds 1–6, 9, 10 and
15–24
(2.84), 264 (3.37), 211 (3.71). 1H NMR (C5D5N, 400 MHz):
d
(3H, s, H-1a, b, c), 2.99 (2H, t, J ¼ 7.6 Hz, H-10a, b), 3.24 (1H, dd,
J ¼ 8.0, 7.6 Hz, H-11a), 3.48 (1H, dd, J ¼ 7.6, 7.6 Hz, H-11b), 4.23 (2H,
t, J ¼ 4.4 Hz, H-4a, b), 4.27 (1H, t, J ¼ 7.0 Hz, Fmoc CH), 4.44 (1H, dd,
J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.59 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2),
4.78–4.83 (1H, m, H-8), 5.04 (1H, q, J ¼ 15.6 Hz, H-5), 7.15 (1H, t,
J ¼ 7.4 Hz, Ar CH), 7.22–7.32 (11H, m, Ar CH, Fmoc Ar CH), 7.41 (2H, t,
J ¼ 7.4 Hz, Fmoc Ar CH), 7.62 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.85
(2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 9.12 (1H, d, J ¼ 8.4 Hz, NH-6), 9.22
To a solution of L-amino acid (1.0 mmol) with L- or D-phenyl-
alaninol (1.0 mmol) in CH2Cl2 (10 mL), respectively, were added
successively coupling agents HBTU (1.5 mmol) and DIEA (1.5 mmol).
The reaction mixture was stirred for 6 h at room temperature,
concentrated, esterified with acetic anhydride (150 mL/1 mmol) in
pyridine (1 mL) and purified by column chromatography (Si-Gel)
using CHCl3 to afford the products.
(1H, d, J ¼ 8.4 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
¼ 20.7 (C-1),
d
5.1.1.1. (S)-2-((S)-2-Benzamido-3-phenylpropanamido)-3-phenylpropyl
acetate (1). Yield: 85%, [
37.5 (C-10), 39.3 (C-11), 47.7 (Fmoc CH), 50.1 (C-5), 57.3 (C-8), 65.3
(C-4), 66.7 (Fmoc CH2), 120.4, 125.6, 125.7, 127.5, 128.0 (Fmoc Ar
CH), 126.7 (C-40), 126.9 (C-400), 128.7 (C-20, 60), 128.8 (C-200, 600), 129.7
(C-30, 50), 129.8 (C-300, 500), 138.4 (C-10, 100), 141.6, 144.6 (Fmoc Ar quat
a
]D ¼ ꢁ26.1ꢂ (c 0.1, CH3OH). 1H NMR (C5D5N,
400 MHz):
d
¼ 2.00 (3H, s, H-1a, b, c), 2.99 (2H, dd, J ¼ 2.8, 2.8 Hz, H-
10a, b), 3.32 (1H, dd, J ¼ 7.6, 7.6 Hz, H-11a), 3.51 (1H, dd, J ¼ 7.6, 7.6 Hz,

C.-T. Yen et al. / European Journal of Medicinal Chemistry 44 (2009) 1933–1940
1937
C), 157.0 (Fmoc CO), 170.6 (C-2), 172.2 (C-7). HRESI-MS m/z
585.2520 [M þ Na]þ (calcd for C35H34N2O5Na 585.2522).
5.1.1.9. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
(1-trityl-1H-imidazol-4-yl)propanamido)-3-phenylpropyl
acetate
nm (log ):
MeOH
(15). Yield: 88%, [
a
]D ¼ ꢁ3.4ꢂ (c 0.1, CH3OH). UV
l
max
3
5.1.1.6. (R)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
299 (3.62), 289 (3.63), 265 (br, 4.17), 255 (4.28), 246 (4.12), 214
phenylpropan-amido)-3-phenylpropyl acetate (6). Yield: 86%,
(4.53). 1H NMR (C5D5N, 400 MHz):
d
¼ 1.93 (3H, s, H-1a, b, c), 3.05
MeOH
[
a
]D ¼ þ17.5ꢂ (c 0.1, CH3OH). UV
l
max
nm (log
3
): 300 (2.83), 288
¼ 1.89
(2H, dddd, J ¼ 7.6, 7.6, 7.2, 6.8 Hz, H-10a, b), 3.37 (1H, dd, J ¼ 8.4,
8.4 Hz, H-11a), 3.52 (1H, dd, J ¼ 5.2, 4.8 Hz, H-11b), 4.27 (2H, dd,
J ¼ 6.8, 6.0 Hz, H-4a, b), 4.35 (1H, dd, J ¼ 4.8, 4.4 Hz, Fmoc CH), 4.42
(1H, dd, J ¼ 7.6, 7.2 Hz, Fmoc CH2), 4.50 (1H, dd, J ¼ 7.6, 7.6 Hz, Fmoc
CH2), 4.81–4.87 (1H, m, H-8), 5.19–5.24 (1H, m, H-5), 6.93 (1H, s,
histidine CH), 7.15 (1H, t, J ¼ 7.2 Hz, Ar CH), 7.22–7.29 (19H, m, Ar
CH, trityl CH), 7.34 (1H, s, histidine CH), 7.39 (2H, t, J ¼ 7.6 Hz, Fmoc
Ar CH), 7.65 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.84 (2H, d, J ¼ 7.6 Hz,
Fmoc Ar CH), 8.87 (1H, d, J ¼ 8.0 Hz, NH-6), 9.12 (1H, d, J ¼ 8.0 Hz,
(2.84), 264 (3.37), 211 (3.71). 1H NMR (C5D5N, 400 MHz):
d
(3H, s, H-1a, b, c), 2.95 (2H, dd, J ¼ 2.4, 2.4 Hz, H-10a, b), 3.20 (1H,
dd, J ¼ 8.0, 8.0 Hz, H-11a), 3.39 (1H, dd, J ¼ 6.4, 6.4 Hz, H-11b), 4.23–
4.27 (2H, m, H-4a, b), 4.31 (1H, dd, J ¼ 4.4, 4.4 Hz, Fmoc CH), 4.39
(1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.57 (1H, dd, J ¼ 7.2, 6.8 Hz, Fmoc
CH2), 4.81–4.89 (1H, m, H-8), 5.04 (1H, q, J ¼ 15.6 Hz, H-5), 7.22–
7.32 (12H, m, Ar CH, Fmoc Ar CH), 7.40 (2H, t, J ¼ 7.4 Hz, Fmoc Ar
CH), 7.60 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.84 (2H, d, J ¼ 7.6 Hz, Fmoc
Ar CH), 9.07 (1H, d, J ¼ 8.4 Hz, NH-6), 9.17 (1H, d, J ¼ 8.4 Hz, NH-9).
NH-9). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.6 (C-1), 32.2 (C-11), 37.7
13C NMR (C5D5N, 100 MHz):
d
¼ 20.5 (C-1), 37.7 (C-10), 39.2 (C-11),
(C-10), 47.7 (Fmoc CH), 50.2 (C-5), 56.2 (C-8), 65.1 (C-4), 66.9 (Fmoc
CH2), 75.4 (Trityl quat C), 119.8, 129.8 (histidine CH), 120.4, 125.8,
127.5, 128.0 (Fmoc Ar CH), 126.8 (C-40), 128.2 (C-20, 60), 128.4, 130.1,
138.4, 138.8, 143.1 (trityl Ar CH), 128.8 (C-30, 50), 138.3 (C-10), 141.6,
144.6 (Fmoc Ar quat C), 157.1 (Fmoc CO), 170.7 (C-2), 172.5 (C-7).
HRESI-MS m/z 817.6645 [M þ Na]þ (calcd for C51H46N4O5Na
817.6650).
47.7 (Fmoc CH), 50.0 (C-5), 57.3 (C-8), 65.3 (C-4), 66.6 (Fmoc CH2),
120.4, 125.6, 125.7, 127.5, 128.0 (Fmoc Ar CH), 126.9 (C-40, 400), 128.7
(C-20, 60), 128.9 (C-200, 600), 129.7 (C-30, 50), 129.9 (C-300, 500), 138.4 (C-
10), 138.5 (C-100), 141.6, 144.6 (Fmoc Ar quat C), 157.0 (Fmoc CO),
170.6 (C-2), 172.2 (C-7). HRESI-MS m/z 585.2522 [M þ Na]þ (calcd
for C35H34N2O5Na 585.2522).
5.1.1.7. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(4-
5.1.1.10. (R)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-
tert-butoxy-phenyl)propanamido)-3-phenylpropyl acetate (9). Yield:
3-(1-trityl-1H-imidazol-4-yl)propanamido)-3-phenylpropyl acetate
MeOH
MeOH
90%, [
a
]D ¼ ꢁ35.8ꢂ (c 0.1, CH3OH). UV
l
nm (log
3): 299 (3.81),
(16). Yield: 88%, [
a
]D ¼ þ1.3ꢂ (c 0.1, CH3OH). UV
l
max
nm (log 3):
max
289 (3.78), 264 (4.38), 214 (4.56). 1H NMR (C5D5N, 400 MHz):
299 (3.62), 289 (3.63), 265 (br, 4.17), 255 (4.28), 246 (4.12), 214
d
¼ 1.25 (9H, s, t-butyl CH3), 2.01 (3H, s, H-1a, b, c), 3.00 (2H, t,
(4.53). 1H NMR (C5D5N, 400 MHz):
d
¼ 1.94 (3H, s, H-1a, b, c), 3.02
J ¼ 6.6 Hz, H-10a, b), 3.23 (1H, dd, J ¼ 7.6, 7.6 Hz, H-11a), 3.48 (1H, dd,
J ¼ 6.8, 6.8 Hz, H-11b), 4.22–4.29 (3H, br m, H-4a, b, Fmoc CH), 4.44
(1H, dd, J ¼ 7.6, 7.2 Hz, Fmoc CH2), 4.59 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc
CH2), 4.80–4.86 (1H, m, H-8), 5.02 (1H, q, J ¼ 15.8 Hz, H-5), 7.01 (2H,
d, J ¼ 8.0 Hz, Ar CH), 7.15 (1H, t, J ¼ 7.2 Hz, Ar CH), 7.26 (4H, dd,
J ¼ 3.2, 3.2 Hz, Ar CH), 7.33 (2H, t, J ¼ 7.8 Hz, Fmoc Ar CH), 7.41 (2H, t,
J ¼ 7.4 Hz, Fmoc Ar CH), 7.64 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.85 (2H,
d, J ¼ 7.6 Hz, Fmoc Ar CH), 9.09 (1H, d, J ¼ 8.8 Hz, NH-6), 9.24 (1H, d,
(2H, d, J ¼ 7.2 Hz, H-10a, b), 3.24 (1H, dd, J ¼ 8.2, 7.6 Hz, H-11a), 3.42
(1H, dd, J ¼ 5.2, 4.8 Hz, H-11b), 4.28 (2H, dd, J ¼ 7.0, 7.0 Hz, H-4a, b),
4.36 (1H, dd, J ¼ 4.8, 4.4 Hz, Fmoc CH), 4.44 (1H, dd, J ¼ 7.6, 7.2 Hz,
Fmoc CH2), 4.48 (1H, dd, J ¼ 7.6, 7.6 Hz, Fmoc CH2), 4.82–4.88 (1H,
m, H-8), 5.22 (1H, q, J ¼ 15.8 Hz, H-5), 6.77 (1H, s, histidine CH), 7.11
(1H, t, J ¼ 7.2 Hz, Ar CH), 7.24–7.42 (22H, m, Ar CH, Trityl CH,
histidine CH, Fmoc Ar CH), 7.64 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.83
(2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.75 (1H, d, J ¼ 8.0 Hz, NH-6), 9.12
J ¼ 8.0 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.7 (C-1), 28.8 (t-
(1H, d, J ¼ 8.0 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
¼ 20.1 (C-1),
d
butyl CH3), 37.5 (C-10), 38.5 (C-11), 47.7 (Fmoc CH), 50.1 (C-5), 57.4
(C-8), 65.2 (C-4), 66.7 (Fmoc CH2), 78.0 (t-butyl quat C), 120.4, 125.6,
125.7, 127.5, 128.0 (Fmoc Ar CH), 124.3 (C-200, 600), 126.7 (C-40), 128.8
(C-20, 60), 129.7 (C-30, 50), 130.3 (C-300, 500), 133.0 (C-100), 138.4 (C-10),
141.6, 144.6 (Fmoc Ar quat C), 154.6 (C-400), 157.0 (Fmoc CO), 170.7
(C-2), 172.3 (C-7). HRESI-MS m/z 657.2936 [M þ Na]þ (calcd for
C39H42N2O6Na 657.2940).
31.6 (C-11), 37.4 (C-10), 47.2 (Fmoc CH), 49.7 (C-5), 55.6 (C-8), 65.1
(C-4), 66.5 (Fmoc CH2), 75.0 (trityl quat C), 119.3, 129.2 (histidine
CH), 119.8, 125.3, 126.2, 128.3 (Fmoc Ar CH), 127.0 (C-40), 128.2 (C-20,
60), 128.4, 130.1, 138.4, 138.8, 143.1 (trityl Ar CH), 128.8 (C-30, 50),
138.3 (C-10), 141.6, 144.6 (Fmoc Ar quat C), 157.1 (Fmoc CO),170.1 (C-
2), 171.8 (C-7). HRESI-MS m/z 817.6652 [M þ Na]þ (calcd for
C51H46N4O5Na 817.6650).
5.1.1.8. (R)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(4-
5.1.1.11. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-
tert-butoxy-phenyl)propanamido)-3-phenylpropyl acetate (10). Yield:
3-(1H-indol-3-yl)propanamido)-3-phenylpropyl acetate (17). Yield:
MeOH
MeOH
93%, [
a
]D ¼ þ15.5ꢂ (c 0.1, CH3OH). UV
l
nm (log
3): 299 (3.81),
90%, [
a
]D ¼ ꢁ1.2ꢂ (c 0.1, CH3OH). UV
l
nm (log 3): 264 (4.27),
¼ 1.93 (3H, s, H-1a, b, c),
max
max
289 (3.78), 264 (4.38), 214 (4.56). 1H NMR (C5D5N, 400 MHz):
209 (4.86). 1H NMR (C5D5N, 400 MHz):
d
d
¼ 1.24 (9H, s, t-butyl CH3), 1.89 (3H, s, H-1a, b, c), 2.96 (2H, dd,
2.95 (2H, dddd, J ¼ 7.6, 7.2, 6.8, 6.8 Hz, H-10a, b), 3.60 (1H, dd,
J ¼ 7.2, 6.8 Hz, H-11a), 3.77 (1H, dd, J ¼ 7.6, 7.6 Hz, H-11b), 4.20 (2H,
d, J ¼ 4.8 Hz, H-4a, b), 4.29 (1H, t, J ¼ 7.2 Hz, Fmoc CH), 4.47 (1H, dd,
J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.60 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2),
4.80–4.85 (1H, m, H-8), 5.20 (1H, q, J ¼ 15.2 Hz, H-5), 7.13 (2H, q,
J ¼ 16.0 Hz, Trp Ar CH), 7.25 (5H, d, J ¼ 7.2, Ar CH), 7.29 (2H, t,
J ¼ 7.0 Hz, Trp Ar CH), 7.39 (2H, t, J ¼ 7.6 Hz, Fmoc Ar CH), 7.43 (1H, s,
Trp CH), 7.54 (1H, d, J ¼ 8.4 Hz, Fmoc Ar CH), 7.63 (2H, d, J ¼ 7.6 Hz,),
7.85 (3H, t, J ¼ 8.0 Hz, Fmoc Ar CH), 9.07 (1H, d, J ¼ 8.0 Hz, NH-6),
9.22 (1H, d, J ¼ 8.4 Hz, NH-9), 11.9 (1H, s, Trp NH). 13C NMR (C5D5N,
J ¼ 7.2, 7.2 Hz, H-10a, b), 3.19 (1H, dd, J ¼ 8.0, 8.0 Hz, H-11a), 3.39 (1H,
dd, J ¼ 6.8, 6.8 Hz, H-11b), 4.23–4.31 (3H, m, H-4a, b, Fmoc CH), 4.40
(1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.57 (1H, dd, J ¼ 7.2, 6.8 Hz, Fmoc
CH2), 4.84–4.88 (1H, m, H-8), 5.02 (1H, q, J ¼ 15.8 Hz, H-5), 7.04 (2H,
d, J ¼ 8.4 Hz, Ar CH), 7.25 (4H, dd, J ¼ 8.0, 7.6 Hz, Ar CH), 7.33 (3H, d,
J ¼ 4.4 Hz, Ar CH, Fmoc Ar CH), 7.40 (2H, t, J ¼ 7.6 Hz, Fmoc Ar CH),
7.62 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.84 (2H, d, J ¼ 7.6 Hz, Fmoc Ar
CH), 9.08 (1H, d, J ¼ 8.4 Hz, NH-6), 9.20 (1H, d, J ¼ 8.0 Hz, NH-9). 13C
NMR (C5D5N, 100 MHz):
d
¼ 20.5 (C-1), 28.8 (t-butyl CH3), 37.8 (C-
10), 38.7 (C-11), 47.7 (Fmoc CH), 50.0 (C-5), 57.4 (C-8), 65.3 (C-4),
66.6 (Fmoc CH2), 78.0 (t-butyl quat C), 120.4, 125.6, 125.7, 127.5, 128.0
(Fmoc Ar CH), 124.3 (C-200, 600), 126.8 (C-40), 128.9 (C-20, 60), 129.7 (C-
30, 50), 130.4 (C-300, 500), 133.0 (C-100), 138.4 (C-10), 141.6, 144.6 (Fmoc
Ar quat C), 154.6 (C-400), 157.0 (Fmoc CO), 170.7 (C-2), 172.3
(C-7). HRESI-MS m/z 657.2936 [M þ Na]þ (calcd for C39H42N2O6Na
657.2940).
100 MHz):
d
¼ 20.6 (C-1), 29.4 (C-11), 37.5 (C-10), 47.7 (Fmoc CH),
50.1 (C-5), 56.9 (C-8), 65.2 (C-4), 66.7 (Fmoc CH2), 111.3, 111.9, 114.2,
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), 126.8 (C-40), 128.8 (C-20, 60), 129.7 (C-30, 50),
138.5 (C-10), 141.6,144.6 (Fmoc Ar quat C),157.1 (Fmoc CO),170.7 (C-
2), 172.5 (C-7). HRESI-MS m/z 624.2470 [M þ Na]þ (calcd for
C37H325N3O5Na 624.2474).

1938
C.-T. Yen et al. / European Journal of Medicinal Chemistry 44 (2009) 1933–1940
MeOH
5.1.1.12. (R)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-
[
a
]
¼ ꢁ5.9ꢂ (c 0.1, CH3OH). UV
l
nm (log
3): 263 (4.24), 208
D
max
3-(1H-indol-3-yl)propanamido)-3-phenylpropyl acetate (18). Yield:
(4.84). 1H NMR (C5D5N, 400 MHz):
d
¼ 1.07 (6H, dd, J ¼ 3.6, 3.2 Hz, H-
MeOH
90%, [
a]
¼ þ9.9ꢂ (c 0.1, CH3OH). UV
l
nm (log
3
): 264 (4.42),
12, 13), 2.03 (3H, s, H-1a, b, c), 2.33–2.41 (1H, m, H-11), 3.00 (2H, t,
J ¼ 6.4 Hz, H-10a, b), 4.32 (3H, dddd, J ¼ 7.2, 6.8, 6.8, 6.8 Hz, H-8,
Fmoc CH2, Fmoc CH), 4.42 (1H, dd, J ¼ 4.4, 4.4 Hz, H-4a), 4.55 (1H,
ddd, J ¼ 4.0, 2.8, 2.4 Hz, H-4b), 4.71 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2),
4.86–4.74 (1H, m, H-5), 7.22–7.29 (3H, m, Ar CH), 7.32 (2H, d,
J ¼ 7.2 Hz, Fmoc Ar CH), 7.40 (2H, ddd, J ¼ 4.0, 3.6, 3.6 Hz, Ar CH), 7.69
(2H, dd, J ¼ 2.4, 2.0 Hz, Fmoc Ar CH), 7.85 (2H, d, J ¼ 7.6 Hz, Fmoc Ar
CH), 8.89 (1H, d, J ¼ 9.2 Hz, NH-6), 9.21 (1H, d, J ¼ 8.4 Hz, NH-9). 13C
D
max
209 (4.96). 1H NMR (C5D5N, 400 Hz):
d
¼ 1.88 (3H, s, H-1a, b, c), 2.89
(2H, d, J ¼ 6.8 Hz, H-10a, b), 3.55 (1H, dd, J ¼ 7.6, 7.2 Hz, H-11a), 3.70
(1H, dd, J ¼ 6.8, 6.8 Hz, H-11b), 4.22 (2H, d, J ¼ 4.8 Hz, H-4a, b), 4.28
(1H, t, J ¼ 7.2 Hz, Fmoc CH), 4.43 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2),
4.57 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.85–4.90 (1H, m, H-8), 5.18
(1H,q, J ¼ 15.6 Hz, H-5), 7.17 (2H, q, J ¼ 13.2 Hz, Trp Ar CH), 7.22-7.38
(8H, m, Ar CH, Trp Ar CH), 7.40 (2H, t, J ¼ 7.6 Hz, Fmoc Ar CH), 7.41
(1H, s, Trp CH), 7.55 (1H, d, J ¼ 8.0 Hz, Fmoc Ar CH), 7.61 (2H, d,
J ¼ 7.6 Hz,), 7.83 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.89 (1H,
d, J ¼ 8.0 Hz, Fmoc Ar CH), 9.06 (1H, d, J ¼ 8.4 Hz, NH-6), 9.20 (1H, d,
J ¼ 8.4 Hz, NH-9), 11.9 (1H, s, Trp NH). 13C NMR (C5D5N, 100 MHz):
NMR (C5D5N, 100 MHz):
d
¼ 18.9 (C-12), 19.6 (C-13), 20.7 (C-1), 31.5
(C-11), 37.8 (C-10), 47.8 (Fmoc CH), 49.9 (C-5), 61.7 (C-8), 65.3 (C-4),
66.6 (Fmoc CH2), 120.4, 125.7, 127.5, 128.0 (Fmoc Ar CH), 126.8 (C-40),
128.8 (C-20, 60), 129.7 (C-30, 50), 138.3 (C-10), 141.7, 144.5, 144.7 (Fmoc
Ar quat C), 157.4 (Fmoc CO), 170.7 (C-2), 172.4 (C-7). HRESI-MS m/z
537.2518 [M þ Na]þ (calcd for C31H34N2O5Na 537.2522).
d
¼ 20.5 (C-1), 29.5 (C-11), 37.6 (C-10), 47.7 (Fmoc CH), 49.9 (C-5),
57.1 (C-8), 65.2 (C-4), 66.6 (Fmoc CH2),111.4,112.0,121.7,124.0,128.6,
135.8 (Trp Ar CH),120.4,125.7,125.8,127.5,128.0 (Fmoc Ar CH),126.8
(C-40),128.8 (C-20, 60),129.7 (C-30, 50),138.5 (C-10),141.6,144.6 (Fmoc
Ar quat C), 157.1 (Fmoc CO), 170.6 (C-2), 172.8 (C-7). HRESI-MS m/z
624.2471 [M þ Na]þ (calcd for C37H325N3O5Na 624.2474).
5.1.1.16. (R)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-
3-methylbutan-amido)-3-phenylpropyl acetate (22). Yield: 94%,
MeOH
[a
]D ¼ þ13.3ꢂ (c 0.1, CH3OH). UV
l
nm (log 3): 263 (4.16), 208
max
(4.64). 1H NMR (C5D5N, 400 MHz):
d
¼ 0.95 (6H, dd, J ¼ 6.8, 6.8 Hz, H-
5.1.1.13. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
12, 13), 1.91 (3H, s, H-1a, b, c), 2.24–2.32 (1H, m, H-11), 3.04 (2H, d,
J ¼ 7.2 Hz, H-10a, b), 4.30–4.37 (3H, m, H-8, Fmoc CH2, Fmoc CH),
4.52 (2H, m, H-4a, b), 4.68 (1H, dd, J ¼ 7.6, 7.6 Hz, Fmoc CH2), 4.89–
4.97 (1H, m, H-5), 7.23–7.27 (3H, m, Ar CH), 7.32 (2H, t, Fmoc Ar CH),
7.37–7.42 (4H, m, Fmoc Ar CH, Ar CH), 7.67 (2H, t, J ¼ 6.4 Hz, Fmoc Ar
CH), 7.84 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 8.82 (1H, d, J ¼ 8.8 Hz, NH-
(naphthalen-2-yl) propanamido)-3-phenylpropyl acetate (19). Yield:
MeOH
83%, [
a
]D ¼ ꢁ20.0ꢂ (c 0.1, CH3OH). UV
l
max
nm (log
3): 264 (3.38),
224 (5.11), 210 (5.04). 1H NMR (C5D5N, 400 MHz):
d
¼ 1.82 (3H, s, H-
1a, b, c), 2.98 (2H, dddd, J ¼ 7.2, 7.2, 7.2, 6.8 Hz, H-10a, b), 3.39 (1H,
dd, J ¼ 7.6, 7.6 Hz, H-11a), 3.63 (1H, dd, J ¼ 8.0, 7.6 Hz, H-11b), 4.22
(2H, d, J ¼ 5.2, H-4a, b), 4.26 (1H, t, J ¼ 7.0 Hz, Fmoc CH), 4.45 (1H,
dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.58 (1H, dd, J ¼ 7.6, 7.2 Hz, Fmoc CH2),
4.78–4.82 (1H, m, H-8), 5.14 (1H, q, J ¼ 15.8 Hz, H-5), 7.15 (2H, q,
J ¼ 15.4 Hz, Ar CH), 7.21–7.25 (4H, m, Ar CH), 7.29 (2H, d, J ¼ 7.2 Hz,
Fmoc Ar CH), 7.39 (2H, t, J ¼ 7.6 Hz, Fmoc Ar CH), 7.46 (3H, m,
naphthylalanine Ar CH), 7.60 (2H, d, J ¼ 7.6 Hz, Fmoc Ar CH), 7.76
(3H, d, J ¼ 7.6 Hz, naphthylalanine Ar CH), 7.84 (2H, d, J ¼ 7.2 Hz,
Fmoc Ar CH), 9.26 (1H, d, J ¼ 8.4 Hz, NH-6), 9.33 (1H, d, J ¼ 8.4 Hz,
6), 9.12 (1H, d, J ¼ 8.4 Hz, NH-9). 13C NMR (C5D5N,100 MHz):
¼ 18.5
d
(C-12),19.7 (C-13), 20.5 (C-1), 31.6 (C-11), 37.8 (C-10), 47.8 (Fmoc CH),
49.9 (C-5), 61.6 (C-8), 65.7 (C-4), 66.5 (Fmoc CH2), 120.4, 125.6, 125.7,
127.5, 128.0 (Fmoc Ar CH), 126.8 (C-40), 128.9 (C-20, 60), 129.7 (C-30,
50), 138.6 (C-10),141.6, 144.5,144.7 (Fmoc Ar quat C), 157.4 (Fmoc CO),
170.6 (C-2), 172.4 (C-7). HRESI-MS m/z 537.2520 [M þ Na]þ (calcd for
C31H34N2O5Na 537.2522).
NH-9). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.5 (C-1), 37.5 (C-10), 39.5
5.1.1.17. (9H-Fluoren-9-yl)methyl (2S,3S)-1-((S)-1-hydroxy-3-phenyl-
propan-2-ylamino)-3-methyl-1-oxopentan-2-ylcarbamate (23). Yield:
(C-11), 47.7 (Fmoc CH), 50.2 (C-5), 57.4 (C-8), 65.2 (C-4), 66.7 (Fmoc
CH2), 120.4, 125.5, 125.8, 127.4, 128.0 (Fmoc Ar CH), 126.3 (C-40, 400),
128.2 (C-20, 60), 128.4 (C-200, 600), 132.8 (C-30, 50), 134.0 (C-300, 500),
138.4 (C-10, 100), 141.6, 144.5 (Fmoc Ar quat C), 157.0 (Fmoc CO), 170.5
(C-2), 172.1 (C-7). HRESI-MS m/z 635.2519 [M þ Na]þ (calcd for
C39H36N2O5Na 635.2522).
89%, [
a
]D ¼ ꢁ7.4ꢂ (c 0.1, CH3OH). UV lmMaexOH nm (log
3): 263 (3.59), 207
(4.22). 1H NMR (C5D5N, 400 MHz):
d
¼ 0.79 (3H, t, J ¼ 7.4 Hz, H-13a, b,
c), 1.06 (3H, d, J ¼ 6.8 Hz, H-14a, b, c), 1.31 (1H, quint, H-12a), 1.70–
1.77 (1H, m, H-12b), 2.05 (3H, s, H-1a, b, c), 2.14–2.19 (1H, m, H-11),
3.01 (2H, t, J ¼ 6.6 Hz, H-10a, b), 4.32 (2H, ddd, J ¼ 7.2, 6.8, 6.8 Hz,
Fmoc CH2, Fmoc CH), 4.43 (1H, dd, J ¼ 4.4, 4.4 Hz, H-8), 4.57 (2H, dd,
J ¼ 9.2, 8.0 Hz, H-4a, b), 4.72 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.88–
4.92 (1H, m, H-5), 7.14 (1H, t, J ¼ 7.2 Hz, Ar CH), 7.23–7.28 (4H, m, Ar
CH), 7.33 (2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 7.40 (2H, ddd, J ¼ 3.6, 3.6,
3.6 Hz, Fmoc Ar CH), 7.69 (2H, dd, J ¼ 3.6, 3.6 Hz, Fmoc Ar CH), 7.85
(2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 8.90 (1H, d, J ¼ 9.2 Hz, NH-6), 9.24
5.1.1.14. (R)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-
(naphthalen-2-yl) propanamido)-3-phenylpropyl acetate (20). Yield:
85%, [
a
]D ¼ þ7.9ꢂ (c 0.1, CH3OH). UV lmMaexOH nm (log
3
): 264 (3.22), 224
(5.04), 210 (4.86).1H NMR (C5D5N, 400 MHz):
d
¼ 1.88 (3H, s, H-1a, b,
c), 2.92 (2H, dddd, J ¼ 7.2, 7.2, 7.2, 6.8 Hz, H-10a, b), 3.36 (1H, dd,
J ¼ 8.0, 7.6 Hz, H-11a), 3.55 (1H, dd, J ¼ 6.8, 6.8 Hz, H-11b), 4.24 (2H,
m, H-4a, b), 4.30 (1H, dd, J ¼ 7.2, 6.4 Hz, Fmoc CH), 4.39 (1H, dd,
J ¼ 7.2, 6.8 Hz, Fmoc CH2), 4.55 (1H, dd, J ¼ 7.2, 6.8 Hz, Fmoc CH2),
4.83–4.88 (1H, m, H-8), 5.14 (1H, q, J ¼ 15.6 Hz, H-5), 7.17–7.24 (8H, m,
Fmoc Ar CH, Ar CH, naphthylalanine Ar CH), 7.38 (2H, t, J ¼ 7.6 Hz,
Fmoc Ar CH), 7.44 (3H, m, naphthylalanine Ar CH), 7.56 (2H, d,
J ¼ 7.6 Hz, Fmoc Ar CH), 7.76 (3H, m, naphthylalanine Ar CH), 7.82
(2H, d, J ¼ 7.2 Hz, Fmoc ArCH), 9.22 (1H, d, J ¼ 8.8 Hz, NH-6), 9.26 (1H,
(1H, d, J ¼ 8.4 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
¼ 11.0 (C-13),
d
15.8 (C-14), 20.7 (C-1), 25.3 (C-12), 37.3 (C-10), 37.8 (C-11), 47.8 (Fmoc
CH), 49.9 (C-5), 60.4 (C-8), 65.3 (C-4), 66.6 (Fmoc CH2), 120.4, 125.7,
127.5, 128.0 (Fmoc Ar CH), 126.8, 128.8, 129.7, 138.3 (Ar CH), 141.7,
144.5, 144.8 (Fmoc Ar quat C), 157.3 (Fmoc CO), 170.7 (C-2), 172.5
(C-7). HRESI-MS m/z 551.2520 [M þ Na]þ (calcd for C32H36N2O5Na
551.2522).
d, J ¼ 8.0 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.5 (C-1), 37.6
5.1.1.18. (9H-Fluoren-9-yl)methyl (2S,3S)-1-((R)-1-hydroxy-3-phenyl-
propan-2-ylamino)-3-methyl-1-oxopentan-2-ylcarbamate (24). Yield:
(C-10), 39.4 (C-11), 47.6 (Fmoc CH), 50.0 (C-5), 57.3 (C-8), 65.2 (C-4),
66.6 (Fmoc CH2),120.4,125.5,125.7,127.4, 128.0 (Fmoc Ar CH),126.8,
128.8, 129.7, 138.5 (Ar CH), 125.9, 126.4, 128.3, 128.5, 132.8, 134.0
(naphthylalanine Ar CH), 141.6, 144.6 (Fmoc Ar quat C), 157.0 (Fmoc
CO), 170.6 (C-2), 172.3 (C-7). HRESI-MS m/z 635.2518 [M þ Na]þ
(calcd for C39H36N2O5Na 635.2522).
89%, [
a
]D ¼ þ6.4ꢂ (c 0.1, CH3OH). UV lmMaexOH nm (log
3): 263 (3.80), 207
(4.38). 1H NMR (C5D5N, 400 MHz):
d
¼ 0.75 (3H, t, J ¼ 7.4 Hz, H-13a,
b,c), 0.90 (3H, d, J ¼ 6.8 Hz, H-14a, b, c), 1.22 (1H, quint, H-12a), 1.61–
1.68 (1H, m, H-12b), 1.91 (3H, s, H-1a, b, c), 2.04–2.08 (1H, m, H-11),
3.05 (2H, t, J ¼ 7.6 Hz, H-10a, b), 4.30–4.38 (3H, m, H-8, Fmoc CH2,
Fmoc CH), 4.50–4.57 (2H, m, H-4a, b), 4.69 (1H, dd, J ¼ 7.2, 7.2 Hz,
Fmoc CH2), 4.92–4.96 (1H, m, H-5), 7.14 (1H, t, J ¼ 7.2 Hz, Ar CH), 7.23-
7.24 (3H, m, Ar CH), 7.33 (2H, d, J ¼ 7.4 Hz, Fmoc Ar CH), 7.40 (4H, m,
5.1.1.15. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-
3-methylbutan-amido)-3-phenylpropyl acetate (21). Yield: 92%,

C.-T. Yen et al. / European Journal of Medicinal Chemistry 44 (2009) 1933–1940
1939
Fmoc Ar CH, Ar CH), 7.67 (2H, t, J ¼ 7.2 Hz, Fmoc Ar CH), 7.84 (2H, d,
J ¼ 7.6 Hz, Fmoc Ar CH), 8.84 (1H, d, J ¼ 9.2 Hz, NH-6), 9.14 (1H, d,
b), 3.20 (1H, dd, J ¼ 8.0, 8.0 Hz, H-11a), 3.43 (1H, dd, J ¼ 7.6, 7.6 Hz,
H-11b), 3.94 (2H, d, J ¼ 6.4, H-4a, b), 3.98 (1H, dd, J ¼ 5.2, 5.2 Hz, H-
8), 4.89 (1H, m, H-5), 7.20–7.29 (8H, m, Ar CH), 7.42 (2H, d,
J ¼ 8.0 Hz, Ar CH), 8.70 (1H,d, J ¼ 8.4 Hz, NH-3). 13C NMR (C5D5N,
J ¼ 8.4 Hz, NH-9). 13C NMR (C5D5N,100 MHz):
¼ 11.1 (C-13), 15.8 (C-
d
14), 20.6 (C-1), 25.2 (C-12), 37.5 (C-10), 37.7 (C-11), 47.8 (Fmoc CH),
49.9 (C-5), 60.4 (C-8), 65.7 (C-4), 66.5 (Fmoc CH2), 120.4,125.6, 125.8,
127.5, 128.0 (Fmoc Ar CH), 126.8, 128.8, 129.7, 138.7 (Ar CH), 141.2,
144.5, 144.8 (Fmoc Ar quat C), 157.3 (Fmoc CO), 170.6 (C-2), 172.5 (C-
7). HRESI-MS m/z 551.2520 [M þ Na]þ (calcd for C32H36N2O5Na
551.2522).
100 MHz):
d
¼ 20.7 (C-1), 37.6 (C-10), 41.5 (C-11), 53.2 (C-5), 57.1 (C-
8), 63.0 (C-4), 126.5 (C-40), 126.8 (C-400), 128.7 (C-20, 60), 128.8 (C-200,
600), 129.9 (C-30, 50, 300, 500), 138.9 (C-10), 139.6 (C-100), 170.6 (C-2),
174.0 (C-7).
5.1.3.2. (R)-2-((S)-2-Amino-3-phenylpropanamido)-3-phenylpropyl
5.1.2. General procedure for synthesis of compounds 11 and 12
To a solution of compounds 9 and 10 in TFA/TIS/H2O ¼ 95/2.5/2.5
acetate (8). Yield: 80%, [
400 MHz):
a
]D ¼ þ5.9ꢂ (c 0.1, CH3OH). 1H NMR (C5D5N,
d
¼ 1.89 (3H, s, H-1a, b, c), 2.93 (1H, dd, J ¼ 8.4, 8.4 Hz, H-
(100
mL/mg), respectively, was stirred at room temperature for 1 h. The
10a), 3.11 (1H, dd, J ¼ 7.2, 6.8 Hz, H-10b), 3.22 (1H, dd, J ¼ 7.2, 6.8 Hz,
H-11a), 3.40 (1H, dd, J ¼ 4.8, 4.4 Hz, H-11b), 3.85 (2H, dd, J ¼ 4.8,
4.4 Hz, H-4a, b), 3.93 (1H, dd, J ¼ 5.2, 4.4 Hz, H-5), 4.70 (1H, m, H-8),
7.22–7.30 (8H, m, Ar CH), 7.42 (2H, d, J ¼ 8.4 Hz, Ar CH), 8.54 (1H, d,
solvent was evaporated under vacuum and the reaction mixture was
purified by suction (MeOH as solvent) to give the desired products.
5.1.2.1. (S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(4-
hydroxyphenyl) propanamido)-3-phenylpropyl acetate (11). Yield: 93%,
J ¼ 8.4 Hz, NH-3). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.5 (C-1), 37.7
(C-10), 41.9 (C-11), 53.1 (C-5), 57.3 (C-8), 63.0 (C-4), 126.5 (C-40),
126.7 (C-400), 128.7 (C-20, 60), 128.8 (C-200, 600), 129.9 (C-30, 50), 130.0
(C-300, 500), 139.2 (C-10), 139.6 (C-100), 170.6 (C-2), 174.6 (C-7).
MeOH
[a
]D ¼ ꢁ12.3ꢂ (c 0.1, CH3OH). UV
l
max
nm (log 3): 264 (3.88), 208
(4.50). 1H NMR (C5D5N, 400 MHz): 2.04 (3H, s, H-1a, b, c), 3.01 (2H,
dddd, J ¼ 7.6, 7.2, 7.2, 6.8 Hz, H-10a, b), 3.23 (1H, dd, J ¼ 7.6, 7.6 Hz, H-
11a), 3.46 (1H, dd, J ¼ 7.6, 7.2 Hz, H-11b), 4.22–4.31 (3H, m, H-4a, b,
Fmoc CH), 4.44 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2), 4.61 (1H, dd, J ¼ 7.2,
6.8 Hz, Fmoc CH2), 4.81–4.85 (1H, m, H-8), 5.04 (1H,q, J ¼ 15.4 Hz, H-
5), 7.09 (2H, d, J ¼ 8.0 Hz, Ar CH), 7.15 (1H, t, J ¼ 7.4 Hz, Ar CH), 7.23–
7.28 (2H, m, Ar CH), 7.31 (2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 7.39 (2H, t,
J ¼ 7.4 Hz, Fmoc Ar CH), 7.64 (2H, dd, J ¼ 4.0, 3.6 Hz, Fmoc Ar CH), 7.84
(2H, d, J ¼ 8.0 Hz, Fmoc Ar CH), 9.09 (1H, d, J ¼ 8.4 Hz, NH-6), 9.26 (1H,
5.1.3.3. (S)-2-((S)-2-Amino-3-(4-hydroxyphenyl)propanamido)-3-
phenylpropyl acetate (13). Yield: 82%, 1H NMR (C5D5N, 400 MHz):
d
¼ 1.98 (3H, s, H-1a, b, c), 2.89 (1H, d, J ¼ 8.4, 8.4 Hz, H-11a), 2.97 (2H,
t, J ¼ 8.6 Hz, H-10a, b), 3.31 (1H, dd, J ¼ 4.8, 4.8 Hz, H-11b), 3.84 (1H,
dd, J ¼ 4.8, 4.8 Hz, H-8), 4.26 (2H, dddd, J ¼ 6.0, 5.6, 4.8, 4.8 Hz, H-4a,
b), 4.76–4.85 (2H, m, H-5, 8), 7.04 (2H, d, J ¼ 8.0 Hz, Ar CH), 7.23–7.32
(7H, m, Ar CH), 8.55 (1H, d, J ¼ 8.8 Hz, NH-6). 13C NMR (C5D5N,
d, J ¼ 8.0 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.7 (C-1), 37.5
100 MHz):
d
¼ 20.6 (C-1), 28.8 (t-butyl CH3), 37.7 (C-10), 41.2 (C-11),
(C-10), 38.6 (C-11), 47.7 (Fmoc CH), 50.0 (C-5), 57.7 (C-8), 65.3 (C-4),
66.7 (Fmoc CH2), 116.1 (C-200, 600), 120.4, 125.6, 125.8, 127.5, 128.0
(Fmoc Ar CH), 126.7 (C-40), 128.4 (C-100), 128.8 (C-20, 60), 128.9 (C-200,
600), 129.7 (C-30, 50), 131.0 (C-300, 500), 138.5 (C-10), 141.6, 144.6 (Fmoc Ar
quat C), 157.0 (Fmoc CO), 157.6 (C-400), 170.7 (C-2), 172.4 (C-7). HRESI-
MS m/z 601.2317 [M þ Na]þ (calcd for C35H34N2O6Na 601.2314).
49.5 (C-5), 57.3 (C-8), 65.4 (C-4), 78.1 (t-butyl quat C),124.3 (C-200, 600),
126.8 (C-40), 128.8 (C-20, 60),129.7 (C-30, 50), 130.3 (C-300, 500), 133.7 (C-
100), 138.4 (C-10), 154.5 (C-400), 170.7 (C-2), 175.1 (C-7). HRESI-MS m/z
356.2520 [M þ Na]þ (calcd for C20H24N2O4Na 356.2522).
5.1.3.4. (R)-2-((S)-2-Amino-3-(4-hydroxyphenyl)propanamido)-3-
phenylpropyl acetate (14). Yield: 83%,
d
¼ 1.89 (3H, s, H-1a, b, c), 2.93
5.1.2.2. (R)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(4-
hydroxyphenyl) propanamido)-3-phenylpropyl acetate (12). Yield: 85%,
(1H, dd, J ¼ 8.8, 8.6 Hz, H-11a), 3.18 (2H, t, J ¼ 8.6 Hz, H-10a, b), 3.39
(1H, dd, J ¼ 4.6, 4.4 Hz, H-11b), 3.83 (1H, dd, J ¼ 4.8, 4.8 Hz, H-8),
3.92 (2H, t, J ¼ 4.2 Hz, H-4a, b), 4.68–4.72 (2H, m, H-5, 8), 7.06 (2H,
d, J ¼ 8.4 Hz, Ar CH), 7.25 (4H, dd, J ¼ 8.0, 7.6 Hz, Ar CH), 7.33 (1H, d,
J ¼ 6.6 Hz, Ar CH), 7.43 (2H, d, J ¼ 7.4 Hz, Fmoc Ar CH), 8.46 (1H, d,
MeOH
[a
]D ¼ þ15.3ꢂ (c 0.1, CH3OH). UV
l
max
nm (log 3): 263 (3.81), 208
(4.34). 1H NMR (C5D5N, 400 MHz): 1.90 (3H, s, H-1a, b, c), 2.99 (2H, t,
J ¼ 7.6 Hz, H-10a, b), 3.20 (1H, dd, J ¼ 8.0, 8.0 Hz, H-11a), 3.38 (1H, dd,
J ¼ 6.8, 6.4 Hz, H-11b), 4.24–4.32 (3H, m, H-4a, b; Fmoc CH), 4.39 (1H,
dd, J ¼ 7.2, 6.8 Hz, Fmoc CH2), 4.59 (1H, dd, J ¼ 7.2, 7.2 Hz, Fmoc CH2),
4.84–4.89 (1H, m, H-8), 5.02 (1H,q, J ¼ 15.6 Hz, H-5), 7.10 (2H, d,
J ¼ 8.0 Hz, Ar CH), 7.22–7.27 (3H, m, Ar CH), 7.31 (2H, d, J ¼ 7.2 Hz, Fmoc
Ar CH), 7.38 (2H, t, J ¼ 7.4 Hz, Fmoc Ar CH), 7.62 (2H, d, J ¼ 7.6 Hz, Fmoc
Ar CH), 7.83 (2H, d, J ¼ 7.2 Hz, Fmoc Ar CH), 9.02 (1H, d, J ¼ 8.4 Hz, NH-
J ¼ 8.8 Hz, NH-6). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.5 (C-1), 37.8
(C-10), 41.2 (C-11), 50.0 (C-5), 57.4 (C-8), 65.4 (C-4), 124.3 (C-200, 600),
126.8 (C-40), 128.9 (C-20, 60), 129.7 (C-30, 50), 130.4 (C-300, 500), 133.8
(C-100), 138.4 (C-10), 154.6 (C-400), 170.7 (C-2), 174.9 (C-7). HRESI-MS
m/z 356.2520 [M þ Na]þ (calcd for C20H24N2O4Na 356.2522).
6), 9.18 (1H, d, J ¼ 8.4 Hz, NH-9). 13C NMR (C5D5N, 100 MHz):
d
¼ 20.5
5.2. Biological assay
(C-1), 37.7 (C-10), 38.6 (C-11), 47.7 (Fmoc CH), 50.0 (C-5), 57.6 (C-8),
65.3 (C-4), 66.6 (Fmoc CH2), 116.1 (C-200, 600), 120.4, 125.6, 125.8, 127.5,
128.0 (Fmoc Ar CH), 126.8 (C-40), 128.5 (C-100), 128.9 (C-20, 60), 129.8 (C-
30, 50), 130.3 (C-300, 500), 131.0 (C-300, 500), 138.5 (C-10), 141.6, 144.6 (Fmoc
Ar quat C),157.0 (Fmoc CO),157.7 (C-400),170.6 (C-2),172.4 (C-7). HRESI-
MS m/z 601.2318 [M þ Na]þ (calcd for C35H34N2O6Na 601.2314). HRESI-
MS m/z 624.2317 [M þ Na]þ (calcd for C37H34N3O5Na 624.2314).
5.2.1. Preparation of human neutrophils
Blood was taken from healthy human donors (20–32 years old)
by venipuncture, using a protocol approved by the institutional
review board at Chang Gung Memorial Hospital. Neutrophils were
isolated with a standard method of dextran sedimentation prior to
centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of
erythrocytes [1,17]. Purified neutrophils that contained >98% viable
cells, as determined by the trypan blue exclusion method, were
resuspended in calcium (Ca2þ)-free HBSS buffer at pH 7.4, and were
maintained at 4 ꢂC before use.
5.1.3. General procedure for synthesis of compounds 7, 8, 13 and 14
To a solution of compounds 5, 6, 11 and 12 in 20% piperidine/
CH2Cl2 (1 mL), respectively, was stirred at room temperature for
1 h. The solvent was evaporated under vacuum and the reaction
mixture was purified by column chromatography on silica gel to
give the desired products.
5.2.2. Measurement of Oꢃ2ꢁ generation
The assay of Oꢃ2ꢁ generation was based on the SOD-inhibitable
reduction of ferricytochrome c [1,17]. In brief, after supplementa-
tion with 0.5 mg/mL ferricytochrome c and 1 mM Ca2þ, neutrophils
were equilibrated at 37 ꢂC for 2 min and incubated with drugs for
5 min. Cells were activated with 100 nM FMLP for 10 min. When
5.1.3.1. (S)-2-((S)-2-Amino-3-phenylpropanamido)-3-phenylpropyl
acetate (7). Yield: 80%, [
400 MHz):
a
]
¼ ꢁ6.5ꢂ (c 0.1, CH3OH). 1H NMR (C5D5N,
D
d
¼ 1.99 (3H, s, H-1a, b, c), 2.99 (2H, t, J ¼ 7.6 Hz, H-10a,

1940
C.-T. Yen et al. / European Journal of Medicinal Chemistry 44 (2009) 1933–1940
FMLP was used as a stimulant, CB (1
m
g/mL) was incubated for
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The investigation was supported by research grants to P. W.
Hsieh from the National Science Council of the Republic of China
(NSC95-2320-B-037-002) in Taiwan.