Design and synthesis of tryptophan containing dipeptide derivatives as formyl peptide receptor 1 antagonist

Article abstract of DOI:10.1039/c3ob40215k

Our previous studies identified an Fmoc-(S,R)-tryptophan-containing dipeptide derivative, 1, which selectively inhibited neutrophil elastase release induced by formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) in human neutrophils. In an attempt to improve pharmacological activity, a series of tryptophan-containing dipeptides were synthesized and their pharmacological activities were investigated in human neutrophils. Of these, five compounds 3, 6, 19a, 24a, and 24b exhibited potent and dual inhibitory effects on FMLP-induced superoxide anion (O₂^-) generation and neutrophil elastase release in neutrophils with IC₅₀ values of 0.23/0.60, 1.88/2.47, 1.87/3.60, 0.12/0.37, and 1.32/1.03 μM, respectively. Further studies indicated that inhibition of superoxide production in human neutrophils by these dipeptides was associated with the selective inhibition of formyl peptide receptor 1 (FPR1). Furthermore, the results of structure-activity relationship studies concluded that the fragment N-benzoyl-Trp-Phe-OMe (3) was most suitable as a core structure for interaction with FPR1, and may be approved as a lead for the development of new drugs in the treatment of neutrophilic inflammatory diseases. As some of the synthesized compounds exhibited separable conformational isomers, and showed diverse bioactivities, the conformation analysis of these compounds is also discussed herein. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40215k

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Design and synthesis of tryptophan containing
dipeptide derivatives as formyl peptide receptor 1
Cite this: Org. Biomol. Chem., 2013, 11,
3742
antagonist†
Tsong-Long Hwang,a Chih-Hao Hung,a Ching-Yun Hsu,b Yin-Ting Huang,a
Yu-Chi Tsaic and Pei-Wen Hsieh*a
Our previous studies identified an Fmoc-(S,R)-tryptophan-containing dipeptide derivative, 1, which selec-
tively inhibited neutrophil elastase release induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)
in human neutrophils. In an attempt to improve pharmacological activity, a series of tryptophan-contain-
ing dipeptides were synthesized and their pharmacological activities were investigated in human neutro-
phils. Of these, five compounds 3, 6, 19a, 24a, and 24b exhibited potent and dual inhibitory effects on
FMLP-induced superoxide anion (O2˙−) generation and neutrophil elastase release in neutrophils with
IC50 values of 0.23/0.60, 1.88/2.47, 1.87/3.60, 0.12/0.37, and 1.32/1.03 μM, respectively. Further studies
indicated that inhibition of superoxide production in human neutrophils by these dipeptides was associ-
ated with the selective inhibition of formyl peptide receptor 1 (FPR1). Furthermore, the results of struc-
ture–activity relationship studies concluded that the fragment N-benzoyl-Trp-Phe-OMe (3) was most
suitable as a core structure for interaction with FPR1, and may be approved as a lead for the develop-
ment of new drugs in the treatment of neutrophilic inflammatory diseases. As some of the synthesized
compounds exhibited separable conformational isomers, and showed diverse bioactivities, the confor-
mation analysis of these compounds is also discussed herein.
Received 31st January 2013,
Accepted 10th April 2013
DOI: 10.1039/c3ob40215k
www.rsc.org/obc
(O2˙−) through activated nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase, which is a predominant mechan-
1. Introduction
Neutrophils are a major type of blood leukocyte, and are essen- ism in human neutrophils.4,5 Several studies showed that
tial for host defense against invading bacteria, fungi, and oxidant stress enhances many pathological processes in
viruses via phagocytosis. However, abnormal and excessive various inflammatory diseases and leads to tissue damage.6
neutrophil activation releases reactive oxygen species (ROS) In contrast, neutrophil elastase (NE), a basic 30 kDa single
and elastase-induced oxidative damage, and causes inflamma- polypeptide glycoprotein, is stored in the azurophil granules of
tory responses in many chronic diseases, e.g. chronic obstruc- neutrophils in its active form and is released during phago-
tive pulmonary disease (COPD), bronchiectasis, acute cytosis.2,7,8 However, excessive NE release can directly induce
respiratory distress syndrome, acute phases of rheumatoid cytotoxicity or degradation of adhesion molecules, thereby
arthritis, and acute lung injury.1–3 Of these, ROS are generated contributing to multiple tissue damage and subsequent pro-
via diverse intracellular pathways: especially, the respiratory gression of inflammatory diseases.9 Thus, modulation of the
burst, which catalyzes the formation of superoxide anion respiratory burst pathway and degranulation of neutrophils in
physiological conditions potentiating these functions are a way
of controlling these syndromes.1 As only a few agents have
aGraduate Institute of Natural Products, School of Traditional Chinese Medicine,
been used to modulate neutrophil proinflammatory responses,
College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
there is still great interest in novel small-molecular therapeutic
E-mail: pewehs@mail.cgu.edu.tw; Fax: +886-3-211-8643;
agents.
Tel: +886-3-211-8800, ext. 3105
bResearch Center for Industry of Human Ecology, Chang Gung University of Science
The formyl peptide receptors (FPRs), including FPR1, FPR2,
and FPR3, are members of the G-protein coupled receptor
family, involved in host defense and sensing cellular dys-
function.9 N-Formyl-methionyl-leucyl-phenylalanine (FMLP),
the most studied phagocyte chemoattractant and a prototype
for microbe-derived formylated peptides, is able to activate
and Technology, Taoyuan 33302, Taiwan
cGraduate Institute of Natural Products, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
†Electronic supplementary information (ESI) available: Additional information
of the purity using HPLC analysis of the target compounds. See DOI:
10.1039/c3ob40215k
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phagocytes via the FPRs.10–12 FRP1 and FPR2 are two members
of the FPRs found in human neutrophils.13 FPR1 is function-
ally expressed on neutrophils and macrophages, modulating
the innate immune response. Furthermore, FPR1 stimulates
proinflammatory neutrophil functions, and mediates neutro-
philic inflammatory diseases.14 Therefore, an FPR1 antagonist
may prove to be a target for generating agents in the treatment
of neutrophilic inflammatory disease. However, recent studies
indicated that FPR1 is not only involved in infection and the
inflammatory process, but also plays a role in promoting
tumor progression.15 In particular, FPR1 is able to interact
with endogenous annexin AI and then transactivate EGFR in
glioblastoma cells to mediate cell migration and growth.13,15
Therefore, FPR1 is also a therapeutic target for treating human
glioblastoma.
Table 1 Inhibitory effects of N-(N-aroyl-L-tryptophanyl)-D-phenylalanine methyl
esters and their analogs on O2˙− generation and elastase release by human neu-
trophils in response to fMLP/CB
Anti-inflammationa (μM)
Aurantiamides (also known as Matijing-Su or MTS) are
dipeptide analogs with an N-benzoylphenylalanine and a phe-
nylalaninol in their structures, and are major compounds in
Polygonum chinensis, Dichondra repens, Zanthoxylum dissitum,
and Aspergillus penicilloides.16–19 Aurantiamides and their
analogs have been recognized to have diverse biological activi-
ties, e.g. antibacterial,20,21 anti-inflammatory,16,18,21,22 analge-
sic,21 antioxidant,23 anti-HIV,18 anti-hepatitis B virus (anti-
HBV),24,25 anti-hepatitis C virus (anti-HCV),26 and cytotoxic
R1
R2
O2˙− generation NE release
EFB-1b
0.16 0.01
Sivelestatb
0.046 0.020
1
3
4
5
6
7
8
9
10
11a
11b
15a
15b
>20c
0.23 0.02
>30
1.70 0.60c
0.60 0.07
>30
H
H
H
H
H
H
H
H
Phenyl
4-Fluorophenyl
4-Chlorophenyl
4-Methylphenyl
4-Methoxyphenyl >30
Benzyl
Benzylmethyl
Benzyloxyl
>30
1.88 0.29
10.40 4.61
2.47 0.25
>30
10.17 4.53
5.71 0.39
12.54 4.87
8.92 4.84
9.11 0.76
22.11 4.96
>30
effects.26
A previous structure–activity relationship study
10.10 3.05
17.43 1.85
8.81 1.22
18.67 4.02
13.11 0.50
>30
enabled us to identify an Fmoc-(S,R)-dipeptide derivative, 1
(Fig. 1), which selectively inhibited neutrophil elastase release
induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)
in human neutrophils with an IC50 value of 1.7 0.6 μM.22 In
this study, 1 was chosen as the model structure in an attempt
to obtain more potent anti-inflammatory agents. Accordingly,
the route of structural modification included: (1) the effect of
replacement of the N-Fmoc moiety with different N-substi-
tutions, e.g. N-substituted or non-substituted benzoyl, N-Cbz,
and N-nicotinoyl; (2) the effects of β-homotryptophan or
5-hydroxyl-tryptophan on bioactivity; (3) para-substitution
effect on the phenyl of the phenylalanine residue,24,25 as well
as replacement of the phenyl ring of phenylalanine with a satu-
rated cyclohexyl ring; and (4) substitution effect on the C-termi-
OH Phenyl
OH Phenyl
H
H
Pyridinyl
Pyridinyl
>30
a The IC50 values are presented as mean SD. (n = 3). b Sivelestat and
EFB-1 were used as positive controls in the present study.28 c The
biological data of 1 were from the literature directly.22
report on the synthetic dipeptide analogs, as well as their
pharmacologic data and structure–activity relationships (SARs).
2. Chemistry
nal. All synthetic compounds (Tables 1–3) were evaluated for To optimize 1, a series of dipeptide analogs with a tryptophan
their in vitro inhibitory effects on O2˙− generation and elastase scaffold were designed and synthesized according to the proto-
release induced by FMLP in human neutrophils. Herein, we col outlined in Schemes 1–5. Initially, N-substituted-L-trypto-
phan or N-substituted-5-hydroxy-L-tryptophan were synthesized
by dissolving L-tryptophan or 5-hydroxy-L-tryptophan in 2 N
NaOH aqueous solutions, and then slowly treated with corres-
ponding benzoyl chlorides, phenylacetyl chloride, cinnamoyl
chloride, or nicotinoyl chloride, respectively, to yield inter-
mediates. The intermediates were subsequently reacted with D-
phenylalanine methyl ester to obtain N-(N-substituted-L-trypto-
phanyl)-D-phenylalanine methyl esters (3–10), N-(N-benzoyl-5-
hydroxy-L-tryptophanyl)-D-phenylalanine methyl esters (11a
and 11b), and N-(N-nicotinoyl-5-hydroxy-L-tryptophanyl)-D-
phenylalanine methyl esters (15a and 15b) (Schemes 1 and 2).
To study dipeptides with para-substituted phenylalanines, the
corresponding Boc-D-phenylalanine derivatives were deprotected
Fig. 1 The chemical structure of 1.
by treatment with 20% TFA–dichloromethane (DCM) solution,
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Table 2 Inhibitory effects of N-(N-benzoyl-L-tryptophanyl)-para-substituted-D-
phenylalanine methyl esters and N-(N-benzoyl-L-tryptophanyl)-3-cyclohexyl-D-
alanine methyl esters on O2˙− generation and elastase release by human neu-
trophils in response to fMLP/CB
Table 3 Inhibitory effects of N-(N-benzoyl-L-tryptophanyl)-D-phenylalanine
analogs/derivatives on O2˙− generation and elastase release by human neutro-
phils in response to fMLP/CB
Anti-inflammationa (μM)
Anti-inflammationa (μM)
R3
O2˙− generation NE release
n
R1
R4
O2˙− generation NE release
EFB-1b
Sivelestatb
3
18a
18b
19a
19b
20a
20b
21a
21b
22a
22b
23a
0.16 0.01
0.046 0.020
0.60 0.07
EFB-1b
Sivelestatb
3
27a
27b
12a
12b
13a
13b
0.16 0.01
Benzyl
4-Nitrobenzyl
0.23 0.02
18.99 2.70
13.08 0.40
1.87 0.22
18.83 5.80
5.40 1.50
14.01 1.21
4.41 0.27
5.26 0.84
6.82 3.09
15.97 0.83
0.046 0.020
11.17 0.46
15.39 0.54
3.60 0.05
23.99 2.02
11.49 2.20
19.37 0.71
4.31 0.52
12.41 3.53
2.41 1.60
7.20 3.36
21.05 2.92
8.76 2.33
0.37 0.04
1.03 0.02
0
0
0
0
0
0
0
1
H
H
H
COOCH3
CONHCH2CN 4.23 2.44
CONHCH2CN 10.51 4.23
0.23 0.02
0.60 0.07
4.69 0.80
14.51 2.87
>30
>30
>30
4-Methylbenzyl
4-Fluorobenzyl
4-Chlorobenzyl
4-Bromobenzyl
OH CH2OH
OH CH2OH
H
H
H
>30
>30
>30
>30
>30
CH2OH
CH2OH
COOCH3
>30
14.60 4.57
30
a The IC50 values are presented as mean SD (n = 3). b Sivelestat and
4-Trifluoromethylbenzyl 17.25 1.94
3.16 0.52
EFB-1 were used as positive controls in the present study.28
23b
24a
24b
Cyclohexylmethyl
0.12 0.02
1.32 0.14
a The IC50 values are presented as mean SD (n = 3). b Sivelestat and
EFB-1 were used as positive controls in the present study.28
Table 4 Temperature dependence of the 1H NMR chemical shift (ppm K−1) of
the amide protons of isomers (in DMSO-d6)
and then esterified by refluxing in methanol–c-H2SO4 solutions
to yield D-phenylalanine methyl esters, which were sub-
sequently coupled with N-benzoyl-L-tryptophan as described
to afford compounds 18a–23a and 18b–23b (Scheme 3). In
addition, N-benzoyl-L-tryptophan was also coupled with
3-cyclohexyl-D-alanine to afford compounds 24a and 24b
(Scheme 3).
The dipeptide nitriles were synthesized using Boc-D-phenyl-
alanine coupled with aminoacetonitrile in DCM, and then
deprotected to yield the intermediate, D-phenylalanyl-glycine-
nitrile (26).27 The intermediates were subsequently reacted
with N-benzoyl-L-tryptophan to obtain target dipeptide nitriles
(27a and 27b) (Scheme 4). Similarly, N-benzoyl-L-tryptophan
and N-benzoyl-5-hydroxy-L-tryptophan were also coupled with
D-phenylalaninol to afford the dipeptide alcohols (12a–13a and
12b–13b) (Scheme 1).22
dδ/dT(303–313 K)
dδ/dT(313–323 K)
(ppm K−1
)
(ppm K−1
)
Compounds
C1ONH
C1′ONH
C1ONH
C1′ONH
11a
11b
12a
12b
13a
13b
15a
15b
18a
18b
19a
19b
20a
20b
21a
21b
22a
22b
23a
23b
24a
24b
27a
27b
0.009
0.007
0.006
0.008
0.005
0.006
0.005
0.006
0.008
0.007
0.010
0.005
0.009
0.008
0.009
0.007
0.009
0.007
0.008
0.007
0.008
0.006
0.009
0.005
0.005
0.007
0.009
0.010
0.009
0.007
0.010
0.009
0.006
0.006
0.008
0.008
0.009
0.006
0.005
0.006
0.006
0.006
0.006
0.006
0.006
0.007
0.005
0.007
0.008
0.009
0.005
0.005
0.005
0.006
0.006
0.005
0.007
0.007
0.005
0.006
0.005
0.007
0.008
0.008
0.007
0.007
0.007
0.007
0.009
0.005
0.009
0.005
0.006
0.005
0.008
0.006
0.007
0.007
0.007
0.007
0.005
0.006
0.006
0.008
0.005
0.005
0.006
0.006
0.005
0.006
0.005
0.005
0.006
0.008
0.005
0.006
The synthesis of the target compound of N-(N-benzoyl-L-
β-homotryptophanyl)-D-phenylalanine methyl esters (30) is
illustrated in Scheme 5. Briefly, Fmoc-L-β-homotryptophan (28)
was deprotected by 5% piperidine in DCM solution, and then
the N-benzoyl group was introduced and coupled with
D-phenylalanine methyl ester as described. All synthesized
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Scheme 1 The route for the preparation of N-(N-aroyl-L-tryptophanyl)-D-phenylalanine methyl esters and their analogs. Reagents and conditions: (a) 2.0 N NaOH,
aroyl chlorides, 20 h; (b) D-phenylalanine methyl ester, HBTU, DIEA, DCM, 6 h; (c) 2.0 N NaOH, benzoyl chloride, 20 h; (d) D-phenylalaninol, HBTU, DIEA, DCM, 6 h.
compounds, in Tables 1–3, were fully characterized using spec-
troscopic data. The spectroscopic data of the new compounds
are shown in the Experimental section.
3. Results and discussion
All synthesized compounds and positive controls (EFB-1 and
sivelestat) were subjected to O2˙− generation and neutrophil
elastase release using FMLP as an inducer in human neutro-
phils.28 Of these synthesized compounds, five compounds 3,
Scheme 2 The route for the preparation of N-(N-nictotinoyl-L-tryptophanyl)-D-
6, 19a, 24a, and 24b exhibited the most potent and dual
inhibitory effects on FMLP-induced O2˙− generation and
neutrophil elastase release in neutrophils with IC50 values of
phenylalanine methyl esters. Reagents and conditions: (a) nicotinoyl chloride,
pyridine, 20 h; (b) 1.0 M LiOH, THF, 1 h; (c) D-phenylalanine methyl ester, HBTU,
DIEA, DCM, 6 h.
Scheme 3 The route for the synthesis of N-(N-benzoyl-L-tryptophanyl)-para-substituted-D-phenylalanine methyl esters and N-(N-benzoyl-L-tryptophanyl)-3-cyclo-
hexyl-D-alanine methyl esters. Reagents and conditions: (a) 20% TFA (TFA–DCM = 1 : 4), 30 min; (b) MeOH–c-H2SO4, reflux, 2 h; (c) N-benzoyl-L-tryptophan, HBTU,
DIEA, DCM, 6 h.
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Scheme 4 The route for the preparation of N-(N-benzoyl-L-tryptophanyl)-D-phenylalanine-glycine-nitriles. Reagents and conditions: (a) aminoacetonitrile, HBTU,
DIEA, DCM, 6 h; (b) 20% TFA (TFA–DCM = 1 : 4), 30 min; (c) N-benzoyl-L-tryptophan, HBTU, DIEA, DCM, 6 h.
Scheme 5 The route for the preparation of N-(N-benzoyl-L-β-homotryptophanyl)-D-phenylalanine methyl ester. Reagents and conditions: (a) 5% piperidine (piper-
idine–DCM = 1 : 19), 6 h; (b) 2.0 N NaOH, benzoyl chloride; (c) D-phenylalanine methyl ester, HBTU, DIEA, DCM, 6 h.
0.23/0.60, 1.88/2.47, 1.87/3.60, 0.12/0.37, and 1.32/1.03 μM, favorable than other substitutions (Tables 1 and 2). Conversely,
respectively.
replacement of the unsaturated phenyl ring of the phenyl-
As previously reported,22 1 has a selective inhibitory effect alanine residue with a saturated cyclohexyl ring (24a) led to
on neutrophil elastase release. In addition, another report slightly improved inhibitory effects on O2˙− generation and
suggested that N-substituted benzoyl MTSs showed potent neutrophil elastase release in human neutrophils (Table 2).
anti-HBV activities.24,25 In the present study, the N-Fmoc
5-Hydroxytryptophan is a monooxygenase metabolite of
moiety in 1 was replaced by the N-benzoyl group to obtain tryptophan with notable antioxidant properties.29 However,
compound 3, which resulted in augmented effects on the inhi- when the tryptophan moieties in 3, 13a, and 13b were replaced
bition of O2˙− generation and neutrophil elastase release. with 5-hydroxytryptophan (11a, 11b, 12a and 12b), respectively,
Therefore, we further explored the structure–activity relation- inhibition of O2˙− generation and neutrophil elastase release
ships (SARs) of the N-benzoyl groups and their analogs. The was not decreased (Tables 1 and 3), which indicated that a
results in Table 1 show that the activities of compounds with hydroxyl group cannot be tolerated. Furthermore, the activi-
para-substitution of benzoyl (4–7), phenylacetyl (8), phenylpro- ties were also reduced or inhibited when the tryptophan
panoyl (9), and nicotinoyl (15a and 15b) groups in the N-termi- moieties were replaced with β-homotryptophan (30), which
nal were dramatically decreased than non-substitution of suggested that the space between aromatic rings or hydrogen
benzoyl in the N-terminal one (3) (Table 1). The introduction bonding interaction may play a critical role in bioactivity
of para-substituting groups on the phenyl ring of the D-phenyl- (Table 3).
alanine residue (18a–23b) also resulted in a dramatic deterio-
Exploration of the relationship between activity and the
ration in inhibitory activities compared to the unsubstituted substitutions, e.g. methyl ester, hydroxymethyl, and cyano-
residue 3 (Table 2). Moreover, a comparison of the bioactivities methyl amide at the C-terminal, was carried out and is
of compounds with a para-substitution in the benzene ring of reported in Table 3. These results showed that the activities
D-phenylalanine residue (18a–23b, and 4–7) suggested that a were attenuated by the replacement of a methyl ester in 2
methyl group at the para-position (6 and 19a) was more with a cyanomethyl amide (27a and 27b). Moreover, the
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Table 5 Inhibitory effects of compounds 3, 6, 24a, and 24b on O2˙− gener-
ation and elastase release by human neutrophils in response to fMLP/CB and
WKYMVm/CB
substitution of a hydroxymethyl moiety at the C-terminal
significantly decreased these activities.
The synthesized compounds, such as compounds 11, 12,
13, 15, 18–24, and 27, exhibited separable conformational
isomers (isomer a and isomer b). Of these, isomer a was the
major isomer, whereas isomer b was the minor isomer. It is
worth noting that isomer a was more potent than isomer b
except for compounds 18a/18b and 23a/23b. Accordingly, we
hypothesized that racemization, intramolecular hydrogen
bonding, or amide bond conformation exchange may promote
the generation of isomers. Racemization is an exchangeable
process between L-amino acids and D-amino acids. To deter-
mine the configurations of the synthesized compounds, four
components, 18a, 18b, 27a, and 27b, were hydrolyzed and
reacted with Marfey’s reagent, respectively, and then analyzed
by HPLC.30 The results showed that all configurations of
amino acid residues in each isomer structure were assigned as
S/R. These results suggested that racemization did not mediate
isomerization in these compounds.
The formation of intramolecular hydrogen bonding plays
an important role in molecular structure and physicochemical
properties, e.g. structural conformation, membrane per-
meability, and lipophilic property.31 Recent studies have
suggested that the existence of intramolecular hydrogen
bonding could be determined by the proton chemical shift of
hydrogen bonding donors using NMR studies, which showed
temperature dependence.32,33 The results showed that the
values for temperature coefficients (dδ/dT) of amide NHs were
in excess of 0.004 ppm K−1, and indicated the absence of intra-
molecular hydrogen bonding in these isomers (Table 4).32
Amide E/Z form isomerization is an exchange process in
biological systems.34 Importantly, they showed that character-
istic mirror image CD spectra in E/Z form isomers at ca.
220 nm.35 To determine the conformation of the isomers,
seven components, 3, 19a, 19b, 20a, 20b, 22a, and 22b, were
determined by their CD spectra in methanol, respectively. The
results showed that isomers a (19a, 20a, and 22a) and 3 with a
positive Cotton effect near 220 nm indicated the presence of Z
form amide bonds at the N-benzoyl moiety; moreover, isomers
b (19b, 20b, and 22b) with a negative band at ca. 220 nm indi-
cated that they were E form isomers.35
FMLP/CBa (μM)
O2˙−
WKYMVm/CBa (μM)
O2˙−
Compounds generation
NE release generation
NE release
CycHb
WRW4b
3
6
24a
0.04 0.01
2.26 0.48
0.23 0.02
1.88 0.29
0.12 0.02
1.32 0.14
0.04 0.01 2.02 0.18
1.14 0.27 0.38 0.01
0.60 0.07 4.83 0.66
2.47 0.25 6.55 0.59
0.37 0.04 1.58 0.04
1.03 0.02 4.27 0.31
0.17 0.01
0.59 0.09
4.61 0.72
1.96 0.11
1.62 0.04
1.06 0.05
24b
a The IC50 values are presented as mean SD. (n = 3). b Cyclosporin
H (CycH) and WRW4 were used as positive controls.
controls, cyclosporine H (CycH, a selective antagonist of FPR1)
and WRW4 (a selective antagonist of FPR2), were chosen to
determine their inhibitory effects on O2˙− generation and elas-
tase release induced by WKYMVM in human neutrophils. The
results showed that they selectively inhibited the neutrophil
respiratory burst induced by FMLP, indicating that these com-
pounds could be specific FPR1 antagonists (Table 5). More-
over, although 24a was more potent than 3 in inhibiting the
neutrophil respiratory burst and neutrophil elastase release
induced by FMLP, 24a was less specific than 3, suggesting that
the fragment N-benzoyl-Trp-Phe-OMe (3) was more suitable as
a core structure for interaction with FPR1.
4. Conclusion
In this study, we synthesized thirty-three dipeptide derivatives
and compared their inhibitory effects on superoxide gener-
ation induced by FMLP and WKYMVM, respectively, in human
neutrophils. The results of structure–activity relationship
studies concluded that the fragment N-benzoyl-Trp-Phe-OMe
(3) was most suitable as a core structure for interaction with
FPR1. Additionally, most of the isomers a were potent than
isomers b, and they may include a Z form amide bond at the
N-benzoyl moeity. In conclusion, compound 3 had a more
stable Z form conformation at the N-benzoyl moeity and
showed more selectivity to FPR1, whereas 3 may be approved
as a candidate for the development of new agents in the treat-
ment of neutrophilic inflammatory diseases and human
glioblastoma.
As a specific FPR1 antagonist showed an inhibitory effect
on O2˙− generation induced by FMLP, but was unable to sup-
press O2˙− generation induced by WKYMVM in human neutro-
phils,10 this method could be used to identify novel specific
FPR1 antagonists.10 In 2010, Movitz and his co-workers
showed that a peptide with a Trp-Phe fragment in the C-termi-
nal was able to selectively bind to the FPR1 receptor; however,
this dipeptide alone was unable to inhibit the neutrophil res-
piratory burst induced by FMLP, although it connected with a
Gln-Ala fragment and formed a tetrapeptide.13 In the present
5. Experimental section
5.1. Materials and methods
study, the bioactive Trp-Phe dipeptide derivatives not only All commercial chemicals and solvents were of reagent grade
inhibited the neutrophil respiratory burst, but also suppressed and were used without further treatment unless otherwise
neutrophil elastase release induced by FMLP. These results noted. Reactions were detected by TLC using Merck 60 F254
differ from a previous conclusion.13 Furthermore, the most silica gel aluminum backed plates and were recorded under
potent compounds (3, 6, 24a, and 24b) and two positive ultraviolet irradiation (254 and 365 nm). The UV spectra were
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obtained on a Hitachi U-3010 spectrophotometer, and IR (1H, d, J = 8.0 Hz), 8.04 (2H, dd, J = 8.0 Hz), 7.87 (1H, d, J =
spectra were measured on a Jasco FT/IR-410 and a Jasco FT/ 7.6 Hz), 7.52 (1H, d J = 8.0 Hz, H-7), 7.32 (1H, s), 7.26–7.13
IR-4100 spectrophotometer. CD spectra were measured on a (7H, m), 7.02 (2H, t, J = 8.6 Hz), 5.66 (1H, ddd, J = 14.6, 7.2, 7.2
Jasco J-715 dichroism and an optical rotator dispersion Hz), 5.24 (1H, ddd, J = 14.2, 7.2, 7.2 Hz), 3.75 (1H, dd, J = 14.4,
spectrometer. Flash column chromatography was performed 6.8 Hz), 3.61 (1H, dd, J = 14.4, 6.8 Hz), 3.51 (3H, s), 3.20
with silica gel (Silicycle, 70–230 mesh or 230–400 mesh, MN). (1H, dd, J = 14.2, 7.2 Hz), 3.09 (1H, dd, J = 14.2, 7.2 Hz).
The purities of the synthesized compounds were determined 13C NMR (C5D5N) δ 174.4 (s), 174.0 (s), 168.4 (s), 166.4 (s, JC–F
by HPLC using a Jasco PU-1580 intelligent HPLC pump and a 248.0 Hz), 139.2 (2 × C, s), 133.5 (s), 132.2 (2 × C, d, JC–F
=
=
Jasco AS 1555-10 intelligent sampler with an Ascentis® C-18 9.0 Hz), 131.5 (2 × C, d), 130.5 (2 × C, d), 130.4 (s), 128.8 (d),
analytical column (Supelco, 5 m, 4.6 mm × 250 mm). Detection 126.1 (d), 123.5 (d), 121.0 (2 × C, d), 117.0 (2 × C, d, JC–F
=
was conducted at 248 nm in a Jassco UV-1575 UV-Vis detector. 22.0 Hz), 113.7 (d), 112.9 (s), 57.1 (d), 56.1 (d), 53.6 (q), 39.7 (t),
Purities of all the tested components were found to be more 30.7 (t). ESI-MS (m/z, %): 510 [M + Na]+ (100), 488 [M + 1]+ (37).
than 96% unless otherwise stated. The NMR spectra were HRESI-MS m/z 510.1802 [M + Na]+ (calcd for C28H26FN3O4Na
obtained on a Bruker AVANCE-400 MHz FT-NMR spectrometer. 510.1805).
Chemical shifts were internally referenced to the solvent
5.1.1.3. N-(N-4-Chlorobenzoyl-L-tryptophanyl)-D-phenyl-
signals in TMS. Low-resolution EI-MS were recorded on a alanine methyl ester (5). 23% yield. White powder, mp
1
Quattro GC/MS spectrometer having a direct inlet system, and 191–193 °C. H NMR (C5D5N) δ 11.82 (1H, s), 9.48 (1H, d, J =
low-resolution and high-resolution ESI-MS spectra on a Bruker 7.6 Hz), 9.18 (1H, d, J = 8.0 Hz), 7.96 (2H, d, J = 8.4), 7.85 (1H,
Daltonics APEX II 30e spectrometer.
d, J = 8.0 Hz), 7.51 (1H, d, J = 8.0 Hz), 7.28 (1H, s), 7.26–7.12
5.1.1. General procedure for the synthesis of N-(N-aroyl-L- (9H, m), 5.65 (1H, ddd, J = 14.6, 6.8, 6.8 Hz), 5.24 (1H, ddd, J =
tryptophanyl)-D-phenylalanine methyl esters (3–7) and their 14.0, 7.6, 7.6 Hz), 3.74 (1H, dd, J = 14.4, 6.8 Hz), 3.59 (1H, dd,
analogs (8–10, 11a, and 11b). To a mixture solution of L-trypto- J = 14.2, 7.2 Hz), 3.50 (3H, s), 3.20 (1H, dd, J = 13.8, 7.6 Hz),
phan (2a, 1.0 equiv.) or 5-hydroxy-L-tryptophan (2b, 1.0 equiv.) 3.08 (1H, dd, J = 13.6, 7.6 Hz). 13C NMR (C5D5N) δ 172.8 (s),
in 2 N NaOH aqueous solution was added suitable acyl chlor- 172.5 (s), 166.9 (s), 137.7 (s), 137.3 (s), 135.7 (s), 134.2 (s), 130.0
ides (1.1 equiv.), respectively. The reaction mixture was stirred (2 × C, d), 129.9 (2 × C, d), 129.0 (2 × C, d), 128.9 (s), 128.8 (2 ×
at room temperature for 3.0 h, and then 1 N HCl solution was C, d), 127.3 (d), 124.5 (d), 122.0 (d), 119.5 (2 × C, d), 112.2 (d),
added and the pH values were adjusted to 1–2. The mixture 111.3 (s), 55.6 (d), 54.6 (d), 52.0 (q), 38.2 (t), 29.1 (t). ESI-MS
solution was further partitioned by chloroform, and the (m/z, %): 526 [M + Na]+ (100), 504 [M + 1]+ (33). HR-ESI-MS m/z
organic layer was evaporated at reduced pressure to yield inter- 526.1506 [M + Na]+ (calcd for C28H26ClN3O4Na 526.1509).
mediates. The intermediates and D-phenylalanine methyl ester
5.1.1.4. N-(N-4-Methylbenzoyl-L-tryptophanyl)-D-phenyl-
(1.0 mmole) were dissolved in DCM and then HBTU (2.0 alanine methyl ester (6). 39% yield. White powder, mp
1
equiv.) and DIEA (1.5 equiv.) were added. The reaction mixture 169–171 °C. H NMR (C5D5N) δ 11.77 (1H, s), 9.41 (1H, d, J =
was stirred for 6 hours at room temperature, concentrated, and 8.0 Hz), 8.98 (1H, d, J = 8.0 Hz), 8.00 (2H, d, J = 8.0 Hz), 7.89
purified by silica gel column chromatography using a mixture (1H, d, J = 7.6 Hz), 7.54 (1H, d, J = 8.4 Hz), 7.33 (1H, s),
of n-hexane–ethyl acetate (6 : 4) or n-hexane–acetone (7 : 3), 7.30–7.14 (7H, m), 7.05 (2H, d, J = 7.6 Hz), 5.69 (1H, ddd, J =
respectively, to afford products (3–10, 11a, and 11b).
5.1.1.1. N-(N-Benzoyl-L-tryptophanyl)-D-phenylalanine methyl
14.4, 6.8, 6.8 Hz), 5.23 (1H, ddd, J = 14.0, 7.2, 7.2 Hz), 3.79
(1H, dd, J = 14.6, 6.8 Hz), 3.65 (1H, dd, J = 14.4, 6.8 Hz), 3.51
ester (3). 52% yield. White powder, mp 174–176 °C. CD (3H, s), 3.21 (1H, dd, J = 13.8, 6.8 Hz), 3.09 (1H, dd, J = 13.6,
(c 0.11 mM, MeOH) nm (mdeg) 261 (−0.43), 253 (−0.40), 239 7.2 Hz), 2.13 (3H, s). 13C NMR (C5D5N) δ 174.5 (s), 174.0 (s),
1
(−0.01), 223 (0.46) nm. H NMR (C5D5N) δ 11.79 (1H, s, NH), 169.5 (s), 143.4 (s), 139.2 (2 × C, s), 134.3 (s), 131.5 (2 × C, d),
9.41 (1H, d, J = 8.0 Hz, C1ONH), 9.06 (1H, d, J = 8.0 Hz, NH), 130.9 (2 × C, d), 130.5 (2 × C, d), 130.4 (s), 129.8 (2 × C, d),
8.06 (2H, d, J = 7.2 Hz), 7.88 (1H, d, J = 8.0 Hz), 7.54 (1H, d, J = 128.8 (d), 126.1 (d), 123.5 (d), 121.0 (2 × C, d), 113.7 (d), 112.9
8.0 Hz), 7.36–7.12 (11H, m), 5.67 (1H, ddd, J = 14.4, 7.2, (s), 57.0 (d), 56.2 (d), 53.6 (q), 39.7 (t), 30.7 (t), 22.9 (q). ESI-MS
7.2 Hz), 5.23 (1H, ddd, J = 14.0, 7.2, 7.2 Hz), 3.77 (1H, dd, J = (m/z, %): 506 [M + Na]+ (99), 484 [M + 1]+ (100). HR-ESI-MS m/z
14.6, 7.2 Hz), 3.63 (1H, dd, J = 14.4, 6.8 Hz), 3.50 (3H, s), 3.20 506.2059 [M + Na]+ (calcd for C29H29N3O4Na 506.2056).
(1H, dd, J = 13.6, 7.2 Hz), 3.09 (1H, dd, J = 13.6, 7.2 Hz). 13C
5.1.1.5. N-(N-4-Methoxylbenzoyl-L-tryptophanyl)-D-phenyl-
NMR (C5D5N) δ 172.9 (s), 172.5 (s), 168.0 (s), 137.7 (3 × C, s), alanine methyl ester (7). 47% yield. White powder, mp
1
131.6 (d), 130.0 (2 × C, d), 129.0 (2 × C, d), 128.9 (s), 128.7 (2 × 151–153 °C. H NMR (C5D5N) δ 11.80 (1H, s), 9.39 (1H, d, J =
C, d), 128.2 (2 × C, d), 127.3 (d), 124.6 (d), 122.0 (d), 119.5 (2 × 8.0 Hz), 8.92 (1H, d, J = 8.0 Hz), 8.09 (2H, d, J = 8.4 Hz), 7.99
C, d), 112.2 (d), 111.4 (s), 55.5 (d), 54.6 (d), 52.0 (q), 38.2 (t), (1H, d, J = 8.0 Hz), 7.53 (1H, d, J = 8.0 Hz), 7.33 (1H, s),
29.20 (t). ESI-MS (m/z, %): 492 [M + Na]+ (100), 470 [M + 1]+ 7.33–7.13 (7H, m), 6.88 (2H, d, J = 8.4 Hz), 5.69 (1H, ddd, J =
(44). HR-ESI-MS m/z 492.1901 [M
C28H27N3O4Na 492.1899).
+
Na]+ (calcd for 14.4, 6.8, 6.8 Hz), 5.23 (1H, ddd, J = 14.0, 7.2, 7.2 Hz), 3.78 (1H,
dd, J = 14.2, 6.8 Hz), 3.64 (1H, dd, J = 14.6, 6.8 Hz), 3.59
(3H, s), 3.50 (3H, s), 3.20 (1H, dd, J = 13.6, 7.2 Hz), 3.09 (1H,
5.1.1.2. N-(N-4-Fluorobenzoyl-L-tryptophanyl)-D-phenylalanine
methyl ester (4). 31% yield. White powder, mp 191–193 °C. dd, J = 13.6, 7.6 Hz). 13C NMR (C5D5N) δ 174.6 (s), 174.0 (s),
1H NMR (C5D5N) δ 11.81 (1H, s), 9.48 (1H, d, J = 8.0 Hz), 9.11 169.0 (s), 164.1 (s), 139.2 (2 × C, s), 131.6 (2 × C, d), 131.5
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(2 × C, d), 130.5 (2 × C, d), 130.4 (s), 129.4 (s), 128.8 (d), dd, J = 14.2, 7.2 Hz), 5.23 (1H, dd, J = 14.2, 7.2 Hz), 3.72 (1H,
126.1 (d), 123.5 (d), 121.0 (2 × C, d), 115.6 (2 × C, d), 113.7 (d), dd, J = 14.2, 7.2 Hz), 3.59 (1H, dd, J = 14.2, 7.2 Hz), 3.47
113.0 (s), 57.0 (q), 56.9 (d), 56.1 (d), 53.6 (q), 39.03 (t), 30.7 (t). (3H, s), 3.15 (1H, dd, J = 13.6, 7.2 Hz), 3.05 (1H, dd, J = 13.6,
ESI-MS (m/z, %): 522 [M + Na]+ (100), 500 [M + 1]+ (38). 7.2 Hz). 13C NMR (CDCl3) δ 172.7 (s), 172.3 (s), 167.7 (s), 152.4
HR-ESI-MS m/z 522.2007 [M + Na]+ (calcd for C29H29N3O5Na (s), 137.4 (s), 135.5 (s), 132.2 (s), 131.4 (d), 129.8 (s), 129.8 (2 ×
522.2005).
C, d), 128.8 (2 × C, d), 128.5 (2 × C, d), 128.0 (2 × C, d), 127.1
(d), 125.0 (d), 112.8 (d), 112.6 (d), 110.3 (s), 103.9 (d), 55.5 (d),
5.1.1.6. N-(N-Phenylacetyl-L-tryptophanyl)-D-phenylalanine
methyl ester (8). 43% yield. White powder, mp 182–184 °C. 54.5 (d), 51.8 (q), 38.0 (t), 29.2 (t). ESI-MS (m/z, %): 508 [M +
1H NMR (C5D5N) δ 11.76 (1H, s), 9.34 (1H, d, J = 8.0 Hz), 8.98 Na]+ (100). HR-ESI-MS m/z 508.1850 [M + Na]+ (calcd for
(1H, d, J = 8.4 Hz), 7.81 (1H, d, J = 8.0 Hz), 7.53 (1H, d, J = 8.4 C28H27N3O5Na 508.1848). 11b: 17% yield. White powder, mp
1
Hz), 7.33 (2H, d, J = 7.2 Hz), 7.27–7.13 (11H, m), 5.50 (1H, ddd, 110–112 °C. H NMR (CDCl3) δ 11.53 (1H, s, NH), 9.41 (1H, d,
J = 14.6, 6.8, 6.8 Hz), 5.19 (1H, ddd, J = 14.0, 7.2, 7.2 Hz), J = 8.0 Hz, NH), 9.11 (1H, d, J = 8.0 Hz, NH), 8.10 (2H, d, J = 8.0
3.75–3.46 (4H, m), 3.49 (3H, s), 3.17 (1H, dd, J = 13.6, 6.8 Hz), Hz), 7.64 (1H, d, J = 1.6 Hz), 7.43 (1H, d, J = 8.0 Hz), 7.37 (1H,
3.05 (1H, dd, J = 13.8, 6.8 Hz). 13C NMR (C5D5N) δ 172.8 (s), t, J = 7.6 Hz), 7.30 (2H, t, J = 7.6 Hz), 7.26 (2H, t, J = 7.6 Hz),
172.5 (s), 171.1 (s), 137.7 (s), 137.6 (s), 136.9 (s), 130.0 (2 × 7.19–7.13 (4H, m), 7.08 (1H, t, J = 8.0 Hz), 5.65 (1H, dd, J =
C, d), 129.9 (2 × C, d), 129.0 (2 × C, d), 128.9 (s), 128.9 (2 × 14.4, 7.2 Hz), 5.21 (1H, dd, J = 14.2, 7.2 Hz), 3.74 (1H, dd, J =
C, d), 127.3 (d), 127.0 (d), 124.5 (d), 121.9 (d), 119.5 (d), 119.4 14.4, 7.2 Hz), 3.61 (1H, dd, J = 14.4, 7.2 Hz), 3.55 (3H, s), 3.27
(d), 112.1 (d), 111.2 (s), 54.9 (d), 54.6 (d), 52.0 (q), 43.5 (t), (1H, dd, J = 13.6, 7.2 Hz), 3.15 (1H, dd, J = 13.6, 7.2 Hz). 13C
38.2 (t), 29.3 (t). ESI-MS (m/z, %): 506 [M + Na]+ (100), 484 NMR (CDCl3) δ 172.6 (s), 172.3 (s), 167.8 (s), 152.3 (s), 137.5 (s),
[M + 1]+ (87). HR-ESI-MS m/z 506.2060 [M + Na]+ (calcd 135.5 (s), 132.2 (s), 131.4 (d), 129.8 (2 × C, d), 129.7 (s), 128.7 (2
for C29H29N3O4Na 506.2056).
× C, d), 128.6 (2 × C, d), 128.1 (2 × C, d), 127.0 (d), 125.0 (d),
112.8 (d), 112.5 (d), 110.3 (s), 103.9 (d), 55.2 (d), 54.5 (d), 51.9
5.1.1.7. N-(N-Phenylpropanoyl-L-tryptophanyl)-D-phenyl-
alanine methyl ester (9). 24% yield. White powder, mp (q), 38.2 (t), 28.9 (t). ESI-MS (m/z, %): 508 [M + Na]+ (100).
1
159–161 °C. H NMR (C5D5N) δ 11.78 (1H, s), 9.27 (1H, d, J = HR-ESI-MS m/z 508.1851 [M + Na]+ (calcd for C28H27N3O5Na
8.0 Hz), 8.88 (1H, d, J = 8.4 Hz), 7.85 (1H, d, J = 7.6 Hz), 7.55 508.1848).
(1H, d, J = 7.6 Hz), 7.27–7.12 (13H, m), 5.52 (1H, ddd, J = 14.8,
5.1.2. General procedure for the synthesis of N-(N-benzoyl-
7.2, 7.2 Hz), 5.21 (1H, ddd, J = 14.0, 7.2, 7.2 Hz), 3.65 (1H, dd. L-tryptophanyl)-D-phenylalaninol derivatives (12a–13a and
J = 14.4, 6.8 Hz), 3.50 (3H, s), 3.50–3.45 (1H, m), 3.17 (1H, J = 12b–13b). N-Benzoyl-L-tryptophan and N-benzoyl-5-hydroxy-L-
13.6, 6.8 Hz), 3.09–2.97 (3H, m), 2.67–2.61 (2H, m). 13C NMR tryptophan were obtained by a similar procedure as described.
(C5D5N) δ 174.4 (s), 174.0 (s), 173.9 (s), 143.7 (s), 139.2 (2 × C, s), Dissolved N-benzoyl-L-tryptophan (1.0 equiv.) or N-benzoyl-5-
131.5 (2 × C, d), 130.5 (2 × C, d), 130.4 (d), 130.4 (d), 130.3 (2 × hydroxy-L-tryptophan (1.0 equiv.) in DCM, respectively, and
C, d), 128.8 (s), 128.0 (d), 126.0 (d), 123.4 (d), 121.0 (d), 120.9 then D-phenylalaninol (1.0 equiv.), HBTU (2.0 equiv.) and DIEA
(d), 113.6 (d), 112.9 (s), 56.4 (d), 56.0 (d), 53.5 (q), 39.7 (2 × C, (1.5 equiv.) were added. The reaction mixture was stirred at
t), 33.7 (t), 30.8 (t). ESI-MS (m/z, %): 520 [M + Na]+ (100), 498 room temperature for 6.0 h, and purified by silica gel column
[M + 1]+ (39). HR-ESI-MS m/z 520.2215 [M + Na]+ (calcd for chromatography using ethyl acetate or MeOH–CHCl3 (1 : 20) to
C29H29N3O4Na 520.2212).
afford the mixtures 12a/b and 13a/b. The mixture was further
purified by HPLC (mobile phase: 35% acetonitrile + 0.3% TFA)
5.1.1.8. N-(N-Benzyloxycarbonyl-L-tryptophanyl)-D-phenyl-
alanine methyl ester (10). 67% yield. White powder, mp to afford products.
1
147–149 °C. H NMR (C5D5N) δ 11.76 (1H, s), 9.32 (1H, d, J =
5.1.2.1. N-(N-Benzoyl-L-tryptophanyl)-5-hydroxy-D-phenylala-
7.6 Hz), 8.63 (1H, d, J = 8.4 Hz), 7.88 (1H, d, J = 7.6 Hz), 7.54 ninol (12a and 12b). 12a: 16% yield. White powder, mp
1
(1H, d, J = 8.4 Hz), 7.32–7.14 (13H, m), 5.25–5.13 (4H, m), 3.72 175–177 °C. H NMR (C5D5N) δ 11.77 (1H, s, NH), 8.97 (1H, d,
(1H, dd, J = 14.4, 7.2 Hz), 3.55 (1H, dd, J = 14.8, 7.2 Hz), 3.49 J = 8.0 Hz, NH), 8.88 (1H, d, J = 8.0 Hz, NH), 8.05 (2H, d, J =
(3H, s), 3.17 (1H, dd, J = 13.6, 7.2 Hz), 3.07 (1H, dd, J = 13.8, 7.2 Hz), 7.89 (1H, d, J = 8.0 Hz), 7.51 (1H, d, J = 8.0 Hz),
7.2 Hz). 13C NMR (C5D5N) δ 172.9 (s), 172.5 (s), 157.2 (s), 137.9 7.36–7.32 (4H, m), 7.28–7.22 (5H, m), 7.19 (1H, m), 7.09 (1H, t,
(s), 137.7 (2 × C, s), 130.0 (2 × C, d), 129.0 (2 × C, d), 129.0 (d), J = 7.6 Hz), 5.58 (1H, dd, J = 14.4, 6.8 Hz), 5.50 (1H, br.s, OH),
128.9 (2 × C, d), 128.9 (s), 128.3 (2 × C, d), 127.3 (s), 124.6 (d), 4.75 (1H, m), 3.91 (2H, m), 3.75 (1H, dd, J = 14.4, 6.8 Hz), 3.64
122.0 (d), 119.5 (2 × C, d), 112.2 (d), 111.3 (s), 66.6 (t), 57.0 (d), (1H, dd, J = 14.4, 6.8 Hz), 3.15 (1H, dd, J = 13.4, 7.2 Hz), 3.03
54.5 (d), 52.0 (q), 38.2 (t), 29.7 (t). ESI-MS (m/z, %): 522 (1H, dd, J = 13.4, 7.2 Hz). 13C NMR (C5D5N) δ 172.7 (s), 167.9
[M + Na]+ (100), 500 [M + 1]+ (30). HR-ESI-MS m/z 522.2008 (s), 139.8 (s), 137.7 (s), 135.7 (s), 131.5 (d), 130.2 (d), 129.0 (s),
[M + Na]+ (calcd for C29H29N3O5Na 522.2005).
5.1.1.9. N-(N-Benzoyl-5-hydroxy-L-tryptophanyl)-D-phenyl-
128.9 (2 × C, d), 128.7 (2 × C, d), 128.2 (2 × C, d), 126.7 (d),
124.6 (d), 121.9 (d), 119.5 (d), 119.4 (d), 112.1 (d), 111.5 (s),
alanine methyl esters (11a and 11b). 11a: 23% yield. White 63.2 (t), 55.9 (d), 53.8 (d), 37.7 (t), 29.4 (t). ESI-MS (m/z, %): 464
1
powder, mp 112–114 °C. H NMR (CDCl3) δ 11.54 (1H, s, NH), [M + Na]+ (100), 442 [M + 1]+ (5). HR-ESI-MS m/z 464.1947 [M +
9.39 (1H, d, J = 8.0 Hz, NH), 9.04 (1H, d, J = 8.0 Hz, NH), Na]+ (calcd for C27H27N3O3Na 464.1950). 12b: 11% yield. White
1
8.06 (2H, d, J = 7.2 Hz), 7.74 (1H, d, J = 2.0 Hz), 7.47 (1H, d, powder, mp 173–175 °C. H NMR (C5D5N) δ 11.78 (1H, s, NH),
J = 8.8 Hz), 7.35 (1H, t, J = 7.2 Hz), 7.29–7.13 (9H, m), 5.65 (1H, 9.07 (1H, d, J = 7.6 Hz, NH), 8.87 (1H, d, J = 8.0 Hz, NH), 8.09
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(2H, d, J = 7.6 Hz), 7.85 (1H, d, J = 8.0 Hz), 7.52 (1H, br.s), 7.48 combined aqueous layer was neutralized with 1.0 N HCl solu-
(1H, d, J = 8.0 Hz), 7.29 (2H, t, J = 7.6 Hz), 7.27 (2H, t, J = tion, followed by extraction with ethyl acetate three times. The
7.6 Hz), 7.22–7.15 (3H, m), 7.09 (2H, t, J = 8.6 Hz), 5.62 (1H, combined organic layer was dried with anhydrous magnesium
dd, J = 14.4, 7.2 Hz), 4.71 (1H, m), 3.87 (2H, m), 3.80 (1H, dd, sulfate and evaporated to yield N-nicotinoyl-L-tryptophan.
J = 14.4, 7.2 Hz), 3.68 (1H, dd, J = 14.4, 7.2 Hz), 3.21 (1H, dd, D-Phenylalanine methyl ester (1.0 mmole) and N-nicotinoyl-L-
J = 13.6, 6.8 Hz), 3.09 (1H, dd, J = 13.6, 6.8 Hz). 13C NMR tryptophan were dissolved in DCM, and then HBTU (2.0
(C5D5N) δ 172.5 (s), 167.8 (s), 139.6 (s), 137.5 (s), 135.5 (s), equiv.) and DIEA (1.5 equiv.) were added. The reaction mixture
131.4 (d), 129.9 (2 × C, d), 128.7 (s), 128.6 (2 × C, d), 128.5 (2 × was stirred for 6 hours at room temperature, concentrated, and
C, d), 128.1 (2 × C, d), 126.4 (d), 124.4 (d), 121.7 (d), 119.3 (d), purified by silica gel column chromatography using ethyl
119.2 (d), 111.9 (d), 111.4 (s), 63.1 (t), 55.5 (d), 53.8 (d), 37.6 (t), acetate to afford the products (15a and 15b). 15a: 30% yield.
28.8 (t). ESI-MS (m/z, %): 464 [M + Na]+ (100), 442 [M + 1]+ (37). White powder, mp 161–163 °C. 1H NMR (C5D5N) δ 8.88 (1H, s),
HR-ESI-MS m/z 464.1947 [M + Na]+ (calcd for C27H27N3O3Na 8.66 (1H, d, J = 4.4 Hz), 8.21 (1H, s, NH), 7.98 (1H, d, J =
464.1950).
8.0 Hz), 7.72 (1H, d, J = 7.6 Hz), 7.35 (1H, d, J = 7.6 Hz), 7.31
(1H, dd, J = 7.8, 2.0 Hz), 7.23–7.12 (5H, m), 7.08 (1H, d, J =
5.1.2.2. N-(N-Benzoyl-L-tryptophanyl)-D-phenylalaninol (13a
and 13b). 13a: 53% yield. White powder, mp 151–153 °C. 1H 7.6 Hz), 6.85 (1H, d, J = 6.8 Hz), 6.35 (1H, d, J = 8.0 Hz, NH),
NMR (C5D5N) δ 11.53 (1H, s, NH), 8.96 (1H, d, J = 7.6 Hz, NH), 4.95 (1H, ddd, J = 14.4, 7.6, 7.6 Hz), 4.79 (1H, dd, J = 13.6,
8.82 (1H, d, J = 8.0 Hz, NH), 8.07 (2H, d, J = 7.6 Hz), 7.76 (1H, 6.0 Hz), 3.63 (3H, s), 3.40 (1H, dd, J = 14.4, 7.6 Hz), 3.23 (1H,
s), 7.46 (1H, d, J = 8.4 Hz), 7.34–7.13 (10H, m), 5.95 (1H, br.s, dd, J = 14.4, 7.6 Hz), 2.93 (1H, dd, J = 13.6, 6.0 Hz), 2.82 (1H,
OH), 5.56 (1H, dd, J = 14.0, 7.2 Hz), 4.73 (1H, m), 3.91 (1H, dd, dd, J = 13.6, 6.0 Hz). 13C NMR (C5D5N) δ 172.0 (s), 171.4 (s),
J = 10.8, 5.2 Hz), 3.85 (1H, dd, J = 10.8, 5.2 Hz), 3.70 (1H, dd, 165.8 (s), 152.7 (d), 148.6 (d), 136.6 (s), 136.0 (d), 135.6 (s),
J = 14.0, 7.2 Hz), 3.60 (1H, dd, J = 14.0, 7.2 Hz), 3.11 (1H, dd, J = 129.9 (s), 129.5 (2 × C, d), 129.0 (2 × C, d), 127.9 (s), 127.6 (d),
13.6, 7.2 Hz), 3.00 (1H, dd, J = 13.6, 7.2 Hz). 13C NMR (C5D5N) 123.8 (d), 123.6 (d), 122.8 (d), 120.3 (d), 119.1 (d), 111.8 (d),
δ 172.5 (s), 167.6 (s), 152.4 (s), 139.5 (s), 135.5 (s), 132.2 (s), 110.7 (d), 54.8 (d), 53.7 (d), 52.7 (q), 37.9 (t), 28.8 (t). ESI-MS
131.4 (d), 130.1 (2 × C, d), 129.8 (s), 128.7 (2 × C, d), 128.5 (2 × (m/z, %): 471 [M + 1]+ (100). HR-ESI-MS m/z 493.1850 [M + Na]+
C, d), 128.0 (2 × C, d), 125.0 (d), 112.8 (d), 112.5 (d), 110.5 (s), (calcd for C27H26N4O4Na 493.1852). 15b: 6% yield. White
1
104.0 (d), 62.8 (t), 55.8 (d), 53.6 (d), 37.5 (t), 29.5 (t). ESI-MS powder, mp 130–132 °C. H NMR (C5D5N) δ 8.88 (1H, s), 8.69
(m/z, %): 480 [M + Na]+ (100), 458 [M + 1]+ (18). HR-ESI-MS m/z (1H, d, J = 4.0 Hz), 8.25 (1H, s, NH), 7.99 (1H, dd, J = 6.0,
480.1897 [M + Na]+ (calcd for C27H27N3O4Na 480.1899). 13b: 2.0 Hz), 7.73 (1H, d, J = 8.0 Hz), 7.35–7.31 (2H, m), 7.20 (1H, t,
1
25% yield. White powder, mp 120–122 °C. H NMR (C5D5N) δ J = 8.0 Hz), 7.13–7.06 (6H, m), 6.85 (2H, d, J = 6.8 Hz), 6.30
11.53 (1H, s, NH), 9.05 (1H, d, J = 8.0 Hz, NH), 8.76 (1H, d, J = (1H, d, J = 7.6 Hz, NH), 4.92 (1H, dd, J = 14.6, 7.6 Hz), 4.73
8.0 Hz, NH), 8.10 (2H, d, J = 7.6 Hz), 7.71 (1H, s), 7.55 (1H, s), (1H, dd, J = 13.6, 6.4 Hz), 3.65 (3H, s), 3.45 (1H, dd, J = 14.6,
7.46 (1H, d, J = 8.0 Hz), 7.38–7.35 (3H, m), 7.30 (2H, t, J = 7.6 7.6 Hz), 3.21 (1H, dd, J = 14.6, 7.6 Hz), 3.01 (1H, dd, J = 13.6,
Hz), 7.18–7.14 (4H, m), 7.08 (1H, d, J = 8.0 Hz), 5.90 (1H, br.s, 6.4 Hz), 2.92 (1H, dd, J = 13.6, 6.4 Hz). 13C NMR (C5D5N) δ
OH), 5.59 (1H, dd, J = 14.2, 7.2 Hz), 4.69 (1H, m), 3.88 (1H, dd, 171.8 (s), 171.1 (s), 165.7 (s), 152.8 (d), 148.6 (d), 136.7 (s),
J = 10.4, 5.2 Hz), 3.82 (1H, dd, J = 10.4, 5.2 Hz), 3.74 (1H, dd, 135.9 (d), 135.5 (s), 129.9 (s), 129.5 (2 × C, d), 128.9 (2 × C, d),
J = 14.2, 7.2 Hz), 3.64 (1H, dd, J = 14.2, 7.2 Hz), 3.20 (1H, dd, 127.8 (s), 127.5 (d), 124.0 (d), 123.8 (d), 122.8 (d), 120.4 (d),
J = 13.6, 7.2 Hz), 3.07 (1H, dd, J = 13.6, 7.2 Hz). 13C NMR 119.2 (d), 111.8 (d), 110.7 (d), 54.5 (d), 53.9 (d), 52.7 (q),
(C5D5N) δ 172.4 (s), 167.7 (s), 152.3 (s), 139.6 (s), 135.5 (s), 38.2 (t), 28.7 (t). ESI-MS (m/z, %): 471 [M + 1]+ (100). HR-ESI-MS
132.2 (s), 131.4 (d), 129.9 (2 × C, d), 129.8 (s), 128.6 (2 × C, d), m/z 493.1849 [M + Na]+ (calcd for C27H26N4O4Na 493.1852).
128.5 (2 × C, d), 128.1 (2 × C, d), 126.4 (d), 125.0 (d), 112.8 (d),
5.1.4. General procedure for the synthesis of N-(N-benzoyl-
112.5 (d), 110.6 (s), 104.0 (d), 63.8 (t), 55.5 (d), 53.7 (d), 37.6 (t), L-tryptophanyl)-para-substituted-D-phenylalanine methyl esters
28.9 (t). ESI-MS (m/z, %): 480 [M + Na]+ (100), 458 [M + 1]+ (82). (18a–23a and 18b–23b). para-Substituted-D-phenylalanine
HR-ESI-MS m/z 480.1897 [M + Na]+ (calcd for C27H27N3O4Na methyl esters (17a–17f) were synthesized through deprotecting
480.1899).
corresponding N-Boc-D-phenylalanine derivatives (16a–16f),
5.1.3. Preparation of N-(N-nicotinoyl-L-tryptophanyl)-D- respectively, in 20% trifluroacetic acid (TFA) solution
phenylalanine methyl esters (15a and 15b). To a mixture solu- (TFA–DCM = 1 : 4), and then esterified through refluxing
tion of L-tryptophan methyl ester (1.0 equiv.) in pyridine was methanol (50 ml)–c-H2SO4 (1.0 ml) for 2 h. The resulting mix-
added nicotinoyl chlorides (1.1 equiv.). The reaction mixture tures were subsequently neutralized by ammonium water, par-
was stirred at room temperature for 16.0 h and then evapor- titioned between ice water (100 ml) and chloroform, and
ated and purified by silica gel column chromatography using a concentrated the organic layers. Subsequently, para-substi-
mixture of MeOH–CHCl3 (1 : 20) to afford N-nicotinoyl-L-trypto- tuted-D-phenylalanine methyl esters (17a–17f, 1.0 equiv.) and
phan methyl ester. N-Nicotinoyl-L-tryptophan methyl ester was N-benzoyl-L-tryptophan (1.0 equiv.), which were synthesized by
further dissolved in 10 mL of 1.0 M LiOH solution and then a procedure similar to that described, were dissolved in DCM,
added to the mixture for hydrolysis. Upon completion, the respectively, and then HBTU (2.0 equiv.) and DIEA (1.5 equiv.)
reaction mixture was partitioned three times with ethyl acetate were added. The reaction mixture was stirred for 6 hours at
and saturated sodium bicarbonate aqueous solution. The room temperature, concentrated, and purified by silica gel
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column chromatography using a mixture of n-hexane–ethyl J = 8.0 Hz, NH), 8.09 (2H, d, J = 8.0 Hz), 7.78 (2H, d, J = 8.0 Hz),
acetate (6 : 4), respectively, to afford the products (18a–23a and 7.48 (1H, d, J = 8.0 Hz), 7.46 (1H, d, J = 1.6 Hz), 7.38 (1H, t, J =
18b–23b).
8.0 Hz), 7.30 (2H, t, J = 8.0 Hz), 7.22–7.15 (3H, m), 7.07 (1H, d,
J = 8.0 Hz), 6.92 (2H, d, J = 7.6 Hz), 5.70 (1H, dd, J = 14.4,
5.1.4.1. N-(N-Benzoyl-L-tryptophanyl)-para-nitro-D-phenyl-
alanine methyl esters (18a and 18b). 18a: 42% yield. White 7.2 Hz), 5.24 (1H, dd, J = 13.8, 7.2 Hz), 3.80 (1H, dd, J = 14.4,
1
powder, mp 240–242 °C. H NMR (C5D5N) δ 11.91 (1H, s, NH), 7.2 Hz), 3.65 (1H, dd, J = 14.4, 7.2 Hz), 3.58 (3H, s), 3.27 (1H,
9.76 (1H, d, J = 8.0 Hz, NH), 9.24 (1H, d, J = 8.0 Hz, NH), 8.09 dd, J = 13.8, 7.2 Hz), 3.14 (1H, dd, J = 13.8, 7.2 Hz), 2.04 (3H,
(2H, d, J = 8.0 Hz), 7.98 (2H, d, J = 8.0 Hz), 7.90 (1H, d, J = s). 13C NMR (C5D5N) δ 172.7 (s), 172.5 (s), 167.9 (s), 137.5 (s),
8.0 Hz), 7.56 (1H, d, J = 8.0 Hz), 7.45 (1H, s), 7.36 (1H, t, J = 136.2 (s), 135.3 (s), 134.3 (s), 131.4 (d), 129.7 (2 × C, d), 129.4
8.0 Hz), 7.30–7.24 (3H, m), 7.17–7.14 (3H, m), 5.66 (1H, dd, J = (2 × C, d), 128.7 (s), 128.5 (2 × C, d), 128.1 (2 × C, d), 124.4 (d),
14.2, 7.2 Hz), 5.31 (1H, dd, J = 13.8, 7.2 Hz), 3.80 (1H, dd, J = 121.7 (d), 119.2 (2 × C, d), 111.9 (d), 111.2 (s), 55.1 (d), 54.6 (d),
14.2, 7.2 Hz), 3.62 (1H, dd, J = 14.2, 7.2 Hz), 3.55 (3H, s), 3.22 51.9 (q), 37.8 (t), 28.6 (t), 20.9 (q). ESI-MS (m/z, %): 506 [M +
(1H, dd, J = 13.8, 7.2 Hz), 3.15 (1H, dd, J = 13.8, 7.2 Hz). 13C Na]+ (100). HR-ESI-MS m/z 506.2060 [M + Na]+ (calcd for
NMR (C5D5N) δ 172.8 (s), 171.7 (s), 167.9 (s), 147.1 (s), 145.4 C29H29N3O4Na 506.2056).
(s), 137.5 (s), 135.4 (s), 131.5 (d), 130.7 (2 × C, d), 128.6 (s),
5.1.4.3. N-(N-Benzoyl-L-tryptophanyl)-para-fluoro-D-phenyl-
128.6 (2 × C, d), 128.1 (2 × C, d), 124.5 (d), 123.7 (2 × C, d), alanine methyl esters (20a and 20b). 20a: 45% yield. White
121.9 (d), 119.4 (d), 119.3 (d), 112.1 (d), 111.2 (s), 55.5 (d), 53.8 powder, mp 178–180 °C. CD (c 0.12 mM, MeOH) nm (mdeg)
(d), 52.1 (q), 37.5 (t), 28.9 (t). ESI-MS (m/z, %): 537 [M + Na]+ 232 (−0.76), 223 (0.68) nm. 1H NMR (C5D5N) δ 11.84 (1H, s,
(100). HR-ESI-MS m/z 537.1754 [M
+
Na]+ (calcd for NH), 9.40 (1H, d, J = 7.6 Hz, NH), 9.15 (1H, d, J = 7.6 Hz, NH),
C28H26N4O6Na 537.1750). 18b: 8% yield. White powder, mp 8.07 (2H, d, J = 7.6 Hz), 7.90 (2H, d, J = 7.6 Hz), 7.54 (1H, d, J =
1
182–184 °C. H NMR (C5D5N) δ 11.81 (1H, s, NH), 9.72 (1H, d, 7.6 Hz), 7.40 (1H, s), 7.35 (1H, t, J = 7.2 Hz), 7.29–7.25 (3H, m),
J = 8.0 Hz, NH), 9.29 (1H, d, J = 8.0 Hz, NH), 8.10 (2H, d, J = 8.0 7.25–7.20 (3H, m), 7.15 (1H, t, J = 7.6 Hz), 7.08 (1H, d, J = 6.4
Hz), 7.97 (2H, d, J = 8.0 Hz), 7.74 (1H, d, J = 8.0 Hz), 7.49 (1H, Hz), 6.89 (2H, t, J = 8.0 Hz), 5.68 (1H, dd, J = 14.2, 6.8 Hz), 5.19
d, J = 8.0 Hz), 7.44 (1H, s), 7.41–7.37 (3H, m), 7.32 (2H, t, J = (1H, dd, J = 13.6, 6.8 Hz), 3.79 (1H, dd, J = 14.2, 6.8 Hz), 3.65
7.6 Hz), 7.20 (1H, t, J = 8.0 Hz), 7.05 (1H, t, J = 8.0 Hz), 5.65 (1H, dd, J = 14.2, 6.8 Hz), 3.58 (3H, s), 3.27 (1H, dd, J = 13.8,
(1H, dd, J = 14.8, 7.2 Hz), 5.31 (1H, dd, J = 13.6, 8.0 Hz), 3.77 6.8 Hz), 3.13 (1H, dd, J = 13.6, 6.8 Hz). 13C NMR (C5D5N) δ
(1H, dd, J = 14.8, 7.2 Hz), 3.62 (3H, s), 3.61 (1H, dd, J = 14.8, 172.7 (s), 172.1 (s), 167.9 (s), 162.1 (s, JC–F = 242.2 Hz), 137.5
7.2 Hz), 3.39 (1H, dd, J = 13.6, 8.0 Hz), 3.21 (1H, dd, J = 13.6, (s), 135.4 (s), 133.4 (s, JC–F = 3.0 Hz), 131.4 (d), 128.7 (s), 128.5
8.0 Hz). 13C NMR (C5D5N) δ 173.0 (s), 171.9 (s), 168.0 (s), 147.1 (2 × C, d), 128.1 (2 × C, d), 124.4 (d), 121.9 (d), 119.3 (2 × C, d),
(s), 145.4 (s), 137.5 (s), 135.4 (s), 131.6 (d), 130.9 (2 × C, d), 115.4 (2 × C, d, JC–F = 21.4 Hz), 112.0 (d), 111.2 (s), 55.4 (d),
128.6 (2 × C, d), 128.5 (s), 128.1 (2 × C, d), 124.4 (d), 123.7 (2 × 54.4 (d), 51.9 (q), 37.1 (t), 29.0 (t). ESI-MS (m/z, %): 510 [M +
C, d), 121.7 (d), 119.2 (d), 119.1 (d), 111.9 (d), 111.1 (s), 55.3 Na]+ (100). HR-ESI-MS m/z 510.1808 [M + Na]+ (calcd for
(d), 53.7 (d), 52.2 (q), 37.8 (t), 28.5 (t). ESI-MS (m/z, %): 537 C28H26FN3O4Na 510.1805). 20b: 14% yield. White powder, mp
[M + Na]+ (100). HR-ESI-MS m/z 537.1753 [M + Na]+ (calcd for 184–186 °C. CD (c 0.12 mM, MeOH) nm (mdeg) 233 (0.69), 221
C28H26N4O6Na 537.1750).
5.1.4.2. N-(N-Benzoyl-L-tryptophanyl)-para-methyl-D-phenyl-
(−0.55) nm. 1H NMR (C5D5N) δ 11.80 (1H, s, NH), 9.60 (1H, d, J
= 8.0 Hz, NH), 9.22 (1H, d, J = 8.0 Hz, NH), 8.11 (2H, d, J = 7.6
alanine methyl esters (19a and 19b). 19a: 50% yield. White Hz), 7.77 (2H, d, J = 7.6 Hz), 7.49 (1H, d, J = 7.6 Hz), 7.46 (1H,
powder, mp 185–187 °C. CD (c 0.18 mM, MeOH) nm (mdeg) s), 7.37 (1H, t, J = 7.6 Hz), 7.30 (1H, t, J = 7.6 Hz), 7.25–7.20
1
232 (−1.30), 224 (−0.28) nm. H NMR (C5D5N) δ 11.82 (1H, s, (3H, m), 7.06 (1H, t, J = 7.6 Hz), 6.89 (2H, t, J = 8.0 Hz), 5.68
NH), 9.35 (1H, d, J = 8.0 Hz, NH), 9.07 (1H, d, J = 8.0 Hz, NH), (1H, dd, J = 14.6, 7.2 Hz), 5.22 (1H, dd, J = 13.8, 7.2 Hz), 3.79
8.04 (2H, d, J = 8.0 Hz), 7.87 (2H, d, J = 8.0 Hz), 7.53 (1H, d, J = (1H, dd, J = 14.6, 7.2 Hz), 3.65 (1H, dd, J = 14.6, 7.2 Hz), 3.58
7.6 Hz), 7.36–7.34 (2H, m), 7.28–7.20 (3H, m), 7.14 (1H, t, J = (3H, s), 3.27 (1H, dd, J = 13.8, 7.2 Hz), 3.13 (1H, dd, J = 13.8,
7.6 Hz), 7.08 (1H, d, J = 7.6 Hz), 6.99 (2H, d, J = 7.6 Hz), 5.68 7.2 Hz). 13C NMR (C5D5N) δ 172.8 (s), 172.3 (s), 167.9 (s), 162.1
(1H, dd, J = 14.4, 6.8 Hz), 5.22 (1H, dd, J = 13.8, 7.2 Hz), 3.80 (s, JC–F = 242.0 Hz), 137.5 (s), 135.5 (s), 133.4 (s, JC–F = 3.2 Hz),
(1H, dd, J = 14.4, 6.8 Hz), 3.64 (1H, dd, J = 14.4, 6.8 Hz), 3.52 131.6 (d, JC–F = 8.0 Hz), 131.5 (d), 128.6 (s), 128.5 (2 × C, d),
(3H, s), 3.16 (1H, dd, J = 13.8, 7.2 Hz), 3.07 (1H, dd, J = 13.8, 128.1 (2 × C, d), 124.4 (d), 121.7 (d), 119.2 (d), 119.1 (d), 115.4
7.2 Hz), 2.11 (3H, s). 13C NMR (C5D5N) δ 172.7 (s), 172.4 (s), (2 × C, d, JC–F = 21.0 Hz), 111.9 (d), 111.2 (s), 55.2 (d), 54.4 (d),
167.8 (s), 137.5 (s), 135.4 (s), 134.3 (s), 131.4 (d), 129.7 (2 × C, 52.0 (q), 37.3 (t), 28.6 (t). ESI-MS (m/z, %): 510 [M + Na]+ (100).
d), 129.5 (2 × C, d), 128.7 (s), 128.5 (2 × C, d), 128.0 (2 × C, d), HR-ESI-MS m/z 510.1809 [M + Na]+ (calcd for C28H26FN3O4Na
124.4 (d), 121.8 (d), 119.3 (2 × C, d), 112.0 (d), 111.2 (s), 55.3 510.1805).
(d), 54.5 (d), 51.9 (q), 37.6 (t), 29.0 (t), 20.9 (q). ESI-MS (m/z,
5.1.4.4. N-(N-Benzoyl-L-tryptophanyl)-para-chloro-D-phenyl-
%): 506 [M + Na]+ (100). HR-ESI-MS m/z 506.2059 [M + Na]+ alanine methyl esters (21a and 21b). 21a: 37% yield. White
1
(calcd for C29H29N3O4Na 506.2056). 19b: 16% yield. White powder, mp 190–191 °C. H NMR (C5D5N) δ 11.86 (1H, s, NH),
powder, mp 197–199 °C. CD (c 0.18 mM, MeOH) nm (mdeg) 9.42 (1H, d, J = 8.0 Hz, NH), 9.16 (1H, d, J = 8.0 Hz, NH), 8.07
233 (1.75), 221 (−1.81), 213 (−0.42) nm. 1H NMR (C5D5N) δ (2H, d, J = 8.0 Hz), 7.90 (1H, d, J = 8.0 Hz), 7.54 (1H, d, J =
11.79 (1H, s, NH), 9.52 (1H, d, J = 8.0 Hz, NH), 9.16 (1H, d, 7.6 Hz), 7.40 (1H, d, J = 1.6 Hz), 7.35 (1H, t, J = 7.2 Hz),
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7.29–7.23 (3H, m), 7.19–7.14 (3H, m), 7.02 (2H, d, J = 8.0 Hz), (s), 135.5 (s), 131.8 (2 × C, d), 131.7 (2 × C, d), 131.5 (d), 128.6
5.68 (1H, dd, J = 14.2, 7.2 Hz), 5.18 (1H, dd, J = 14.0, 7.2 Hz), (s), 128.6 (s), 128.6 (2 × C, d), 128.1 (2 × C, d), 124.4 (d), 121.7
3.79 (1H, dd, J = 14.2, 7.2 Hz), 3.64 (1H, dd, J = 14.2, 7.2 Hz), (d), 120.9 (s), 119.2 (d), 119.1 (d), 111.9 (d), 111.2 (s), 55.2 (d),
3.51 (3H, s), 3.11 (1H, dd, J = 14.0, 7.2 Hz), 3.03 (1H, dd, J = 54.2 (d), 52.0 (q), 37.4 (t), 28.6 (t). ESI-MS (m/z, %): 570
14.0, 7.2 Hz). 13C NMR (C5D5N) δ 172.7 (s), 172.0 (s), 167.9 (s), [M + Na]+ (100), 572 [M + 2 + Na]+ (100). HR-ESI-MS m/z
137.5 (s), 136.2 (s), 135.5 (s), 132.6 (s), 131.5 (2 × C, d), 131.4 (2 570.1007 [M + Na]+ (calcd for C28H26BrN3O4Na 570.1004).
× C, d), 128.8 (2 × C, d), 128.7 (s), 128.5 (2 × C, d), 128.1 (2 × C,
5.1.4.6. N-(N-Benzoyl-L-tryptophanyl)-para-trifluoromethyl-D-
d), 124.4 (d), 121.9 (d), 119.3 (2 × C, d), 112.0 (d), 111.2 (s), phenylalanine methyl esters (23a and 23b). 23a: 26% yield.
55.4 (d), 54.2 (d), 51.9 (q), 37.2 (t), 29.0 (t). ESI-MS (m/z, %): White powder, mp 233–235 °C. 1H NMR (C5D5N) δ 11.88 (1H, s,
526 [M + Na]+ (100). HR-ESI-MS m/z 526.1506 [M + Na]+ (calcd NH), 9.50 (1H, d, J = 8.0 Hz, NH), 9.18 (1H, d, J = 8.0 Hz, NH),
for C28H26ClN3O4Na 526.1509). 21b: 9% yield. White powder, 8.08 (2H, d, J = 7.6 Hz), 7.89 (1H, d, J = 7.6 Hz), 7.54 (1H, d, J =
mp 174–176 °C. 1H NMR (C5D5N) δ 11.80 (1H, s, NH), 9.62 7.6 Hz), 7.45 (2H, d, J = 8.0 Hz), 7.42 (1H, d, J = 2.0 Hz), 7.36
(1H, d, J = 8.0 Hz, NH), 9.23 (1H, d, J = 8.0 Hz, NH), 8.11 (2H, (1H, t, J = 7.6 Hz), 7.29–7.23 (3H, m), 7.19 (2H, d, J = 8.0 Hz),
d, J = 8.0 Hz), 7.77 (1H, d, J = 8.0 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.15 (1H, t, J = 7.6 Hz), 5.67 (1H, dd, J = 14.4, 7.2 Hz), 5.23 (1H,
7.46 (1H, br.s), 7.38 (1H, t, J = 8.0 Hz), 7.31 (2H, t, J = 8.0 Hz), dd, J = 14.0, 7.2 Hz), 3.79 (1H, dd, J = 14.4, 7.2 Hz), 3.63 (1H,
7.22–7.19 (3H, m), 7.14 (2H, t, J = 8.0 Hz), 7.06 (1H, d, J = dd, J = 14.4, 7.2 Hz), 3.52 (3H, s), 3.20 (1H, dd, J = 14.0, 7.2
8.0 Hz), 5.68 (1H, dd, J = 14.6, 7.2 Hz), 5.22 (1H, dd, J = 13.8, Hz), 3.14 (1H, dd, J = 14.0, 7.2 Hz). 13C NMR (C5D5N) δ 172.8
7.6 Hz), 3.79 (1H, dd, J = 14.6, 7.2 Hz), 3.64 (1H, dd, J = 14.6, (s), 171.9 (s), 167.6 (s), 142.1 (s), 137.5 (s), 135.4 (s), 131.5 (d),
7.2 Hz), 3.58 (3H, s), 3.26 (1H, dd, J = 13.8, 7.6 Hz), 3.12 (1H, 130.4 (2 × C, d), 128.7 (2 × C, d), 128.6 (s, JC–F = 32.2 Hz), 128.1
dd, J = 13.8, 7.6 Hz). 13C NMR (C5D5N) δ 172.8 (s), 172.2 (s), (2 × C, d), 125.5 (2 × C, d, JC–F = 4.0 Hz), 125.0 (s, JC–F = 270.1
168.0 (s), 137.5 (s), 136.3 (s), 135.5 (s), 132.5 (s), 131.5 (d), Hz), 124.4 (d), 121.9 (d), 119.3 (2 × C, d), 112.0 (d), 111.3 (s),
131.4 (2 × C, d), 128.7 (s), 128.7 (2 × C, d), 128.6 (2 × C, d), 55.4 (d), 54.0 (d), 52.0 (q), 37.5 (t), 29.0 (t). ESI-MS (m/z, %):
124.4 (d), 121.7 (d), 119.2 (d), 119.1 (d), 111.9 (d), 111.2 (s), 560 [M + Na]+ (100). HR-ESI-MS m/z 560.1776 [M + Na]+ (calcd
55.2 (d), 54.2 (d), 52.0 (q), 37.4 (t), 28.6 (t). ESI-MS (m/z, %): for C29H26F3N3O4Na 560.1773). 23b: 9% yield. White powder,
526 [M + Na]+ (100). HR-ESI-MS m/z 526.1511 [M + Na]+ (calcd mp 187–189 °C. 1H NMR (C5D5N) δ 11.81 (1H, s, NH), 9.72
for C28H26ClN3O4Na 526.1509).
5.1.4.5. N-(N-Benzoyl-L-tryptophanyl)-para-bromo-D-phenyl-
(1H, d, J = 8.0 Hz, NH), 9.25 (1H, d, J = 8.0 Hz, NH), 8.12 (2H,
d, J = 8.0 Hz), 7.75 (1H, d, J = 8.0 Hz), 7.49 (1H, d, J = 8.0 Hz),
alanine methyl esters (22a and 22b). 22a: 23% yield. White 7.44 (1H, d, J = 2.0 Hz), 7.43 (2H, d, J = 8.0 Hz), 7.40–7.37 (3H,
powder, mp 190–191 °C. CD (c 0.15 mM, MeOH) nm (mdeg) m), 7.31 (2H, d, J = 8.0 Hz), 7.20 (1H, t, J = 8.0 Hz), 7.05 (1H, t,
232 (−0.41), 223 (0.15) nm. 1H NMR (C5D5N) δ 11.85 (1H, s, J = 8.0 Hz), 5.67 (1H, dd, J = 14.4, 7.2 Hz), 5.25 (1H, dd, J =
NH), 9.40 (1H, d, J = 8.0 Hz, NH), 9.12 (1H, d, J = 8.0 Hz, NH), 14.0, 7.2 Hz), 3.78 (1H, dd, J = 14.4, 7.2 Hz), 3.62 (1H, dd, J =
8.07 (2H, d, J = 7.2 Hz), 7.90 (1H, d, J = 7.6 Hz), 7.54 (1H, d, J = 14.4, 7.2 Hz), 3.60 (3H, s), 3.36 (1H, dd, J = 13.6, 8.0 Hz), 3.21
7.6 Hz), 7.41 (1H, br.s), 7.38–7.32 (3H, m), 7.28 (1H, t, J = 7.2 (1H, dd, J = 13.6, 8.0 Hz). 13C NMR (C5D5N) δ 172.9 (s), 172.0
Hz), 7.24 (1H, t, J = 7.6 Hz), 7.15 (1H, t, J = 8.0 Hz), 6.97 (2H, d, (s), 168.0 (s), 142.2 (s), 137.5 (s), 135.4 (s), 131.5 (d), 130.5 (2 ×
J = 8.0 Hz), 5.67 (1H, dd, J = 14.4, 7.2 Hz), 5.17 (1H, dd, J = C, d), 128.6 (2 × C, d), 128.5 (s, JC–F = 32.0 Hz), 128.1 (2 × C, d),
13.8, 7.2 Hz), 3.79 (1H, dd, J = 14.4, 7.2 Hz), 3.64 (1H, dd, J = 125.5 (2 × C, d, JC–F = 4.0 Hz), 124.9 (s, JC–F = 270.0 Hz), 124.4
14.4, 7.2 Hz), 3.51 (3H, s), 3.09 (1H, dd, J = 13.8, 7.2 Hz), 3.02 (d), 121.9 (d), 119.2 (d), 119.1 (d), 111.9 (d), 111.1 (s), 55.4 (d),
(1H, dd, J = 13.8, 7.2 Hz). 13C NMR (C5D5N) δ 172.7 (s), 172.0 54.0 (d), 52.0 (q), 37.5 (t), 29.0 (t). ESI-MS (m/z, %): 560
(s), 167.8 (s), 137.5 (s), 136.7 (s), 135.4 (s), 131.8 (2 × C, d), [M + Na]+ (100). HR-ESI-MS m/z 560.1775 [M + Na]+ (calcd
131.7 (2 × C, d), 131.5 (d), 128.7 (s), 128.5 (2 × C, d), 128.1 (2 × for C29H26F3N3O4Na 560.1773).
C, d), 124.4 (d), 121.9 (d), 120.9 (s), 119.3 (2 × C, d), 112.0 (d),
5.1.5. Preparation of N-(N-benzoyl-L-tryptophanyl)-3-cyclo-
111.2 (s), 55.4 (d), 54.1 (d), 51.9 (q), 37.2 (t), 29.0 (t). ESI-MS hexyl-D-alanine methyl esters (24a and 24b). Compounds 24a
(m/z, %): 570 [M + Na]+ (100), 572 [M + 2 + Na]+ (100). and 24b were synthesized in 39% and 14%, respectively, yield
HR-ESI-MS m/z 570.1008 [M + Na]+ (calcd for C28H26BrN3O4Na from 3-cyclohexyl-D-alanine (16g) by a similar procedure as
570.1004). 22b: 8% yield. White powder, mp 174–176 °C. CD (c described. 24a: White powder, mp 170–172 °C. 1H NMR
0.18 mM, MeOH) nm (mdeg) 241 (0.49), 232 (0.24), 220 (C5D5N) δ 11.88 (1H, s, NH), 9.45 (1H, d, J = 8.0 Hz, NH), 9.07
1
(−0.75), 214 (−0.28) nm. H NMR (C5D5N) δ 11.81 (1H, s, NH), (1H, d, J = 8.0 Hz, NH), 8.08 (2H, d, J = 8.4 Hz), 7.94 (1H, d, J =
9.62 (1H, d, J = 8.0 Hz, NH), 9.23 (1H, d, J = 8.0 Hz, NH), 8.11 7.6 Hz), 7.56 (1H, d, J = 7.6 Hz), 7.50 (1H, br.s), 7.35 (1H, dt, J =
(2H, d, J = 8.0 Hz), 7.76 (1H, d, J = 7.6 Hz), 7.49 (1H, d, J = 7.6 7.6, 1.2 Hz), 7.29–7.23 (3H, m), 7.14 (1H, t, J = 7.6 Hz), 5.72
Hz), 7.45 (1H, d, J = 1.6 Hz), 7.39 (1H, t, J = 7.6 Hz), 7.32 (2H, t, (1H, dd, J = 14.2, 7.2 Hz), 5.00 (1H, m), 3.86 (1H, dd, J = 14.2,
J = 7.6 Hz), 7.29 (2H, d, J = 7.2 Hz), 7.20 (1H, t, J = 7.6 Hz), 7.14 7.2 Hz), 3.72 (1H, dd, J = 14.2, 7.2 Hz), 3.58 (3H, s), 1.77 (1H,
(2H, d, J = 8.4 Hz), 7.06 (1H, t, J = 7.6 Hz), 5.68 (1H, dd, J = m), 1.66 (2H, m), 1.59–1.48 (4H, m), 1.38 (1H, m), 1.25–0.96
14.6, 7.2 Hz), 5.21 (1H, dd, J = 13.6, 7.6 Hz), 3.79 (1H, dd, J = (3H, m), 0.84–0.71 (2H, m). 13C NMR (C5D5N) δ 173.7 (s), 172.9
14.6, 7.2 Hz), 3.64 (1H, dd, J = 14.6, 7.2 Hz), 3.58 (3H, s), 3.24 (s), 167.7 (s), 137.5 (s), 135.5 (s), 131.4 (s), 128.7 (s), 128.5 (2 ×
(1H, dd, J = 13.6, 7.6 Hz), 3.10 (1H, dd, J = 13.6, 7.6 Hz). 13C C, d), 128.0 (2 × C, d), 124.3 (d), 121.8 (d), 119.3 (d), 119.2 (d),
NMR (C5D5N) δ 172.8 (s), 172.1 (s), 168.0 (s), 137.5 (s), 136.8 112.0 (d), 111.2 (d), 55.4 (d), 51.9 (q), 50.9 (d), 39.4 (t), 34.1 (d),
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33.6 (t), 32.4 (t), 29.2 (t), 26.6 (t), 26.3 (t), 26.1 (t). ESI-MS (m/z, (1H, dd, J = 14.2, 7.6 Hz), 3.62 (1H, dd, J = 14.2, 7.6 Hz), 3.41
%): 498 [M + Na]+ (100). HR-ESI-MS m/z 498.2366 [M + Na]+ (1H, dd, J = 13.6, 8.0 Hz), 3.18 (1H, dd, J = 13.6, 8.0 Hz). 13C
(calcd for C28H33N3O4Na 498.2369). 24b: White powder, mp NMR (C5D5N) δ 172.7 (s), 172.5 (s), 168.0 (s), 137.9 (s), 137.4
1
109–111 °C. H NMR (C5D5N) δ 11.82 (1H, s, NH), 9.67 (1H, d, (s), 135.5 (s), 131.5 (s), 129.8 (2 × C, d), 128.8 (2 × C, d), 128.6
J = 8.0 Hz, NH), 9.27 (1H, d, J = 8.0 Hz, NH), 8.12 (2H, d, J = 8.0 (s), 128.5 (2 × C, d), 128.1 (2 × C, d), 126.9 (d), 124.5 (d), 121.7
Hz), 7.78 (1H, d, J = 7.6 Hz), 7.55 (1H, br.s), 7.49 (1H, d, J = 7.6 (d), 119.2 (d), 119.1 (d), 117.5 (s), 112.0 (s), 111.0 (s), 55.4 (d),
Hz), 7.35 (1H, dt, J = 1.2, 8.0 Hz), 7.28 (2H, dt, J = 1.2, 8.0 Hz), 55.3 (d), 38.4 (t), 28.5 (t), 27.8 (t). ESI-MS (m/z, %): 516 [M +
7.20 (1H, t, J = 7.6 Hz), 7.06 (1H, t, J = 7.6 Hz), 5.71 (1H, dd, J = Na]+ (100). HR-ESI-MS m/z 516.2008 [M + Na]+ (calcd for
14.6, 7.6 Hz), 5.12 (1H, dd, J = 14.6, 7.6 Hz), 3.86 (1H, dd, J = C29H27N5O3Na 516.2011).
14.6, 7.2 Hz), 3.71 (1H, dd, J = 14.6, 7.2 Hz), 3.65 (3H, s), 1.73
5.1.7. Preparation of N-(N-benzoyl-L-β-homotryptophanyl)-
(2H, m), 1.63 (1H, m), 1.54–1.41 (5H, m), 1.13–0.92 (3H, m), D-phenylalanine methyl ester (30). Fmoc-L-β-homotryptophan
0.85–0.70 (2H, m). 13C NMR (C5D5N) δ 173.9 (s), 173.0 (s), (1.0 equiv.) was deprotected with 5% piperidine solution
167.8 (s), 137.5 (s), 135.5 (s), 131.4 (s), 128.7 (s), 128.5 (2 × C, (piperidine–DCM = 1 : 19) for 6.0 h, and then purified by
d), 128.1 (2 × C, d), 124.4 (d), 121.7 (d), 119.2 (2 × C, d), 111.9 C-18 gel column chromatography using a mixture of MeOH to
(d), 111.2 (d), 55.3 (d), 52.0 (q), 50.8 (d), 39.7 (t), 34.2 (d), 33.7 afford L-β-homotryptophan. N-Benzoyl-L-β-homotryptophan
(t), 32.3 (t), 28.6 (t), 26.6 (t), 26.3 (t), 26.1 (t). ESI-MS (m/z, %): was obtained from L-β-homotryptophan by a similar procedure
498 [M + Na]+ (100). HR-ESI-MS m/z 498.2367 [M + Na]+ (calcd as described. Finally, N-benzoyl-L-β-homotryptophan and
for C28H33N3O4Na 498.2369).
D-phenylalanine methyl ester were dissolved in DCM, and then
5.1.6. Preparation of N-(N-benzoyl-L-tryptophanyl)-D- HBTU (2.0 equiv.) and DIEA (1.5 equiv.) were added. The reac-
phenylalanine-glycine-nitriles (27a and 27b). To a mixture tion mixture was stirred for 6 hours at room temperature, con-
solution of Boc-phenylalanine (1.0 equiv.) and aminoaceto- centrated, and purified by silica gel column chromatography
nitrile (1.0 equiv.) in DCM was added HBTU (2.0 equiv.) and using ethyl acetate–n-hexane (1 : 1) to afford products. 30: 75%
1
DIEA (1.5 equiv.). The reaction mixture was stirred at room yield. White powder, mp 170–172 °C. H NMR (C5D5N) δ 11.72
temperature for 6.0 h, and then deprotected and purified by (1H, s, NH), 9.51 (1H, d, J = 7.6 Hz, NH), 9.08 (1H, d, J =
silica gel column chromatography using a mixture of MeOH– 8.0 Hz, NH), 8.16 (2H, d, J = 7.6 Hz), 8.14 (1H, d, J = 8.4 Hz),
CHCl3 (1 : 10) to afford D-phenylalanine-glycine-nitrile (26). 7.54 (1H, d, J = 8.4 Hz), 7.39 (1H, t, J = 7.6 Hz), 7.35–7.32 (4H,
Compound 26 (1.0 equiv.) and N-benzoyl-L-tryptophan (1.0 m), 7.28–7.25 (4H, m), 7.20–7.17 (2H, m), 5.33–5.28 (2H, m),
equiv.) were dissolved in DCM, and then HBTU (2.0 equiv.) 3.60 (3H, s), 3.56 (1H, dd, J = 14.4, 6.8 Hz), 3.42–3.34 (2H, m),
and DIEA (1.5 equiv.) were added. The reaction mixture was 3.20 (1H, dd, J = 14.0, 8.8 Hz), 2.94 (1H, dd, J = 14.8, 6.0 Hz),
stirred for 6 hours at room temperature, concentrated, and 2.85 (1H, dd, J = 14.8, 6.0 Hz). 13C NMR (C5D5N) δ 172.8 (s),
purified by silica gel column chromatography using ethyl 172.3 (s), 167.1 (s), 137.8 (s), 137.6 (s), 136.2 (s), 131.3 (d),
acetate–n-hexane (1 : 1) to afford the mixture of 27a and 27b. 129.5 (2 × C, d), 128.9 (2 × C, d), 128.8 (s), 128.6 (2 × C, d),
The mixture was further purified by HPLC (mobile phase: 65% 127.8 (2 × C, d), 127.1 (d), 124.3 (d), 121.8 (d), 119.6 (d),
MeOH + 0.03% TFA) to afford products. 27a: 49% yield. White 119.3 (d), 112.2 (d), 111.9 (s), 54.3 (d), 52.0 (q), 49.0 (t), 38.6 (t),
1
powder, mp 167–169 °C. H NMR (C5D5N) δ 11.81 (1H, s, NH), 37.8 (t), 30.4 (t). ESI-MS (m/z, %): 506 [M + Na]+ (100). HR-ESI-MS
9.73 (1H, t, J = 5.6 Hz, NH), 9.63 (1H, d, J = 8.4 Hz, NH), 8.39 m/z 506.2057 [M + Na]+ (calcd for C29H29N3O4Na 506.2057).
(1H, d, J = 6.4 Hz, NH), 8.07 (2H, d, J = 7.6 Hz), 7.77 (1H, d, J =
5.2. Biological assays
5.2.1. Preparation of human neutrophils.3 Blood was
8.0 Hz), 7.56 (1H, d, J = 8.0 Hz), 7.35 (1H, t, J = 7.6 Hz),
7.29–7.23 (5H, m), 7.20–7.18 (3H, m), 7.15–7.09 (2H, m),
5.35–5.29 (2H, m), 5.00 (1H, m), 4.47 (2H, d, J = 5.6 Hz), 3.70 taken from healthy human donors (20–32 years old) by veni-
(1H, dd, J = 14.2, 7.6 Hz), 3.61 (1H, dd, J = 14.2, 7.6 Hz), 3.33 puncture, following a protocol approved by the Institutional
(1H, dd, J = 13.6, 8.0 Hz), 3.09 (1H, dd, J = 13.6, 8.0 Hz). 13C Review Board at Chang Gung Memorial Hospital. Neutrophils
NMR (C5D5N) δ 173.2 (s), 172.7 (s), 166.8 (s), 138.1 (s), 137.5 were isolated with a standard method of dextran sedimen-
(s), 135.5 (s), 131.7 (s), 129.7 (2 × C, d), 128.8 (2 × C, d), 128.5 tation prior to centrifugation in a Ficoll Hypaque gradient and
(s), 128.5 (2 × C, d), 128.2 (2 × C, d), 126.9 (d), 124.5 (d), 121.9 the hypotonic lysis of erythrocytes. Purified neutrophils that
(d), 119.3 (d), 119.1 (d), 117.6 (s), 112.0 (s), 110.9 (s), 56.6 (d), contained >98% viable cells, as determined by the trypan blue
55.0 (d), 37.8 (t), 28.4 (t), 28.0 (t). ESI-MS (m/z, %): 516 [M + exclusion method, were resuspended in Ca2+-free HBSS buffer
Na]+ (100). HR-ESI-MS m/z 516.2008 [M + Na]+ (calcd for at pH 7.4 and maintained at 4 °C before use.
C29H27N5O3Na 516.2011). 27b: 7% yield. White powder, mp
5.2.2. Measurement of O2˙− generation.3 The assay of
195–197 °C. H NMR (C5D5N) δ 11.80 (1H, s, NH), 9.76 (1H, d, O2˙− generation was based on the SOD-inhibitable reduction
J = 8.0 Hz, NH), 9.63 (1H, t, J = 5.2 Hz, NH), 9.10 (1H, d, J = 7.6 of ferricytochrome c.3,22 In brief, after supplementation with
Hz, NH), 8.07 (2H, d, J = 8.0 Hz), 7.74 (1H, d, J = 8.0 Hz), 7.49 0.5 mg ml−1 ferricytochrome c and 1 mM Ca2+, neutrophils
(1H, d, J = 8.0 Hz), 7.44 (1H, br.s), 7.37 (1H, t, J = 7.2 Hz), were equilibrated at 37 °C for 2 min and incubated with drugs
7.31–7.25 (4H, m), 7.18–7.14 (3H, m), 7.10–7.04 (2H, m), 5.59 for 5 min. Cells were activated with formyl-L-methionyl-L-
(1H, dd, J = 14.2, 7.2 Hz), 5.18 (1H, dd, J = 14.2, 7.2 Hz), 4.43 leucyl-L-phenylalanine (FMLP, 30 nM) or Trp-Lys-Tyr-Met-Val-D-
(1H, dd, J = 17.2, 5.6 Hz), 4.32 (1H, dd, J = 17.2, 5.6 Hz), 3.74 Met (WKYMVm, 1.5 nM) for 10 min in the pretreatment of
1
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cytochalasin B (CB, 1 μg ml−1) for 3 min. Changes in absor-
bance with the reduction of ferricytochrome c at 550 nm were
9 I. A. Schepetkin, L. N. Kirpotina, J. Tian, A. I. Khlebnikov,
R. D. Ye and M. T. Quinn, Mol. Pharmacol., 2008, 74, 392–402.
continuously monitored in a double-beam, six-cell positioner 10 H. Cevik-Aras, C. Kalderen, A. J. Jensen, T. Oprea,
spectrophotometer with constant stirring (Hitachi U-3010,
C. Dahlgren and H. A. Forsman, Biochem. Pharmacol., 2012,
83, 1655–1662.
Tokyo, Japan). Calculations were based on the differences in
the reactions with and without SOD (100 U ml−1) divided by 11 R. D. Ye, F. Boulay, J. M. Wang, C. Dahlgren, C. Gerard,
the extinction coefficient for the reduction of ferricytochrome
c (ε = 21.1 mM−1/10 mm).
M. Parmentier, C. N. Serhan and P. M. Murphy, Pharmacol.
Rev., 2009, 61, 119–161.
5.2.3. Measurement of elastase release.36 Degranulation of 12 L. N. Kirpotina, A. I. Khlebnilov, I. A. Schepetkin, R. D. Ye,
azurophilic granules was determined by elastase release as
described previously.36 Experiments were performed using
M. Rabiet, M. A. Jutila and M. T. Quinn, Mol. Pharmacol.,
2010, 77, 159–170.
MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase sub- 13 C. Movitz, L. Brive, K. Hellstrand, M. Rabiet and
strate. Briefly, after supplementation with MeO-Suc-Ala-Ala-
C. Dahlgren, J. Biol. Chem., 2010, 285, 14338–14345.
14 A. D. Morley, A. Cook, S. King, B. Robert, S. Lever,
R. Weaver, C. MacDonald, J. Unitt, M. Fagura, T. Phillips,
R. Lewis and M. Wenlock, Bioorg. Med. Chem. Lett., 2011,
21, 6456–6460.
Pro-Val-p-nitroanilide (100 μM), neutrophils (5 × 105 ml−1
)
were equilibrated at 37 °C for 2 min and incubated with drugs
for 5 min. Cells were activated by 30 nM FMLP or 1.5 nM
WKYMVm for 10 min in the pretreatment of 0.5 μg ml−1 CB
for 3 min, and changes in absorbance at 405 nm were continu- 15 M. Liu, J. Zhao, K. Chen, X. Bian, C. Wang, Y. Shi and
ously monitored to assay elastase release. Results are expressed J. M. G. Wang, Int. Immunopharmacol., 2012, 14, 283–288.
as percentage of elastase release in the drug-free control 16 P. L. Tsai, J. P. Wang, C. W. Chang, S. C. Kuo and
group. P. D. Chao, Phytochemistry, 1998, 49, 1663–1666.
5.2.4. Statistical analysis. Results are expressed as the 17 J. L. Songue, D. E. Kouam, T. N. Mpondo and R. L. White,
mean SD. Data were analyzed using GraphPad Prism soft- Molecules, 2012, 17, 13673–13686.
ware (GraphPad Software, San Diego, CA). Statistical analysis 18 K. Isshiki, Y. Asai, S. Tanaka, M. Nishio, T. Uchida,
was performed using a Student’s t-test or one-way analysis of
variance (ANOVA) followed by a Tukey’s multiple comparison
test. A value of p < 0.05 was considered statistically significant.
T. Okuda, S. Kamatsubara and N. Sakurai, Biosci. Biotech-
nol. Biochem., 2011, 65, 1195–1197.
19 Z. J. Ma, Z. K. Meng and P. Zhang, Fitoterapia, 2009, 80,
374–376.
20 E. O. Ferreira, M. J. Salvador, E. M. Pral, S. C. Alfieri,
I. Y. Ito and D. A. A. Dias, Z. Naturforsch., C: Biosci., 2004,
59, 499–505.
Acknowledgements
The investigation was supported by research grants to 21 R. Suhas and D. Channe Gowda, Chem. Biol. Drug Des.,
P. W. Hsieh from the National Science Council of the Republic 2012, 79, 850–862.
of China (NSC100-2320-B-182-016) and the Chang Gung 22 C. T. Yen, T. L. Hwang, Y. C. Wu and P. W. Hsieh,
Medical
Research
Foundation
(CMRPD-180381
and
Eur. J. Med. Chem., 2009, 44, 1933–1940.
CMRPD-180382) in Taiwan.
23 D. Tamokou Jde, D. J. Simo Mpetga, P. Keilah Lunga,
M. Tene, P. Tane and J. R. Kuiate, BMC Complement Altern.
Med., 2012, 12, 99–108.
24 B. Xu, Z. Huang, C. Liu, Z. Cai, W. Pan, P. Cao, X. Hao and
G. Liang, Bioorg. Med. Chem., 2009, 17, 3118–3125.
References
1 F. Dallegri and L. Ottonello, Inflamm. Res., 1997, 46, 25 J. Qiu, B. Xu, Z. Huang, W. Pan, P. Cao, C. Liu, X. Hao,
382–391.
B. Song and G. Liang, Bioorg. Med. Chem., 2011, 19,
5352–5360.
26 C. T. Yen, C. C. Wu, J. C. Lee, S. L. Chen, S. L. Morris-
Natschke, P. W. Hsieh and Y. C. Wu, Eur. J. Med. Chem.,
2010, 45, 2494–2502.
27 R. Loser, K. Schilling, E. Dimmig and M. Gutschow, J. Med.
Chem., 2005, 48, 7688–7707.
28 H. P. Yu, P. W. Hsieh, Y. J. Chang, P. J. Chung, L. M. Kuo
and T. L. Hwang, Free Radicals Biol. Med., 2011, 50,
1737–1748.
29 S. Christen, E. Petterhans and R. Stocker, Proc. Natl. Acad.
Sci. U. S. A., 1990, 87, 2506–2510.
30 P. H. Chuang, P. W. Hsieh, Y. L. Yang, K. F. Hua,
F. R. Chang, J. Shiea, S. H. Wu and Y. C. Wu, J. Nat. Prod.,
2008, 71, 1365–1370.
2 Y. L. Fung and C. C. Silliman, Transfus. Med. Rev., 2009, 23,
266–283.
3 Y. D. Cheng, T. L. Hwang, H. H. Wang, T. L. Pan, C. C. Wu,
W. Y. Chang, Y. T. Liu and P. W. Hsieh, Org. Biomol. Chem.,
2011, 9, 7113–7125.
4 J. P. Tung, J. F. Fraser, P. Wood and Y. L. Fung, BMC
Immunol., 2009, 10, 25–35.
5 J. M. Robinsion, Histochem. Cell Biol., 2009, 131, 465–469.
6 V. Bauer, R. Sotnikova and K. Drabikova, Interdiscip.
Toxicol., 2011, 4, 191–197.
7 W. C. Groutas, D. Dou and K. R. Alliston, Expert Opin. Ther.
Pat., 2011, 21, 339–354.
8 S. D. Shapiro, Am. J. Respir. Cell Mol. Biol., 2002, 26,
266–268.
3754 | Org. Biomol. Chem., 2013, 11, 3742–3755
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
31 B. Kuhn, P. Mohr and M. Stahl, J. Med. Chem., 2010, 53,
2601–2611.
A. V. S. Sarma and I. L. Karle, J. Am. Chem. Soc., 1998, 120,
8448–8460.
32 V. A. Ashwood, M. J. Field, D. C. Horwell, C. Julien-Larose, 34 X. Hu, W. Zhang, I. Carmichael and A. S. Serianni, J. Am.
R. A. Lewthwaite, S. McCleary, M. C. Pritchard, Chem. Soc., 2010, 132, 4641–4652.
J. Raphy and L. Singh, J. Med. Chem., 2001, 44, 35 J. Gawronski, H. Kolbon and M. Kwit, Enantiomer, 2002, 7,
2276–2285. 85–92.
33 D. Ranganathan, V. Haridas, S. Kurur, A. Thomas, 36 P. W. Hsieh, H. P. Yu, Y. J. Chang and T. L. Hwang,
K. P. Madhusudanan, R. Nagaraj, A. C. Kunwar,
Eur. J. Med. Chem., 2010, 45, 3111–3115.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 3742–3755 | 3755