Synthesis and evaluation of myxochelin analogues as antimetastatic agents

Article abstract of DOI:10.1016/j.bmc.2009.02.040

Myxochelin A (1) is an inhibitor of tumor cell invasion produced by the bacterium belonging to the genus Nonomuraea. In order to obtain more potent inhibitors, a series of myxochelin analogues [2 and (S)-3-17] were synthesized through the coupling of lysi

Full text of DOI:10.1016/j.bmc.2009.02.040

Bioorganic & Medicinal Chemistry 17 (2009) 2724–2732
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of myxochelin analogues as antimetastatic agents
Satoshi Miyanaga a, Hiroaki Sakurai b, Ikuo Saiki b, Hiroyasu Onaka a, Yasuhiro Igarashi a,
*
a Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
b Department of Bioscience, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Myxochelin A (1) is an inhibitor of tumor cell invasion produced by the bacterium belonging to the genus
Nonomuraea. In order to obtain more potent inhibitors, a series of myxochelin analogues [2 and (S)-3–17]
were synthesized through the coupling of lysine or diaminoalkane derivatives and appropriately pro-
tected hydroxybenzoate, followed by modification of functional groups and deprotection. These com-
pounds were evaluated for their inhibitory activity against invasion of murine colon 26-L5 carcinoma
cells. Among the synthetic analogues tested, compound (S)-6 which possesses carbamoyl group at C-1
was found to be the most potent antiinvasive agent and is considered to be a promising lead molecule
for the antimetastasis. Compound (S)-6 was also shown to inhibit gelatinase activities of MMP-2 and
MMP-9 and in vivo lung metastasis in mice.
Received 22 January 2009
Revised 16 February 2009
Accepted 19 February 2009
Available online 25 February 2009
Keywords:
Myxochelin
Invasion
Metastasis
Inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Tumor metastasis is the most common cause of death in cancer
patients. It is the process by which a tumor cell leaves the primary
tumor, disseminates to a distant site via the circulatory system,
and establishes a secondary tumor.1 During the metastatic cascade,
translocation of tumor cells across the extracellular matrix barriers,
namely invasion, is an essential step to accomplish the metastasis.
The process of invasion is understood to mainly consist of tumor cell
adhesion, enzymatic degradation of extracellular matrix proteins,
and migration. Although the genetic basis of tumorigenesis can vary
greatly, the steps required for metastasis are similar for all tumor
cells. Therefore, drugs that block metastasis will be useful in treating
cancers of various genetic origins. In our previous screening for anti-
invasive compounds from natural products, distinct inhibitory ef-
fects on tumor cell invasion was found in myxochelin A (1), a
secondary metabolite produced by the actinomycete of the genus
Nonomuraea.2 Compound 1 is composed of three building blocks, a
2.1. Synthesis of myxochelin analogues
The myxochelin analogues were synthesized by the coupling
of lysine or diaminoalkane derivatives and the benzyl-protected
2,3-dihydroxybenzoic acid and the following modification of
functional groups and deprotection. Ten analogues possessing
various types of functionality at C-1 position [(S)-1–9 and (R)-
1] were prepared as shown in Scheme 1. Synthesis of both enan-
tiomers of 1 was described in our previous paper.2 Compound 2
was prepared from 1,5-diaminopentane. Compounds (S)-3, (S)-4
and (S)-5 were synthesized starting from
L-lysine methyl ester.
Compound (S)-6 was prepared from -lysinamide. The nitrile
L
analogue (S)-7 was obtained from the same starting material
through the dehydration of the amide. Compounds (S)-8 and
(S)-9 were prepared by the reported procedure.3 In addition, four
analogues were synthesized to elucidate the involvement of phe-
nolic hydroxyl groups in bioactivity. Compound (S)-10 in which
four phenolic hydroxyl groups of (S)-3 were substituted by
methoxy groups was prepared by the coupling of L-lysine methyl
ester and 2,3-dimethoxybenzoic acid. Compound (S)-11 that
lacked hydroxyl groups in the benzene ring located far from
the C-1 functionality was prepared from a protected L-lysine
diaminoalkane part derived from L-lysine and two identical 2,3-
dihydroxybenzoate units, which are connected by two amide bonds.
This combinatorial structural feature prompted us to synthesize
analogues and assess the structure–activity relationship of this class
of compounds as antiinvasive agents. We herein report the synthesis
of myxochelin analogues, structure–activity relationship and
in vitro and in vivo antitumor activities.
methyl ester (Scheme 2). Similarly, compounds (S)-12 and (S)-
13 lacking hydroxyl groups in the benzene ring located near to
the C-1 functionality were synthesized from protected
methyl ester and -lysinamide, respectively (Schemes 2 and 3).
Compound (S)-14 which had an acetyl group instead of the ben-
zoyl group was prepared from a protected -lysine methyl ester
L-lysine
L
* Corresponding author. Tel.: +81 766567500; fax: +81 766562498.
E-mail address: yas@pu-toyama.ac.jp (Y. Igarashi).
L
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.040

S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
2725
Scheme 1. Reagents: (a) ArCOOH, BOP reagent; (b) H2, Pd/C, MeOH; (c) NaOH; (d) BnONH2, BOP reagent; (e) triphosgene.
Scheme 2. Reagents: (a) BzCl, pyr or ArCOOH, BOP reagent; (b) HCl/MeOH; (c) ArCOOH, BOP reagent or BzCl, pyr; (d) H2, Pd/c, MeOH.
Scheme 3. Reagents: (a) BzCl, pyr; (b) HCl/MeOH; (c) ArCOOH, BOP reagent; (d) H2, Pd/c, MeOH.
Scheme 4. Reagents: (a) ArCOOH, BOP reagent; (b) H2, Pd/C, MeOH.
(Scheme 4). Furthermore, three analogues with different methy-
lene chain length were prepared (Scheme 5). Compounds (S)-15
and (S)-16 which had a shorter methylene chain were synthe-
2.2. Structure–activity relationship of myxochelin analogues
We have previously shown that the inversion of the absolute
configuration at C-2 of 1 from S to R resulted in the decrease in
activity.2 In the present study, a series of analogues with S-config-
uration were designed and synthesized. The synthetic myxochelin
analogues were tested for inhibitory activity against invasion of
murine colon 26-L5 cells into the reconstituted basement mem-
sized from (S)-2,4-diaminobutyric acid and L-ornithine, respec-
tively. Compound (S)-17 which had a longer methylene chain
was synthesized from (S)-2,7-diaminoheptanoate.4 The structures
of synthetic compounds were confirmed by NMR and high reso-
lution ESITOFMS analyses.
Scheme 5. Reagents: (a) ArCOOH, BOP reagent; (b) H2, Pd/C, MeOH.

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S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
brane, Matrigel.5 Cytotoxicity was also examined in the same cell
line. The bioassay results are summarized in Table 1.
A
100
80
60
40
20
0
**
2.2.1. Effect of functional group at C-1 position on antiinvasive
activity
***
75
***
250
Among the analogues with different C-1 functionality, the high-
est activity was shown by compound (S)-6 which has the carbam-
oyl group at C-1. It is noteworthy that, in spite of its increased
potency, the cytotoxicity of (S)-6 was lower than the parent natural
product (S)-1 (Table 1 and Fig. 1). Activities of compounds (S)-5,
(S)-8 and (S)-9 were almost equivalent to that of (S)-1. For the
enantiomer (R)-1, reduction in inhibition of invasion was observed,
indicating that the configuration at C-2 is important for activity.
Compound (S)-3 showed modest activity. Compounds 2, (S)-4
and (S)-7 showed either very weak or no activity at the highest
Control 2.5
7.5
25
(µM)
120
100
80
B
***
concentration (2.5 lM) examined in this study. Interestingly, com-
pound 2 that has no substitution at C-2 was highly toxic and (S)-7
was moderately toxic while other compounds were either weakly
***
***
60
toxic or not toxic at the highest concentration (7.5 lM). Although
40
20
0
it is not clear what kind of physical property of the functional
group (e.g., bulkiness, polarity and acidity) is related, these results
indicate that the C-1 functionality affects the inhibitory activity of
tumor cell invasion and cytotoxicity as well.
Control 0.025 0.075
0.25
(µM)
2.2.2. Effect of hydroxyl groups in benzene rings on
antiinvasive activity
Figure 1. Cytotoxicity (A) and in vitro antiinvasive activity (B) of compound (S)-6
against murine colon 26-L5 cells. The data were expressed as the mean SD.
**p < 0.025, ***p < 0.001 as compared with the control by student’s two-tailed t-test.
Based on the preceding structure–activity relationship informa-
tion, compounds (S)-10–(S)-13 were designed and synthesized to
elucidate the role of hydroxyl groups in benzene rings. At first,
compound (S)-10 in which all hydroxyl groups in (S)-3 have been
substituted with methoxy groups lost antiinvasive activity. This re-
sult indicated that phenolic hydroxyl groups had an essential role
in inhibition of invasion. Then, we examined two analogues (S)-11
and (S)-12 which lacked the hydroxyl groups in one of the aromatic
rings. Compound (S)-11 completely lost the activity, clearly show-
ing that the hydroxyl groups in the benzene ring far from the C-1
Table 1
Antiinvasive activity and cytotoxicity of myxochelin analogues
Compound
R1
R2
R3
n
IC50 (lM)
Invasiona
Cell growthb
(S)-1
(R)-1
2
(S)-3
(S)-4
(S)-5
(S)-6
(S)-7
(S)-8
CH2OH
CH2OH
H
COOMe
COOH
CONHOH
CONH2
CN
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
—
—
—
—
—
—
—
—
—
0.59
2.2
>2.5 (45%)
1.5
>2.5 (30%)
0.98
0.091
>2.5 (45%)
0.73
>7.5 (20%)
>7.5 (30%)
2.7
>7.5 (10%)
>7.5 (0%)
>7.5 (0%)
>7.5 (0%)
7.5
CH2NH2
>7.5 (40%)
(S)-9
OH
OH
—
0.52
>5.6 (0%)
(S)-10
(S)-11
(S)-12
(S)-13
(S)-14
(S)-15
(S)-16
(S)-17
COOMe
COOMe
COOMe
CONH2
—
—
—
—
OMe
H
OH
OH
—
—
—
—
OMe
OH
H
H
—
—
—
—
—
—
—
—
—
1
>2.5 (25%)
>2.5 (0%)
1.0
0.26
2.4
>2.5 (40%)
2.5
1.7
>7.5 (0%)
>7.5 (0%)
>7.5 (0%)
>7.5 (0%)
>7.5 (0%)
>7.5 (13%)
>7.5 (45%)
1.6
2
4
a
Inhibition percentage at 2.5
Inhibition percentage at 5.6
l
l
M is indicated in parentheses.
M for (S)-9 and 7.5 M for other compounds is indicated in parentheses.
b
l

S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
2727
functionality is essential to inhibitory activity. On the other hand,
the activity was retained by the analogue (S)-12 that lacks the phe-
nolic hydroxyl groups near to C-1. The C-1 carbamoyl analogue (S)-
13 was shown again to exhibit higher activity than the ester ana-
logue (S)-12. Furthermore, the benzene ring near to the C-1 func-
tionality is not necessary for inhibitory activity as shown by (S)-14.
zymes in degradation of extracellular matrix by many types of
malignant cancer.6 Therefore, we next examined the inhibitory ef-
fect of (S)-1 and (S)-6 on gelatinase activities. In gelatin-based
zymography assay, (S)-1 showed 35% and 39% inhibition against
MMP-2 and MMP-9 at 2.5
(S)-6 displayed 44% and 33% inhibition against MMP-2 and MMP-
9 at 0.25 M, respectively (Fig. 3B). A similar trend was also found
lM, respectively (Fig. 3A), whereas
l
2.2.3. Effect of length of methylene chain on antiinvasive
activity
on comparing the inhibitory activity of (S)-1 and (S)-6 toward tu-
mor cell invasion. We had no access to the inhibition assay for
other MMPs, however these results suggest that MMP-2 and
MMP-9 are at least the target molecules of (S)-6.
Some inhibitors of tumor cell invasion inhibit MMPs. Such
inhibitors often possess in their structures the chelating-site for a
divalent zinc cation in the catalytic domain of MMPs.7 For example,
ONO-4817 inhibits MMPs by binding to the zinc cation with its
hydroxamic acid moiety.8 In case of myxochelin analogues, the
hydroxamate analogue (S)-5 was 10-fold less active than the car-
bamoyl analogue (S)-6. The hydroxylated benzoyl moiety would
be involved in binding to the zinc cation rather than the C-1 func-
tional group.
The preceding results indicated that both the C-1 functionality
and the phenolic hydroxyl groups located far from C-1 were in-
volved in inhibition against tumor cell invasion. Then, we turned
our attention to the alkyl linker connecting the two parts. Two
methylene shorter analogues, (S)-15 and (S)-16, and a methylene
longer analogue (S)-17 were synthesized and evaluated. Com-
pounds (S)-15 and (S)-16 showed much lower activity than their
counterpart (S)-3. Compound (S)-17 was highly toxic and showed
no invasion inhibition at non-cytotoxic concentrations. These re-
sults confirmed that the methylene chain length of the natural
product is the best for antiinvasive activity.
2.3. Mode of action of myxochelin analogues
2.4. Antimetastatic activity of (S)-6 in lung metastasis model
The process of invasion into the basement membrane is a cas-
cade of sequential complex steps, mainly consisting of tumor cell
adhesion, enzymatic degradation of extracellular matrix proteins,
and migration. To gain insight into the mode of action of myxoch-
elin-type compounds, we assessed their effects on tumor cell adhe-
sion and migration. For this purpose, the natural product (S)-1 and
the most potent analogue (S)-6 were selected. The results revealed
that neither the migration of murine colon 26-L5 cells nor the cell
attachment to extracellular matrix proteins (fibronectin and Mag-
tigel) were inhibited by (S)-1 and (S)-6 (Fig. 2), suggesting that deg-
radation of extracellular matrix must be the target step of these
compounds.
Since compound (S)-6 showed the most potent in vitro activity
and less cytotoxicity, it was regarded as a candidate for in vivo
investigation. To evaluate in vivo antimetastatic effect of (S)-6,
we examined suppression of lung metastasis of colon 26-L5 cells
in mouse.9 Intraperitoneal administration of (S)-6 (50 mg/kg) re-
sulted in a reduction of lung tumor colonies produced by iv injec-
tion of colon 26-L5 cells into the tail vein. The inhibitory effect was
significant (inhibition rate of 35%, p < 0.05), compared to the vehi-
cle control (Fig. 4A). There was no marked body weight loss in the
treated mice during the course of the study (Fig. 4B). The oral
administration of (S)-6 showed tendency to suppress the lung
The process of tumor invasion definitely requires proteolysis of
the extracellular matrix by matrix metalloproteinases (MMPs). In
particular, two gelatinases, MMP-2 and MMP-9 are the key en-
MMP-2
MMP-9
A
120
100
80
60
40
20
0
**
*
**
***
Fibronectin
Matrigel
A
140
120
100
80
Control
0.25
0.75
2.5
60
(S)-1 (µM)
40
20
B
120
100
80
0
Control
120
(S)-1
(S)-6
***
***
B
**
**
100
80
60
40
20
0
60
40
20
0
Control
0.025
2.5
0.25
(S)-6 (µM)
Control
(S)-1
(S)-6
Figure 3. Inhibition of MMP-2 and MMP-9 by compounds (S)-1 (A) and (S)-6 (B).
The data were expressed as the mean SD. *p < 0.01, **p < 0.025, ***p < 0.001 as
compared with the control by student’s two-tailed t-test.
Figure 2. Effects of compounds (S)-1 and (S)-6 at 2.5
and migration (B). The data were expressed as the mean SD.
l
M on tumor cell adhesion (A)

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S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
the reaction mixture was poured into ice-water and extracted with
A
B
300
200
100
0
EtOAc. The organic layer was washed with 0.5 M HCl, saturated
NaHCO3 solution and brine, dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane–EtOAc = 1:0–1:5) to give 18
(222 mg, 99% yield): 1H NMR (400 MHz, CDCl3) d 1.10 (2H, m),
1.26 (4H, m), 3.19 (4H, m), 5.13 (4H, s), 5.15 (4H, s), 7.13–7.48
(24H), 7.73 (2H, m), 7.89 (2H, t, J = 5.1 Hz); HR-ESITOFMS m/z
[MꢁH]ꢁ 733.3284 (calcd for C47H45N2O6, 733.3283).
**
Control
(S)-6
22
20
18
16
Control
4.2.2. 1,5-Bis[(2,3-dihydroxybenzoyl)amino]pentane (2)
(S)-6 (50 mg/kg)
A mixture of compound 18 (134 mg, 0.182 mmol) and 10% Pd/C
(91 mg) in MeOH (45 mL) was stirred under H2 atmosphere at
room temperature. After stirring for 3 h, the mixture was filtered
through Celite and the filtrate was concentrated in vacuo. The res-
idue was purified by preparative HPLC (30% CH3CN in water) and
the collected fraction was concentrated to give 2 (23 mg, 34%
yield): colorless amorphous solid; 1H NMR (400 MHz, CD3OD) d
1.35 (2H, m), 1.56 (4H, m), 3.30 (4H, m), 6.60 (2H, t, J = 7.6 Hz),
6.82 (2H, d, J = 7.3 Hz), 7.10 (2H, d, J = 7.6 Hz); IR mmax 3356,
0
2
4
6
8
10
12
14
Days
3189, 1638, 1579, 1554 cmꢁ1
373.1403 (calcd for C19H21N2O6, 373.1405).
;
HR-ESITOFMS m/z [MꢁH]ꢁ
Figure 4. Inhibition of lung metastasis of colon 26-L5 cells by ip administration of
compound (S)-6. (A) Number of tumor colonies formed on lung 14 days after tumor
inoculation. (B) Body weight change of mice during 14 days. The data were
expressed as the mean SD. **p < 0.025 as compared with the control by student’s
two-tailed t-test.
4.2.3. (S)-2,6-Bis[(2,3-dibenzyloxybenzoyl)amino]hexanoic acid
methyl ester [(S)-19]
In the same manner as described for 18, L-lysine methyl ester
gave (S)-19 in 57% yield: 1H NMR (400 MHz, CDCl3) d 1.04–1.28
(4H, m), 1.38 (1H, m), 1.61 (1H, m), 3.10 (2H, m), 3.59 (3H, s),
4.59 (1H, m), 5.05 (2H, s), 5.14 (1H, d, J = 10.2 Hz), 5.15 (2H, s),
5.16 (1H, d, J = 10.2 Hz), 7.13–7.52 (24H, m), 7.72 (2H, m), 7.85
(1H, t, J = 5.6 Hz), 8.48 (1H, d, J = 7.3 Hz); 13C NMR (100 MHz,
CDCl3) d 22.9, 28.7, 31.5, 52.1, 52.5, 71.22, 71.26, 76.1, 76.3,
116.8, 117.2, 123.25, 123.29, 124.32, 124.37, 126.6, 127.3, 127.6,
127.7, 128.19, 128.23, 128.4, 128.5, 128.63, 128.66, 128.69,
128.74, 136.26, 136.28, 136.3, 136.4, 146.7, 146.9, 151.6, 151.7,
164.9, 164.9, 172.7; HR-ESITOFMS m/z [M+Na]+ 815.3305 (calcd
for C49H48N2O8Na, 815.3303).
metastasis but the effect was not significant (p = 0.55, data not
shown).
3. Conclusion
In summary, we report the synthesis and biological evaluation
of a series of myxochelin analogues. Compound (S)-6 that pos-
sesses the carbamoyl group at C-1 exhibited a sevenfold improve-
ment with regard to the parent natural product (S)-1 and showed
lower cytotoxicity. Several bioassay results revealed that (S)-6
inhibits the process of extracellular matrix degradation by inhibit-
ing the proteolytic activity of at least two MMPs, MMP-2 and
MMP-9. Furthermore, (S)-6 suppressed the lung metastasis of co-
lon 26-L5 cells in mice. Our findings demonstrate that the myxoch-
elin-type compound is a new and interesting scaffold for the
development of antimetastatic agents.
4.2.4. (S)-2,6-Bis[(2,3-dihydroxybenzoyl)amino]hexanoic acid
methyl ester [(S)-3]
In the same manner as described for 2, (S)-19 gave (S)-3 in 64%
yield: colorless amorphous solid; ½a D24
ꢂ
ꢁ 8:0 (c 0.15, MeOH); 1H
NMR (400 MHz, CD3OD) d 1.42 (2H, m), 1.58 (2H, m), 1.79 (1H,
m), 1.91 (1H, m), 3.30 (1H, t, J = 7.1 Hz), 3.63 (3H, s), 4.53 (1H,
dd, J = 8.6, 5.1 Hz), 6.59 (1H, t, J = 7.8 Hz), 6.62 (1H, t, J = 7.8 Hz),
6.82 (1H, d, J = 7.8 Hz), 6.84 (1H, d, J = 7.8 Hz), 7.08 (2H, d,
J = 7.6 Hz), 7.22 (1H, d, J = 8.0 Hz); 13C NMR (100 MHz, CD3OD) d
24.4, 29.9, 32.0, 40.1, 52.8, 53.8, 116.78, 116.83, 118.6, 119.47,
119.49, 119.6, 119.7, 147.29, 147.33, 150.0, 150.3, 171.2, 171.5,
174.3; IR mmax 3346, 1729, 1638, 1585, 1535 cmꢁ1; HR-ESITOFMS
m/z [MꢁH]ꢁ 431.1460 (calcd for C21H23N2O8, 431.1460).
4. Experimental
4.1. General
Optical rotations were measured using a JASCO DIP-3000 polar-
imeter. IR spectra were measured on a Perkin Elmer Spectrum 100.
NMR spectra were obtained on a JEOL LA-400 or a Bruker AVANCE
400 spectrometer. HR-ESITOFMS were recorded on a Bruker micro-
TOF focus. Silica Gel 60-C18 (Nakalai Tesque 250–350 mesh) was
used for ODS column chromatography. HPLC separation was per-
4.2.5. (S)-2,6-Bis[(2,3-dibenzyloxybenzoyl)amino]hexanoic acid
[(S)-20]
To a solution of (S)-19 (50 mg, 0.063 mmol) in MeOH (5 mL)
was added 1 M NaOH (1 mL). After stirring for 2 h at room temper-
ature, the solution was neutralized with 0.5 M HCl and concen-
trated in vacuo. Then, the residue was acidified with 1 M HCl,
extracted with EtOAc, dried over anhydrous Na2SO4 and concen-
trated in vacuo to give (S)-20 (47 mg, 99% yield): 1H NMR
(400 MHz, CDCl3) d 1.15–1.35 (5H), 1.46 (1H, m), 3.11(2H, m),
3.95 (1H, m), 4.99 (2H, s), 5.01 (1H, d, J = 10.8 Hz), 5.05 (1H, d,
J = 10.8 Hz), 5.08 (2H, s), 5.09 (2H, s), 7.04–7.44 (26H); 13C NMR
(100 MHz, CDCl3) d 22.9, 28.8, 30.6, 39.3, 53.0, 71.3, 71.4, 76.4,
76.7, 117.0, 117.6, 123.4, 124.4, 126.0, 127.2, 127.7, 127.8,
128.26, 128.31, 128.6, 126.9, 128.72, 128.8, 128.9, 136.1, 136.27,
formed using
a Cosmosil 5C18-AR-II (Nacalai Tesque Inc.,
20 ꢀ 250 mm) with a photodiode array detector.
4.2. Synthesis of myxochelin analogues
4.2.1. 1,5-Bis[(2,3-dibenzyloxybenzoyl)amino]pentane (18)
To a solution of 1,5-diaminopentane (53 mg, 0.30 mmol), BOP re-
agent (0.464 g, 1.05 mmol) and anhydrous 1-hydroxybenzotriazole
(142 mg, 1.05 mmol) in dry DMF (4 mL) was added 2,3-dibenzyloxy-
benzoic acid10 (200 mg, 0.60 mmol) and N,N-diisopropylethylamine
(0.31 mL, 0.60 mmol). After stirring for 14 h at room temperature,

S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
2729
136.34, 136.3, 136.4, 147.1, 151.68, 151.74, 165.1, 166.2; HR-ESI-
4.2.10. (S)-2,6-Bis[(2,3-dihydroxybenzoyl)amino]hexanenitrile
[(S)-7]
TOFMS m/z [MꢁH]ꢁ 777.3179 (calcd for C48H45N2O8, 777.3181).
In the same manner as described for 2, (S)-2,6-bis[(2,3-dib-
4.2.6. (S)-2,6-Bis[(2,3-dihydroxybenzoyl)amino]hexanoic acid
[(S)-4]
enzyloxybenzoyl) amino]hexanenitrile3 [(S)-23] gave (S)-7 in 38%
yield: colorless amorphous solid; ½a D23
ꢂ
ꢁ 18:1 (c 0.1, MeOH); 1H
In the same manner as described for 2, (S)-20 gave (S)-4 in 61%
NMR (400 MHz, CD3OD) d 1.67 (2H, m), 1.92 (2H, m), 2.01 (2H,
m), 3.40 (2H, dd, J = 13.7, 6.8 Hz), 5.03 (1H, t, J = 7.3 Hz), 6.68
(1H, t, J = 8.0 Hz), 6.69 (1H, t, J = 8.0 Hz), 6.89 (1H, d, J = 7.8 Hz),
6.93 (1H, d, J = 7.8 Hz), 7.17 (1H, d, J = 8.1 Hz), 7.24 (1H, d,
J = 8.0 Hz); 13C NMR (100 MHz, CD3OD) d 24.1, 29.7, 33.2, 40.0,
41.5, 116.2, 116.8, 118.7, 119.2, 119.3, 119.4, 119.5, 119.8, 119.9,
147.4, 147.7, 150.4, 151.1, 171.1, 171.6; IR mmax 3337, 2244,
1638, 1585, 1534 cmꢁ1; HR-ESITOFMS m/z [M+Na]+ 422.1327
(calcd for C20H21N3O6Na, 422.1323).
yield: colorless amorphous solid; ½a D23
ꢂ
þ 5:4 (c 0.13, MeOH); 1H
NMR (400 MHz, CD3OD) d 1.54 (2H, m), 1.67 (2H, m), 1.92 (1H,
m), 2.14 (1H, m), 3.38 (2H, t, J = 6.4 Hz), 4.61 (1H, br s), 6.67 (1H,
t, J = 8.1 Hz), 6.71 (1H, t, J = 7.8 Hz), 6.89 (1H, d, J = 8.1 Hz), 6.92
(1H, d, J = 7.6 Hz), 7.16 (1H, d, J = 7.8 Hz), 7.31 (1H, d, J = 7.8 Hz);
13C NMR (400 MHz, CD3OD) d 24.4, 29.9, 32.4, 40.2, 50.4, 116.8,
117.0, 118.6, 119.4, 119.5, 119.7, 147.2, 147.3, 149.8, 150.2,
170.9, 171.5; IR mmax 3353, 1722, 1639, 1583, 1538 cmꢁ1; HR-ESI-
TOFMS m/z [MꢁH]ꢁ 417.1296 (calcd for C20H21N2O8, 417.1303).
4.2.11. (S)-2,6-Bis[(2,3-dimethoxybenzoyl)amino]hexanoic acid
methyl ester [(S)-10]
4.2.7. (S)-2,6-Bis[(2,3-dibenzyloxybenzoyl)amino]-N-benzyl-
oxyhexanoamide [(S)-21]
In the same manner as described for 18, condensation of L-ly-
Compound (S)-20 (80 mg, 0.10 mmol), BOP reagent (88 mg,
0.20 mmol) and anhydrous 1-hydroxybenzotriazole (27 mg, 0.20 mmol)
weredissolvedindryDMF(2 mL). Tothe mixturewere thenadded O-ben-
zylhydroxylamine hydrochloride (16 mg, 0.10 mmol) and N,N-diisopro-
pylethylamine (0.017 mL, 0.10 mmol). After stirring for 14 h at room
temperature, the reaction mixture was poured into ice-water and ex-
tracted with EtOAc. The organic layer was washed with 0.5 M HCl, satu-
rated NaHCO3 solution and brine, dried over anhydrous Na2SO4, filtered
and concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexane–EtOAc = 10:1–1:3) to give (S)-21 (61 mg 67%
yield): 1H NMR (400 MHz, CDCl3) d 1.08–1.29 (4H, m), 1.60 (1H, m),
2.09 (1H, m), 3.12 (2H, dt, J = 6.3, 6.3 Hz), 4.23 (1H, m), 4.94 (2H, s), 5.04
(2H, s), 5.09 (2H, s), 5.11 (2H, s), 5.13 (2H, s), 7.08–7.49 (29H), 7.64 (2H,
m), 7.89 (1H, br s), 8.44 (1H, d, J = 7.1 Hz), 9.62 (1H, br s); 13C NMR
(100 MHz, CDCl3) d 22.9, 28.7, 30.2, 39.1, 51.0, 71.2, 71.3, 76.1, 76.3,
78.0, 116.8, 117.5, 123.1, 123.2, 124.27, 124.31, 125.9, 127.2, 127.6,
127.7, 128.2, 128.3, 128.4, 128.5, 128.59, 128.61, 128.63, 129.0, 129.1,
135.2, 135.8, 136.2, 136.28, 136.33, 146.7, 146.9, 151.6, 151.7, 164.9,
165.5, 169.1; HR-ESITOFMS m/z [M+Na]+ 906.3725 (calcd for C55H53N3O8-
Na, 906.3725).
sine methyl ester and 2,3-dimethoxybenzoic acid gave (S)-10 in
92% yield: colorless amorphous solid; ½a D22
ꢁ 3:9 (c 1.0, MeOH);
ꢂ
1H NMR (400 MHz, CDCl3) d 1.45 (2H, m), 1.60 (2H, m), 1.79 (1H,
m), 1.94 (1H, m), 3.37 (2H, dd, J = 12.7, 6.4 Hz), 3.68 (3H, s), 3.79
(3H, s), 3.83 (3H, s), 3.86 (3H, s), 3.88 (3H, s), 4.80 (2H, m), 6.94
(1H, d, J = 8.3 Hz), 6.98 (1H, d, J = 8.3 Hz), 7.04 (1H, t, J = 7.8 Hz),
7.06 (1H, t, J = 8.0 Hz), 7.57 (1H, d, J = 8.0 Hz), 7.59 (1H, d,
J = 8.1 Hz); IR mmax 3369, 1740, 1652, 1521 cmꢁ1; HR-ESITOFMS
m/z [M+Na]+ 511.2057 (calcd for C25H32N2O8Na, 511.2051).
e
a
4.2.12. N -Benzoyl-N -tert-butoxycarbonyl-
L-lysine methyl
ester [(S)-24]
a
To a stirred solution of N -tert-butoxycarbonyl-
L-lysine methyl
ester (300 mg, 1.0 mmol) in dry pyridine (3.5 mL) was added ben-
zoyl chloride (211 mg, 1.5 mmol) at 0–5 °C. After stirring at the
same temperature for 15 h, the mixture was poured into ice-water
and extracted with EtOAc. The organic layer was washed with sat-
urated CuSO4 solution and brine, dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane–EtOAc = 1:0–1:1) to give (S)-
24 (380 mg, 99% yield): 1H NMR (400 MHz, CDCl3) d 1.35–1.59 (4H,
m), 1.49 (9H, s), 1.83 (1H, m), 1.98 (1H, m), 3.12 (2H, m), 3.79 (3H,
s), 4.60 (1H, br s), 4.82 (1H, m), 6.75 (1H, br s), 7.45 (2H, t,
J = 7.8 Hz), 7.53 (2H, t, J = 6.8 Hz), 7.83 (1H, d, J = 7.6 Hz); HR-ESI-
TOFMS m/z [M+Na]+ 387.1891 (calcd for C19H28N2O5Na, 387.1890).
4.2.8. (S)-2,6-Bis[(2,3-dihydroxybenzoyl)amino]- N-hydroxy-
hexanoamide [(S)-5]
In the same manner as described for 2, (S)-21 gave (S)-5 in 81%
yield: colorless amorphous solid; ½a D23
ꢂ
ꢁ 6:0 (c 0.13, MeOH); 1H
NMR (400 MHz, CD3OD) d 1.50 (2H, m), 1.67 (2H, m), 1.89 (2H,
m), 3.38 (2H, t, J = 7.3 Hz), 4.50 (1H, t, J = 7.1 Hz), 6.67 (1H, t,
J = 8.1 Hz), 6.69 (1H, t, J = 8.1 Hz), 6.89 (1H, d, J = 7.6 Hz), 6.91
(1H, d, J = 7.6 Hz), 7.17 (1H, d, J = 8.0 Hz), 7.31 (1H, d, J = 7.8 Hz);
13C NMR (100 MHz, CD3OD) d 24.3, 30.0, 32.9, 40.2, 52.5, 116.8,
118.7, 119.5, 119.59, 119.62, 119.7, 147.3, 147.4, 149.8, 150.1,
e
4.2.13. N -Benzoyl-
L-lysine methyl ester [(S)-25]
A solution of compound (S)-24 (350 mg, 0.96 mmol) in 1.5 M
HCl–MeOH (17.5 mL) was stirred for 3 h at room temperature.
The pH of the reaction mixture was adjusted to 8.0 by the addition
of aqueous NH4OH solution. The mixture was then concentrated in
vacuo and the remaining aqueous residue was extracted with
EtOAc. The organic layer was dried over anhydrous Na2SO4, filtered
and concentrated in vacuo to give (S)-25 (168 mg, 66% yield): 1H
NMR (400 MHz, CD3OD) d 1.50 (2H, m), 1.72 (2H, m), 1.90 (1H,
m), 2.02 (1H, m), 2.93 (2H, t, J = 6.3 Hz), 3.34 (3H, s), 4.63 (1H,
m), 7.47 (2H, t, J = 7.3 Hz), 7.55 (1H, t, J = 7.1 Hz), 7.85 (2H, d,
J = 7.3 Hz); HR-ESITOFMS m/z [M+H]+ 265.1555 (calcd for
C14H21N2O3 265.1547).
170.7, 171.2, 171.5; IR m
max 3202, 1635, 1582, 1530 cmꢁ1; HR-ESI-
TOFMS m/z [MꢁH]ꢁ 432.1402 (calcd for C20H22N3O8, 432.1412).
4.2.9. (S)-2,6-Bis[(2,3-dihydroxybenzoyl)amino]hexanoamide
[(S)-6]
In the same manner as described for 2, (S)-2,6-bis[(2,3-dib-
enzyloxybenzoyl)amino]hexanoamide3 [(S)-22] gave (S)-6 in 57%
yield: colorless amorphous solid; ½a D23
ꢂ
ꢁ 7:0 (c 0.1, MeOH); 1H
NMR (400 MHz, CD3OD) d 1.44 (2H, m), 1.51 (2H, m), 1.90 (1H,
m), 1.95 (1H, m), 3.38 (2H, J = 6.6 Hz), 4.58 (1H, dd, J = 8.6,
5.1 Hz), 6.65 (1H, d, J = 7.8 Hz), 6.68 (1H, d, J = 7.8 Hz), 6.90 (2H,
m), 7.17 (1H, d, J = 7.8 Hz), 7.30 (1H, d, J = 7.8 Hz); 13C NMR
(100 MHz, CD3OD) d 24.4, 30.0, 32.9, 40.1, 50.6, 116.9, 117.3,
118.7, 119.32, 119.36, 119.4, 119.7, 147.38, 147.43, 150.3, 150.4,
4.2.14. (S)-6-Benzoylamino-2-[(2,3-dibenzyloxybenzoyl)-
amino]hexanoic acid methyl ester [(S)-26]
In the same manner as described for 18, (S)-25 gave (S)-26 in
36% yield: 1H NMR (400 MHz, CDCl3) d 1.36 (4H, m), 1.90 (2H,
m), 3.19 (1H, m), 3.33 (1H, m), 3.75 (3H, s), 4.70 (1H, m), 5.04
(1H, d, J = 10.5 Hz), 5.06 (1H, d, J = 10.5 Hz), 5.15 (2H, s), 6.91
(1H, d, J = 7.3 Hz), 7.10–7.50 (16H, m), 7.83 (2H, d, J = 8.0 Hz),
7.98 (1H, t, J = 5.8 Hz); 13C NMR (100 MHz, CDCl3) d 22.4, 29.1,
170.9, 171.5, 171.2; IR mmax 3321, 1671, 1637, 1583, 1534 cmꢁ1
;
HR-ESITOFMS m/z [MꢁH]ꢁ 416.1461 (calcd for C20H22N3O7,
416.1463).

2730
S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
31.4, 38.5, 52.4, 52.6, 71.3, 76.4, 116.9, 123.3, 124.4, 127.20,
127.23, 127.7, 128.3, 128.5, 128.68, 128.72, 128.77, 131.6, 136.3,
136.4, 146.7, 151.7, 165.5, 167.3, 173.0; HR-ESITOFMS m/z [MꢁH]ꢁ
579.2513, calcd 579.2501 (for C35H35N2O6).
J = 7.6 Hz), 7.82 (2H, d, J = 7.4 Hz); HR-ESITOFMS m/z [MꢁH]ꢁ
348.1929 (calcd for C18H26N3O4 348.1929).
a
4.2.21. N -Benzoyl-
L-lysinamide [(S)-31]
In the same manner as described for (S)-25, (S)-30 gave (S)-31 in
71% yield: 1H NMR (400 MHz, CD3OD) d 1.47–1.60 (2H, m), 1.71
(2H, m), 1.86 (1H, m), 1.99 (1H, m), 2.94 (2H, m), 4.60 (1H, dd,
J = 9.0, 5.5 Hz), 7.49 (2H, t, J = 7.5 Hz), 7.57 (1H, t, J = 7.5 Hz), 7.89
(2H, d, J = 8.0 Hz); HR-ESITOFMS m/z [M+H]+ 250.1558 (calcd for
C13H20N3O2 250.1550).
4.2.15. (S)-6-Benzoylamino-2-[(2,3-dihydroxybenzoyl)amino]-
hexanoic acid methyl ester [(S)-11]
In the same manner as described for 2, (S)-26 gave (S)-11 in 43%
yield: colorless amorphous solid; ½a D23
ꢂ
ꢁ 4:4 (c 0.1, MeOH); 1H NMR
(400 MHz, CD3OD) d 1.41 (2H, m), 1.56 (2H, m), 1.77 (1H, m), 1.90
(1H, m), 3.27 (2H, t, J = 9.3 Hz), 4.50 (1H, dd, 9.5, 5.1 Hz), 6.57 (1H,
t, J = 7.6 Hz), 6.80 (1H, d, J = 7.6 Hz), 7.08 (1H, d, J = 7.6 Hz), 7.42
(2H, t, J = 7.6 Hz), 7.50 (1H, t, J = 7. 4 Hz), 7.71 (2H, d, J = 7.6 Hz);
13C NMR (100 MHz, CD3OD) d 24.5, 29.9, 31.9, 40.8, 52.7, 54.3,
116.7, 118.6, 119.5, 128.5, 129.5, 132.9, 135.1, 147.4, 150.4,
4.2.22. (S)-2-Benzoylamino-6-[(2,3-dibenzyloxybenzoyl)-
amino]hexanoamide [(S)-32]
In the same manner as described for 18, (S)-31 gave (S)-32 in
49% yield: 1H NMR (400 MHz, CDCl3) d 1.34 (4H, m), 1.86 (2H,
m), 3.15 (1H, m), 3.29 (1H, m), 4.61 (1H, m), 5.01 (1H, d,
J = 10.4 Hz), 5.02 (1H, d, J = 10.4 Hz), 5.12 (2H, s), 6.12 (1H, br s),
6.98 (1H, br s), 7.06–7.64 (16H, m), 7.81 (2H, m), 7.97 (1H, t,
J = 5.7 Hz); HR-ESITOFMS m/z [M+Na]+ 588.2470 (calcd for
C34H35N3O5Na 588.2469).
170.6, 171.5, 174.4; IR
m
max 3348, 1736, 1637, 1535 cmꢁ1; HR-ESI-
TOFMS m/z [M+Na]+ 423.1521 (calcd for C21H24N2O6Na 423.1527).
a
e
4.2.16. N -tert-Butoxycarbonyl-N -(2,3-dibenzyloxybenzoyl)-L-
lysine methyl ester [(S)-27]
a
In the same manner as described for 18, N -tert-butoxycar-
bonyl-
L
-lysine methyl ester gave (S)-27 in 98% yield: 1H NMR
4.2.23. (S)-2-Benzoylamino-6-[(2,3-dihydroxybenzoyl)amino]-
hexanoamide [(S)-13]
(400 MHz, CDCl3) d 1.26 (2H, m), 1.38 (2H, m), 1.45 (9H, s), 1.63–
1.75 (2H, m), 2.98 (2H, m), 3.67 (3H, s), 4.47 (1H, br s), 4.67 (1H,
q, J = 7.6 Hz), 5.12 (1H, d, J = 10.5 Hz), 5.16 (2H, s), 5.19 (1H, d,
J = 10.5 Hz), 7.13–7.47 (13H, m), 7.74 (1H, m), 8.51 (1H, d,
J = 7.6 Hz); HR-ESITOFMS m/z [M+Na]+ 599.2719 (calcd for
C33H40N2O7Na 599.2728).
In the same manner as described for 2, (S)-29 gave (S)-13 in 59%
yield: colorless amorphous solid; ½a D23
ꢂ
ꢁ 4:8 (c 0.1, MeOH); 1H NMR
(400 MHz, CD3OD) d 1.46 (2H, m), 1.59 (2H, m), 1.76 (1H, m), 1.86
(1H, m), 3.30 (2H, t, J = 6.5 Hz), 4.47 (1H, d, J = 9.2, 5.2 Hz), 6.57 (1H,
t, J = 7.8 Hz), 6.81 (1H, d, J = 7.6 Hz), 7.09 (1H, d, J = 7.9 Hz), 7.33
(2H, t, J = 7.4 Hz), 7.42 (1H, t, J = 7.4 Hz), 7.73 (2H, d, J = 7.4 Hz);
13C NMR (100 MHz, CD3OD) d 24.5, 30.1, 32.9, 40.1, 55.0, 116.9,
118.8, 119.2, 119.3, 128.5, 129.5, 132.8, 135.3, 147.5, 150.7,
4.2.17. (S)-2-Amino-6-[(2,3-dibenzyloxybenzoyl)amino]-
hexanoic acid methyl ester [(S)-28]
In the same manner as described for (S)-25, (S)-27 gave (S)-28 in
84% yield: 1H NMR (400 MHz, CD3OD) d 1.25 (3H, m), 1.47 (2H, m),
1.68 (1H, m), 2.67 (2H, m), 3.61 (3H, s), 4.46 (1H, dd, J = 8.3, 5.4 Hz),
5.04 (1H, d, J = 10.5 Hz), 5.05 (1H, d, J = 10.5 Hz), 5.09 (2H, s), 7.05–
7.40 (13H, m); HR-ESITOFMS m/z [M+H]+ 477.2384 (calcd for
C28H33N2O5 477.2384).
170.4, 171.5, 177.3; IR mmax 3310, 1671, 1636, 1578, 1532 cmꢁ1
;
HR-ESITOFMS m/z [M+Na]+ 408.1528 (calcd for C20H23N3O5Na
408.1530).
4.2.24. (S)-2-Acetyl-6-[(2,3-dibenzyloxybenzoyl)amino]-
hexanoic acid methyl ester [(S)-33]
a
In the same manner as described for 18, N -acetyl-
L-lysine
4.2.18. (S)-2-Benzoylamino-6-[(2,3-dibenzyloxybenzoyl)-
amino]hexanoic acid methyl ester [(S)-29]
methyl ester gave (S)-33 in 99% yield: 1H NMR (400 MHz, CDCl3)
d 1.21–1.36 (4H), 1.68 (1H, m), 1.78 (1H, m), 2.06 (3H, s), 3.21
(1H, m), 3.30 (1H, m), 3.71 (3H, s), 4.48 (1H, ddd, J = 7.6, 7.6,
4.4 Hz), 5.07 (1H, d, J = 10.4 Hz), 5.08 (1H, d, J = 10.4 Hz), 5.16
(2H, s), 6.29 (1H, d, J = 7.6 Hz), 7.15–7.17 (2H, m), 7.32–7.49
(10H, m), 7.73 (1H, m), 8.00 (1H, d, J = 5.6 Hz); 13C NMR
(100 MHz, CDCl3) d 22.2, 23.1, 28.9, 31.3, 38.5, 52.1, 52.3, 71.3,
76.4, 117.0, 123.2, 124.4, 127.7, 128.3, 128.71, 128.72, 128.77,
128.80, 136.3, 146.8, 151.7, 165.4, 170.1, 173.0; HR-ESITOFMS m/
z [M+Na]+ 541.2309 (calcd for C30H34N2O6Na 541.2309).
In the same manner as described for 18, (S)-28 gave (S)-29 in
46% yield: 1H NMR (400 MHz, CDCl3) d 1.17 (2H, m), 1.32 (2H,
m), 1.47 (1H, m), 1.64 (1H, m), 2.42 (2H, t, J = 7.1 Hz), 3.64 (3H,
s), 5.02 (4H, s), 7.07–7.44 (18H, m); HR-ESITOFMS m/z [MꢁH]ꢁ
579.2506, calcd 579.2501 (for C35H35N2O6).
4.2.19. (S)-2-Benzoylamino-6-[(2,3-dihydroxybenzoyl)amino]-
hexanoic acid methyl ester [(S)-12]
In the same manner as described for 2, (S)-29 gave (S)-12 in 46%
yield: colorless amorphous solid; ½a D23
ꢂ
ꢁ 9:1 (c 0.1, MeOH); 1H NMR
4.2.25. (S)-2-Acetyl-6-[(2,3-dihydroxybenzoyl)amino]hexanoic
acid methyl ester [(S)-14]
(400 MHz, CD3OD) d 1.43 (2H, m), 1.57 (2H, m), 1.81 (1H, m), 1.89
(1H, m), 3.27 (2H, t, J = 6.6 Hz), 3.62 (3H, s), 4.54 (1H, dd, J = 9.0,
5.1 Hz), 6.59 (1H, t, J = 7.6), 6.83 (1H, d, J = 7.3 Hz), 7.22 (1H, d,
J = 7.8 Hz), 7.37 (2H, t, J = 7.3), 7.39 (1H, t, J = 7.3 Hz), 7.66 (2H, d,
J = 7.6 Hz); 13C NMR (100 MHz, CD3OD) d 24.3, 30.0, 32.0, 40.5,
52.8, 53.8, 116.8, 119.4, 119.5, 119.6, 128.2, 129.5, 132.5, 135.8,
147.4, 150.4, 170.4, 171.2, 174.2; IR mmax 3314, 1730, 1637,
In the same manner as described for 2, (S)-33 gave (S)-14 in 51%
yield: colorless amorphous solid; ½a D24
ꢂ
ꢁ 2:7 (c 0.1, MeOH); 1H NMR
(400 MHz, CD3OD) d 1.36 (2H, m), 1.52 (2H, m), 1.60 (1H, m), 1.75
(1H, m), 3.25 (3H, s), 3.28 (2H, t, J = 6.8 Hz), 3.58 (3H, s), 4.46 (1H,
dd, J = 8.8, 5.2 Hz), 6.60 (1H, t, J = 7.8 Hz), 6.81 (1H, d, J = 7.7 Hz),
7.81 (1H, d, J = 7.9 Hz); 13C NMR (100 MHz, CD3OD) d 22.3, 24.2,
29.9, 32.1, 40.1, 52.6, 53.8, 116.8, 118.7, 119.37, 119.43, 147.5,
150.6, 171.5, 173.4, 174.3; IR mmax 3314, 1733, 1635, 1587,
;
1534 cmꢁ1 HR-ESITOFMS m/z [M+Na]+ 423.1527 (calcd for
C21H24N2O6Na 423.1527).
1537 cmꢁ1
;
HR-ESITOFMS m/z [MꢁH]ꢁ 337.1405 (calcd for
a
e
4.2.20. N -Benzoyl-N -tert-butoxycarbonyl-
In the same manner as described for (S)-24, N -tert-butoxycar-
bonyl-
-lysinamide gave (S)-30 in 34% yield: 1H NMR (400 MHz,
L-lysinamide [(S)-30]
C16H22N2O6 337.1405).
e
L
4.2.26. (S)-2,4-Bis[(2,3-dibenzyloxybenzoyl)amino]butyric acid
methyl ester [(S)-35]
CDCl3) d 1.40 (9H, s), 1.47 (2H, m), 1.56 (2H, m), 1.84 (1H, m),
2.03 (1H, m), 3.13 (2H, m), 4.70 (1H, m), 5.64 (1H, br s), 6.41
(1H, br s), 6.99 (1H, br s), 7.45 (2H, t, J = 7.6 Hz), 7.53 (1H, t,
In the same manner as described for 18, (S)-3411 gave (S)-35 in
46% yield: 1H NMR (400 MHz, CDCl3) d 1.46 (1H, m), 1.90 (1H, m),

S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
2731
3.05 (1H, m), 3.32 (1H, m), 3.61 (3H, s), 4.64 (1H, dt, J = 5.6, 8.4 Hz),
5.08 (1H, d, J = 10.4 Hz), 5.10 (1H, s), 5.11 (1H, s), 5.14 (2H, s), 5.15
(2H, s), 5.21 (1H, J = 10.4 Hz), 7.11–7.47 (24H, m), 7.65–7.70 (2H,
m), 8.03 (1H, t, J = 5.6 Hz), 8.50 (1H, d, J = 7.6 Hz); 13C NMR
(100 MHz, CD3OD) d 32.1, 36.1, 50.6, 52.3, 71.3, 71.4, 76.2, 76.3,
117.0, 117.4, 123.2, 123.4, 124.2, 126.6, 127.6, 127.8, 128.2,
128.3, 128.47, 128.52, 128.65, 128.68, 128.8, 128.9, 136.4, 136.5,
146.8, 147.1, 151.73, 151.75, 165.1, 165.3, 172.3; HR-ESITOFMS
m/z [M+Na]+ 787.2988 (calcd for C47H44N2O8Na 787.2990).
118.7, 119.50, 119.57, 119.65, 119.74, 147.3, 147.4, 150.0, 150.3,
171.1, 171.5, 174.4; IR mmax 3316, 1746, 1635, 1586, 1541 cmꢁ1
;
HR-ESITOFMS m/z [MꢁH]ꢁ 445.1614 (calcd for C22H25N2O8
445.1616).
4.3. Biological activity
4.3.1. Cytotoxicity assay
Cytotoxicity was assayed according to the procedure previously
described,2 with some modifications. Briefly, murine colon 26-L5
carcinoma cells were suspended (1 ꢀ 105 cells/mL) in RPMI med-
4.2.27. (S)-2,4-Bis[(2,3-dihydroxybenzoyl)amino]butyric acid
methyl ester [(S)-15]
ium containing 10% FCS, L-glutamine and the test compound at
In the same manner as described for 2, (S)-35 gave (S)-15 in 31%
various concentrations, and seeded into the wells of a 96-well cul-
yield: colorless amorphous solid; ½a D22
ꢂ
ꢁ 25:9 (c 0.1, MeOH); 1H
ture plate (1 ꢀ 104 cells/100
lL/well). After incubation for 24 h in a
NMR (400 MHz, CD3OD) d 2.16 (1H, m), 2.27 (1H, m), 3.45 (1H,
m), 3.61 (1H, m), 3.70 (3H, s), 4.78 (1H, dd, J = 8.5, 5.0 Hz), 6.71
(1H, t, J = 7.5 Hz), 6.75 (1H, t, J = 8.0 Hz), 6.92 (1H, dd, J = 8.0,
1.0 Hz), 6.96 (1H, dd, J = 7.5, 1.0 Hz), 7.19 (1H, dd, J = 8.0, 1.0 Hz),
7.35 (1H, dd, J = 7.5, 1.0 Hz); 13C NMR (100 MHz, CD3OD) d 32.1,
37.1, 51.8, 53.0, 116.68, 116.70, 118.7, 119.3, 119.6, 119.7, 119.9,
147.4, 150.2, 150.3, 171.4, 171.7, 173.8; IR mmax 3342, 1728,
1638, 1585, 1535 cmꢁ1; HR-ESITOFMS m/z [M+Na]+ 427.1112
(calcd for C19H20N2O8Na 427.1112).
humidified 5% CO2 incubator at 37 °C, the well was washed with
PBS and the cells were fixed with 20% formalin. Then, the wells
were washed with PBS and cells were stained with crystal violet
for 30 min. After washing with water, the crystal violet dye was ex-
tracted with 30% acetic acid and the absorbance at 590 nm was
measured. Solutions of test compounds were prepared in DMSO.
4.3.2. Invasion and migration assays
Invasion assay was carried out according to the procedure pre-
viously described.2 Murine colon 26-L5 carcinoma cells were used.
Solutions of test compounds were prepared in DMSO.
4.2.28. (S)-2,5-Bis[(2,3-dihydroxybenzoyl)amino]pentanoic acid
methyl ester [(S)-16]
In the same manner as described for 2, (S)-2,5-bis[(2,3-dib-
4.3.3. Cell adhesion assay
Cell adhesion assay was performed in 96-well microculture
plates coated with matrix proteins at a concentration of 50
enzyloxybenzoyl)amino]pentanoic acid methyl ester3 [(S)-36] gave
(S)-14 in 24% yield: colorless amorphous solid; ½a D22
ꢂ
ꢁ 2:9 (c 0.1,
lg/
MeOH); 1H NMR (400 MHz, CD3OD) d 1.66 (2H, m), 1.82 (1H, m),
1.95 (1H, m), 3.34 (2H, t, J = 6.8 Hz), 3.64 (3H, s), 4.60 (1H, d, J
=8.8, 5.2 Hz), 6.60 (1H, t, J = 8.0 Hz), 6.62 (1H, t, J = 8.0 Hz), 6.81
(1H, d, J = 8.0 Hz), 6.83 (1H, d, J = 8.0 Hz), 7.10 (1H, d, J = 8.0 Hz),
7.23 (1H, d, J = 8.0 Hz); 13C NMR (100 MHz, CD3OD) d 27.0, 29.9,
39.9, 52.8, 53.7, 116.7, 116.9, 118.7, 119.46, 119.52, 119.7, 147.4,
150.3, 150.5, 171.2, 171.7, 174.1; IR mmax 3376, 1728, 1639, 1587,
mL overnight and then treated with 100 L of RPMI medium con-
l
taining 10% FCS for 1 h to block the remaining protein binding
sites. The matrix proteins used were fibronectin and Matrigel. Mur-
ine colon 26-L5 cells were pre-treated with compounds (S)-1 or
(S)-6 in a humidified 5% CO2 incubator at 37 °C for 24 h. Then,
the cells were harvested and resuspended in RPMI medium con-
taining 10% FCS and L-glutamine, and seeded into a 96-well culture
1537 cmꢁ1
;
HR-ESITOFMS m/z [M+Na]+ 441.1266 (calcd for
plate at a density of 4 ꢀ 104 cells/well. The cells were allowed to
attach to the matrix proteins at 37 °C for 1 h. Non-adherent cells
were removed by vacuum aspiration. Adherent cells were washed
with PBS and fixed with 20% formalin at 4 °C for 1 h. Then, the
wells were washed with PBS and cells were stained with crystal
violet for 30 min. After washing with water, the crystal violet dye
was extracted with 30% acetic acid and the absorbance at
590 nm was measured.
C20H22N2O8Na 441.1268).
4.2.29. (S)-2,7-Bis[(2,3-dibenzyloxybenzoyl)amino]heptanoic
acid methyl ester [(S)-38]
In the same manner as described for 18, (S)-2,7-diaminohepta-
noic acid methyl ester [(S)-37], which was prepared from (S)-2-ami-
no-7-(tert-butoxycarboxamido)heptanoic acid methyl ester4, gave
(S)-38 in 28% yield: 1H NMR (400 MHz, CDCl3) d 1.03–1.20 (6H, m),
1.37 (1H, m), 1.62 (1H, m), 3.17 (2H, dt, J = 5.6, 6.0 Hz), 3.70 (3H, s),
4.61 (1H, ddd, J = 7.6, 7.2, 5.6 Hz), 5.07 (2H, s), 5.11 (1H, d,
J = 10 Hz), 5.150 (2H, s), 5.153 (2H, s), 5.16 (1H, d, J = 10 Hz), 7.12–
7.52 (24H, m), 7.72 (2H, m), 7.90 (1H, t, J = 5.6 Hz), 8.48 (1H, d,
J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 25.3, 26.6, 28.9, 31.8, 39.6,
52.2, 52.7, 71.30, 71.34, 76.1, 76.4, 116.9, 117.3, 123.3, 123.4,
124.43, 124.45, 127.7, 127.8, 128.27, 128.29, 128.48, 128.52,
128.67, 128.68, 128.71, 128.8, 136.32, 136.35, 136.42, 146.8, 148.0,
151.7, 151.8, 165.0, 165.1, 172.8; HR-ESITOFMS m/z [M+Na]+
829.3458 (calcd for C50H50N2O8Na 829.3459).
4.3.4. Gelatin-based zymography assay
Gelatinase inhibition assay was carried out according to the pro-
cedure previously described,12 with some modifications. Condi-
tioned media of murine colon 26-L5 carcinoma cells were
electrophoresed under non-reducing conditions on gelatin zymo-
gram containing 10% SDS–PAGE co-polymerized with 0.1% gelatin
(Sigma Chemical Co.). After electrophoresis, the gels were washed
twice with rinsing buffer (50 mM Tris–HCl, 2.5% Triton X-100,
5 mM CaCl2, 1 lM ZnCl2, 0.05% NaN3) at room temperature for 1 h
to remove SDS. Strips of the gel were incubated with the reaction
buffer for gelatinolytic activity (50 mM Tris–HCl, 5 mM CaCl2,
4.2.30. (S)-2,7-Bis[(2,3-dihydroxybenzoyl)amino]heptanoic acid
methyl ester [(S)-17]
1 lM ZnCl2, 0.05% NaN3) supplemented with various concentrations
of test compound at 37 °C. After incubation for 24 h, the gel strips
were stained with a staining solution (0.1% coomassie brilliant blue,
10% acetic acid, 10% 2-propanol). The locations of the enzymes were
visualized as clear bands on the blue background. Gelatinolytic
activities were quantified using Chemi Dos XRS System (Bio-rad).
In the same manner as described for 2, (S)-38 gave (S)-17 in 54%
yield: colorless amorphous solid; ½a D22
ꢂ
ꢁ 9:1 (c 0.1, MeOH); 1H NMR
(400 MHz, CD3OD) d 1.33 (4H, m), 1.53 (2H, m), 1.75 (1H, m), 1.82
(1H, m), 3.27 (2H, t, J = 7.2 Hz), 3.64 (3H, s), 4.53 (1H, dd, J = 8.8,
5.2 Hz), 6.60 (1H, t, J = 8.0 Hz), 6.63 (1H, t, J = 8.0 Hz), 6.81 (1H,
dd, J = 7.6, 1.2 Hz), 6.84 (1H, dd, J = 8.0, 1.2 Hz), 7.09 (1H, dd,
J = 8.0, 1.2 Hz), 7.23 (1H, dd, J = 7.5, 1.2 Hz); 13C NMR (100 MHz,
CD3OD) d 26.7, 27.5, 30.2, 32.3, 40.3, 52.8, 53.9, 116.8, 116.9,
4.3.5. In vivo lung metastasis
Specific pathogen-free 7-week-old female BALB/c mice were
purchased from Japan SLC, Hamamatsu, Japan. The mice were

2732
S. Miyanaga et al. / Bioorg. Med. Chem. 17 (2009) 2724–2732
maintained in the Laboratory for Animal Experiments, Institute of
Natural Medicine, University of Toyama under laminar air-flow
conditions. Colon 26-L5 cells were harvested with trypsin–EDTA,
washed with RPMI medium and resuspended in cold PBS. BALB/c
mice were given an intravenous injection of colon 26-L5 cells
lL) via the tail vein. Compound (S)-6 (1 mg/mouse/
200 lL in PBS) were administered orally or intraperitoneally once
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given 200 L of PBS. The mice were sacrificed after 14 days after
tumor inoculation. The lungs were fixed in Bouin’s solution and
the tumor colonies were counted under a dissecting microscope.
l
4.3.6. Statistical analysis
The statistical significance of differences between groups was
determined by applying the Student’s two-tailed t-test.
Acknowledgments
Authors thank to Dr. Yoko Ueno for assistance to animal exper-
iments. This research was partly supported by a Grant from Nagase
Science and Technology Foundation awarded to Y. Igarashi.