Y. Kanamori et al. / Tetrahedron Letters xxx (2016) xxx–xxx
3
tested in vitro. As shown in Table 2, 1 showed inhibitory activity
against chymotrypsin with an IC50 value of 33 ± 9 M (n = 3). The
l
inhibitory activity of 1 was much stronger than that of phenyl-
methylsulfonyl fluoride (PMSF) which was used as a positive con-
trol. Moreover, there was no apparent inhibition of elastase or
1
4
trypsin at 100
as symplocamide A, lyngbyastatins 4–10, and molassamide,
has been reported as a strong serine protease inhibitor (IC50
.5 nM–10 M). These compounds share the cyclic structure com-
lM. So far, a series of dolastatin 13 analogs, such
1
5
16
17
2
l
posed of six amino acid residues including 3-amino-6-hydroxy-2-
piperidone, and the previous research revealed the detailed struc-
ture activity relationships of dolastatin 13 type compounds based
1
8
on the X-ray structural analyses. Although the serine protease
inhibitory-activity of urumamide (1) is weaker than them, the
structure of urumamide (1) is completely different from dolastatin
Figure 1. Gross structure of urumamide (1) based on 2D NMR correlations.
1
3 analogs. Therefore, 1 can be expected to be a novel lead for pro-
tease inhibitors.
Table 2
IC50 values of urumamide (1) for serine protease activities in vitro
In conclusion, urumamide (1), a novel cyclic depsipeptide was
isolated from a marine cyanobacterium, Okeania sp. The structure
of 1 was established by spectroscopic analyses, Marfey’s method
and chiral HPLC analyses of acid hydrolysates. The structure of uru-
Serine proteases
IC50 (lM)
Chymotrypsin
Elastase
Trypsin
mamide (1) contains seven L-a-amino acids, one hydroxy acid, D-
Urumamide (1)
PMSF
33 ± 9
480 ± 230
>100
1300
>100
6900
Hiva, and one b-amino acid, (2S,3R)-Map. The stereochemistry of
Map is the same as that of previously reported peptides bearing
Map. Urumamide exhibited weak growth–inhibitory activity
against human cancer cells and inhibited chymotrypsin. To the
best of our knowledge, this is the first report to show that a
Map-containing peptide exhibits chymotrypsin–inhibitory activity.
Other Map-containing peptides may also exhibit the same activity.
follows. The presence of the
isovaleric acid residue (Hiva), was determined on the basis of the
low-field shifted signals of the -position (d 5.29, d 76.4). Mean-
while, the presence of the b-amino acid, Map, was established
based on the chemical shifts at the -position (d 2.46, d 45.7)
and the b-position (d 3.77, d 55.9).
a-hydroxy acid residue, a 2-hydroxy-
a
H
C
a
H
C
Acknowledgement
H
C
The sequences of these partial structures were determined
based on HMBC and NOESY data (Table 1 and Fig. 1). HMBC corre-
lations, NH of Val/C-1 of Pro, NH of Map/C-1 of Val, and NH of Leu/
C-1 of Map, connected the three residues: Val-Map-Leu. Moreover,
three HMBC correlations, H-2 of Hiva/C-1 of N-Me-Ile, N-Me of N-
Me-Ala/C-1 of Hiva, and N-Me of N-Me-Leu/C-1 of N-Me-Ala,
revealed the sequence as follows: N-Me-Ile—Hiva—N-Me-Ala-N-
Me-Leu. Finally, the NOESY correlations, H-2 of Leu/N-Me of N-
Me-Ile, H-2 of N-Me-Leu/N-Me of N-Me-Val, H-2 of N-Me-Val/H-
This work was supported by JSPS KAKENHI Grant Number
6H03285.
1
Supplementary data
Supplementary data ( H, 13C, COSY, NOESY, HMQC and HMBC
1
NMR spectra in CD OD for urumamide (1). HPLC chromatograms
3
for determination of the absolute configurations. A phylogenic tree
of the urumamide-producing cyanobacterium. Detailed experi-
2
of Pro, and H-2 of Pro/NH of Val, allowed us to determine the
gross structure of 1 as shown in Figure 1.
The absolute configuration of 1 was determined as follows. The
stereochemistry of all
form based on the results of Marfey’s analyses. Meanwhile, the
absolute stereochemistry of the -hydroxy acid, Hiva, was deter-
mined to be -form on the basis of chiral HPLC analyses.
a-amino acid residues was assigned to be L-
12
References and notes
a
D
1
With regard to Map, (2R,3R)- and (2S,3R)-Map were synthesized
as described elsewhere.13 Subsequently, the two Maps were
derivatized with
FDLA-(2R,3R)-Map,
-FDLA-(2S,3R)-Map. Meanwhile, we also prepared the
derivative of the Map derived from 1. The HPLC retention time of
the -FDLA derivative of the natural Map matched that of the
authentic standard, -FDLA-(2S,3R)-Map. Therefore, the stereo-
D/
L
-FDLA to afford four authentic standards:
-FDLA-(2R,3R)-Map, -FDLA-(2S,3R)-Map, and
-FDLA
D-
4
.
L
D
L
L
6
.
L
L
7
.
.
chemistry of the Map unit was determined to be 2S,3R.
The growth-inhibitory activities of urumamide against HeLa
and HL60 cells were evaluated by the MTT assay. The cells were
placed in 96-well plates and treated with various concentrations
8
1
of compounds (5–50
for 72 h. As a result, 1 showed weak growth–inhibitory activity
against HeLa and HL60 cells with IC50 values of 18 ± 0.5 M and
3 ± 0.5 M, respectively (n = 3).
In addition, 1 was evaluated for its ability to inhibit serine pro-
teases. The activities of chymotrypsin, elastase and trypsin were
lM for HeLa cells, 5–50 lM for HL60 cells)
1
1
1
l
1
l