Kendarimide A, a novel peptide reversing P-glycoprotein-mediated multidrug resistance in tumor cells, from a marine sponge of Haliclona sp.

Article abstract of DOI:10.1016/j.tet.2003.07.020

A novel modulator of multidrug resistance (MDR) in tumor cells, kendarimide A (1), was isolated from an Indonesian marine sponge of Haliclona sp. Compound 1 reversed MDR in KB-C2 cells mediated by P-glycoprotein (P-gp) at a 6 μM concentration, and the chemical structure of 1 was characterized to be a linear peptide composed of N-methylpyroglutamic acid (pyroMeGlu), N-methylated eight-membered cysteinyl-cysteine (ox-[MeCys-MeCys]) together with many N-methyl amino acid residues. The amino acid sequence of 1 was determined by 2D NMR and FAB MS analysis. The absolute configuration of the amino acid residues in 1 except for the MeCys part was determined to be L-form respectively, based on interpretation of the HPLC analysis of Marfey's derivatives of the hydrolysates of 1 and the synthetic method for the pyroMeGlu residue.

Full text of DOI:10.1016/j.tet.2003.07.020

Tetrahedron 60 (2004) 7053–7059
Kendarimide A, a novel peptide reversing
P-glycoprotein-mediated multidrug resistance in tumor cells,
from a marine sponge of Haliclona sp.
Shunji Aoki,a Liwei Cao,a Kouhei Matsui,a Rachmaniar Rachmat,b Shin-ichi Akiyamac
and Motomasa Kobayashia
,
*
aGraduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita, Osaka 565-0871, Japan
bResearch and Development Centre for Oceanology, LIPI, JL. Pasir Putih I, Ancol Timur, Jakarta 11048, Indonesia
cDepartment of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka,
Kagoshima 890-8520, Japan
Received 15 July 2003; revised 31 July 2003; accepted 31 July 2003
Available online 25 June 2004
Abstract—A novel modulator of multidrug resistance (MDR) in tumor cells, kendarimide A (1), was isolated from an Indonesian marine
sponge of Haliclona sp. Compound 1 reversed MDR in KB-C2 cells mediated by P-glycoprotein (P-gp) at a 6 mM concentration, and the
chemical structure of 1 was characterized to be a linear peptide composed of N-methylpyroglutamic acid (pyroMeGlu), N-methylated eight-
membered cysteinyl-cysteine (ox-[MeCys-MeCys]) together with many N-methyl amino acid residues. The amino acid sequence of 1 was
determined by 2D NMR and FAB MS analysis. The absolute configuration of the amino acid residues in 1 except for the MeCys part was
determined to be L-form respectively, based on interpretation of the HPLC analysis of Marfey’s derivatives of the hydrolysates of 1 and the
synthetic method for the pyroMeGlu residue.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
MDR from the Indonesian gorgonian Briareum excavatum.
We further isolated a peptide named kendarimide A (1)
exhibiting a reversing activity to P-gp-mediated MDR, from
an Indonesian marine sponge. Here we report the detail of
the structure analysis of kendarimide A (1).
Tumor cells, which acquired drug resistance to several
anticancer drugs having unrelated action mechanisms and
chemical structures, are called multidrug resistance (MDR)
cells. In many cases, the major mechanism of MDR is the
overexpression of the membrane glycoproteins as ATP-
dependent pumps, which excrete anticancer drugs in tumor
cells.1 P-glycoprotein (P-gp) encoded by the MDR1 gene,
which belongs to a super family of ATP-binding cassette
(ABC) transporters, has been observed in various MDR cell
lines and is closely related to drug resistance in clinical
treatment. Substances which inhibit the function of P-gp and
increase the concentration of antitumor agents in MDR cell,
are expected to have high potential to realize successful
cancer chemotherapy.2 In the course of our screening of
MDR-modulators from marine organisms, we isolated
agosterols3 as a potent reversing substance to both P-gp-
and multidrug resistance associated protein (MRP1)-
mediated MDR from a marine sponge of Spongia sp. and
brianthein A4 as a reversing substance to P-gp-mediated
2. Results and discussion
The MeOH extract of the titled dried sponge collected at
Sulawesi Island, Indonesia, was partitioned into an H2O–
EtOAc mixture (1:1). The EtOAc-soluble portion, which
showed a reversing activity against P-gp mediated MDR,
was subjected to silica gel flash column chromatography
(CHCl3–MeOH). The active MeOH eluate was further
separated by ODS column (Cosmosil 75C18-OPN, MeOH–
H2O) chromatography. The 90% aq MeOH eluate was
further purified by reversed-phase HPLC (Cosmosil
5C18AR, 90% aq MeCN; Cosmosil 5Ph, 86% aq MeOH)
to furnish kendarimide A (1) (16 mg, 0.0047% from the
dried sponge) as an active substance.
Keywords: Multidrug resistance; Marine sponge; Haliclona sp;
Kendarimide A; N-methyl amino acid.
Kendarimide A (1) was obtained as a colorless powder. The
positive ion FAB MS of 1 gave a quasi-molecular ion
[(MþH)þ] peak at m/z 1631 and the molecular formula of 1
*
Corresponding author. Tel.: þ81-6-6879-8215; fax: þ81-6-6879-8219;
e-mail address: kobayasi@phs.osaka-u.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.07.020

7054
S. Aoki et al. / Tetrahedron 60 (2004) 7053–7059
was determined as C83H135N14O15S2 by HR-FAB MS. The
1
presume the presence of L-pyroMeGlu including the
absolute configuration. Natural depsipeptides containing
pyroGlu, such as didemnins H, D, E, have been isolated
from tunicate,5 while a peptide containing N-methyl-
pyroglutamic acid (pyroMeGlu) has not been reported up
to now.
IR spectrum (nmax 1640 cm21, amide groups) and the H
and 13C NMR spectral data of 1 indicated it to be a highly
N-methylated lipophilic peptide. The 13C NMR and DEPT
spectra of 1 revealed the presence of two phenyl groups, one
oxymethylene carbon (dC 64.60), and 14 amide carbonyl
carbons (dC 175.79, 173.00, 172.88, 172.47, 172.35, 171.72,
170.68, 170.61, 170.51, 170.46, 170.44, 170.11, 170.07 and
From the detailed 2D NMR analysis of 1, the presence of
two continuous N-methylcysteine (MeCys) residues
adjacent to the phenylalaninol residue at the C-terminus
was revealed. Based on the HR-FAB MS analysis of 1 and 2,
the formation of the S–S bond was confirmed. The eight-
membered cysteinyl-cysteine (ox-[Cys-Cys]) connected by
a disulfide bond has been found in a few natural peptides
such as bicyclic malformin,6 catalytic domain of mercuric
ion reductase7 and nicotinic acetylcholine receptor,8 while
compound 1 was the first example of a peptide having
1
167.91). The H NMR spectrum of 1 displayed the signals
ascribable to two amide N-H protons (d 7.37 and 6.46) and
12 N-methyl groups (d 3.29, 3.06, 3.04, 3.03, 3.01, 3.001,
2.998, 2.986, 2.96, 2.95, 2.77 and 2.34). The detailed
analysis of the 2D NMR (COSY, HOHAHA, HMQC,
HMBC) spectra of 1 revealed the existence of 1 equiv. each
of N-methylisoleucine (MeIle), phenylalanine (Phe),
2 equiv. each of N-methylalanine (MeAla) and N-methyl-
cysteine (MeCys), and 6 equiv. of N-methylvaline (MeVal)
together with N- and C-terminal residues.
N-methylated
(ox-[MeCys-MeCys]) connected by a disulfide bond (Fig. 1).
eight-membered
cysteinyl-cysteine
The 1H and 13C NMR signals ascribable to the residue in the
C-terminus were similar to those of phenylalanine except
for the observation of an additional signal ascribable to an
oxymethylene group [(d 3.77, 3.60 (both 1H), dC 64.60)]
instead of the carboxyl group. Then, the residue in the
C-terminus was assigned to be phenylalaninol, while no
correlation was observed between the oxymethylene signal
and the other amino acid residue in the HOHAHA and
HMBC spectra of 1. In order to confirm the presence of the
terminal alcohol moiety, the pyridine–DMF solution of 1
was treated with phthalic anhydride and DMAP to furnish a
phthalic ester 2.
The amino acid sequence of kendarimide A (1) was
examined by NOESY experiment of 1, which gave
sequential information from the correlation between the
amide protons or N-methyl protons and a-protons of the
adjacent residue via the amide bond (Fig. 2). The same
resolution was deduced from the HMBC correlations
between the amide protons or N-methyl protons and its
own the a-protons and carbonyl carbons of the adjacent
residue, which were further correlated with a-protons of the
adjacent residue (Fig. 2).
The amino acid sequence of 1 was further confirmed by
FAB MS analysis of 1. Thus, the positive ion FAB MS of 1
provided many fragment ion peaks containing the
N-terminus, which were consistent with the amino acid
sequence of 1 as shown in Figure 3. The assignment of the
fragment ions of 1 was further confirmed in comparison
with the fragmentation of the phthalic ester 2, which gave a
similar fragmentation in the positive ion FAB MS together
with the quasi-molecular ion ([MþNa]þ) peak at m/z 1802,
while the negative ion FAB MS of 2 did not give the
expected fragment ion peaks containing the C-terminus.
On the other hand, the residue in the N-terminus of 1 was
proposed to be N-methylglutamic acid (MeGlu); however,
the HMBC correlation between N-Me (d 2.77) and d-CO (dC
175.79) assignable to the N-terminal residue indicated the
presence of an intramolecular amide bond. Thus, the residue
in the N-terminus was presumed to be not MeGlu but
N-methylpyroglutamic acid (pyroMeGlu). However, the
signals ascribable to b- and g-protons of the pyroMeGlu
residue were observed to be close to each other and it was
difficult to confirm the structure of pyroMeGlu. Then, we
synthesized a model compound 5 as shown in Scheme 1.
The 1H and 13C signals assignable to the pyroMeGlu part in
5 were closely similar to those of 1. Consequently, we could
In order to determine the absolute configuration of the
amino acid sequence, 1 was hydrolyzed by 6 N HCl at
Scheme 1. Synthesis of L-pyroMeGlu-L-Phe-OEt. Conditions a: H2O, 132 8C, 1.9 kg/cm2 (autoclave), b: NaHCO3, C2H5I, DMF, 98%, c: TFA, CH2Cl2,
d: L-pyroMeGlu, DEPC, TEA, DMF, 0 8C.

S. Aoki et al. / Tetrahedron 60 (2004) 7053–7059
7055
Figure 1. Chemical structure of kendarimide A (1).
110 8C for 20 h. Marfey’s derivatives9 of the resulting
hydrolysates were analyzed by HPLC to reveal the presence
of one each of L-MeGlu, L-Phe, L-MeIle, L-phenylalaninol,
and two MeAla and six L-MeVal. L-PyroMeGlu was
converted to L-MeGlu under this hydrolysis condition.
Thus, Marfey’s method was applicable to determine the
absolute configuration of phenylalaninol. However,
N-methylcysteines existing as an eight-membered disulfide
form (ox-[MeCys-MeCys]) could not be analyzed by
Marfey’s method because of decomposition and racemiza-
tion during acidic hydrolysis. Consequently, we were able to
determine the absolute stereostructure of kendarimide A (1)
except for the absolute configuration of two MeCys. The
absolute configuration of two N-methylcysteine residues is
under investigation by a synthetic method.
Kendarimide A (1) completely reversed the resistance to
colchicine in KB-C2 cells,10 human carcinoma cell line
overexpressing P-gp, at a 6 mM concentration. Thus, the
6 mM of 1 showed a 87% growth inhibition against KB-C2
cells in the presence of 0.1 mg/mL of colchicine, while 1
showed no inhibitory activity against KB-3-1 cells at a
6 mM concentration. Interestingly, cyclosporin A,11 a potent
MDR modulator against P-gp, is also a peptide composed of
Figure 2. 2D NMR correlations for kendarimide A (1).

7056
S. Aoki et al. / Tetrahedron 60 (2004) 7053–7059
Figure 3. Fragment ion peaks observed in positive ion FAB MS for kendarimide A (1).
11 amino acid residues including many N-methyl residues.
The similarity of these compounds may be related to the
MDR reversing activity.
with EtOAc. The organic layer was washed with 0.5 N aq
HCl and brine, dried over MgSO4 and then evaporated in
reduce pressure. The crude product was purified by
reversed-phase HPLC (Mightysil RP-18 4.6f£150 mm,
85% MeCN) to give phthalic ester 2. Compound 2: positive
ion FAB MS: m/z 1802 (MþNa)þ, 1248, 1135, 1022, 937,
824, 711, 598, 485, 400. HR-FAB MS: obsd; m/z
1802.9645. Calcd for C91H138N14NaO18S2; m/z 1802.9653
(MþNa)þ.
3. Experimental
3.1. General
3.1.1. Isolation of kendarimide A (1). The titled dried
sponge (350 g) collected at Sulawesi Island, Indonesia, was
extracted with MeOH (3£1 L) to obtain a MeOH extract
(36 g). The MeOH extract was partitioned into an H2O–
EtOAc mixture (1:1), and the EtOAc layer was evaporated
to afford an EtOAc extract (4.2 g). The EtOAc extract,
which showed MDR reversing activity, was subjected to
silica gel flash column chromatography (CHCl3–MeOH
and hexane–EtOAc). The active fraction was further
separated by ODS column (Cosmosil 75C18-OPN,
MeOH–H2O) chromatography and finally purified by
reversed-phase HPLC (Cosmosil 5C18AR 10f£250 mm,
90% aq MeCN; Cosmosil 5Ph 10f£250 mm, 86% aq
MeOH) to furnish kendarimide A (1) (16 mg, 0.0047% from
the dried sponge).
3.2. Determination of the absolute configurations of
amino acids
(1) Acid hydrolysis of kendarimide A (1): compound 1
(1.0 mg) was treated with 0.5 mL of 6 N aq HCl and heated
at 110 8C for 20 h. The reaction mixture was evaporated and
dried under reduced pressure.
(2) HPLC analysis of Marfey’s derivatives. The H2O
solution (50 mL) of the above hydrolysates was treated
with 350 mL of 1-fluoro-2,4-dinitrophenyl-5-L-leucine
amide (L-FDLA) in acetone (10 mg/mL) and 155 mL of
1 M aq Na2CO3 and the mixture was kept at 37 8C for 6 h.
The 2 aq HCl (77 mL) was added, and the resulting solution
was diluted with 400 mL of MeCN. An aliquot (5 mL) of the
L-FDLA derivatives was analyzed by reversed-phase HPLC
with two different conditions as shown below.
Kendarimide A (1): [a]2D5¼22738 (MeOH, c¼0.3); IR
(KBr) nmax 3354, 3319, 1640 cm21; positive ion FAB MS:
m/z 1631 (MþH)þ, see in Figure 3 for the fragment ion.
HR-FAB MS: obsd; m/z 1631.9708. Calcd for
C83H135N14O15S2; m/z 1631.9743 (MþH)þ. 1H NMR
(600 MHz) and 13C NMR (150 MHz) data in CDCl3 are
given in Table 1.
(a) A linear gradient from 0.1 M aq CH3COONH4 (contain-
ing 0.25% TFA)/MeCN (85:15) to 0.1 M aq CH3COONH4
(containing 0.25% TFA)/MeCN (55:45) over 45 min (flow
rate: 1.0 mL/min) was used to separate the L-FDLA
derivatives, which were detected by UV absorption at
340 nm. The system was allowed to equilibrate for 30 min at
15% MeCN prior to the next analysis. Mightysil RP-18
column (4.6f£150 mm) was used for separation and
maintained at 30 8C.
3.1.2. Phthalic ester 2 of kendarimide A (1). The DMF
(20 mL) solution of 1 (0.5 mg, 0.3 mmol) was treated with
phthalic anhydride (0.2 mg, 3 equiv.), DMAP (0.1 mg,
3 equiv.), and pyridine (5 mL) and stirred for 21 h at room
temperature. After removal of the solvent under reduced
pressure, the residue was dissolved in H2O and extracted
(b) A linear gradient from 0.5% aq TFA/MeCN (70:30) to

S. Aoki et al. / Tetrahedron 60 (2004) 7053–7059
7057
Table 1. NMR data for kendarimide A (1) in CDCl3
d
H (J in Hz)a
C/H no.
dHa (J in Hz)
dC
b
dC
b
C/H no.
pyroMeGlu
1
2
3a
3b
4a
4b
5
NMe
Phe
1
2
3a
3b
4
5/9
6/8
7
NH
MeIle
1
2
3
3-Me
4
4
NMe
0.74, d (6.9)
2.998c, s
17.71m
30.37i
—
3.88, dd (8.7, 4.3)
170.68
63.51
23.21
—
29.24
—
MeVal4
2.31
1.77, m
2.29
1
2
170.51g
58.33
5.13, d (11)
2.33
0.85 (overlapped)
0.81, d (6.6)
2.986,s
3
3-Me
4
NMe
MeAla2
26.96
2.35
19.37k
18.13l
30.00h
175.79
28.94
2.77, s
171.72
49.93
38.63
—
135.43
129.16d
128.53e
127.36
—
1
2
172.47
49.15
14.82
30.71
5.25, dd like
3.09 (overlapped)
2.86 (overlapped)
5.40, q (6.9)
1.24, (d, 6.9)
3.001c, s
3
NMe
MeVal5
7.15, m
7.28, m
7.25, m
1
2
170.61
58.39
27.04
19.27
18.18l
30.02h
5.11, d (11)
2.31
0.87 (overlapped)
0.81, d (6.6)
3.01, s
3
3-Me
6.46, (d, 8.7)
4
Nme
170.07f
56.84
33.21
15.20
24.14
10.69
30.24
5.21, d (11)
2.09, m
0.81, d (6.6)
1.24 (overlapped)
0.84, t (7.7)
2.95, s
MeVal6
1
2
172.88
57.78
27.72
19.50
17.98
30.30
5.28, d (11)
2.27
0.96, d (6.5)
0.77, d (6.5)
2.96, s
3
3-Me
4
Nme
MeCys1
1
2
3a
5
NMe
MeAla1
1
2
172.35
49.31
15.04
30.65
5.42, q (6.9)
1.23, (d, 6.9)
3.06, s
173.00
56.63
38.80
—
3
NMe
5.56, dd like
3.23, dd (14, 12)
2.86 (overlapped)
3.29, s
MeVal1
3b
Nme
1
2
170.44g
58.20j
27.35
19.68
17.66m
30.43
32.50
5.18, d (11)
2.32
0.86 (overlapped)
0.74, d (6.9)
3.04, s
MeCys2
3
3-Me
1
2
3a
167.91
59.25
37.17
—
5.23, dd like
3.33, dd (14, 3.8)
2.85 (overlapped)
2.34, s
4
NMe
3b
Nme
Phenylalaninol
MeVal2
28.01
1
2
170.11f
58.07
5.19, d (11)
2.32
0.85 (overlapped)
0.73, d (6.8)
3.03, s
1a
1b
2
3a
3b
4
5/9
6/8
7
3.77, dd (11, 3.0)
3.60, dd (11, 4.1)
4.28, m
2.95 (overlapped)
2.83 (overlapped)
—
7.21, m
7.26, m
7.20, m
7.37, d (8.7)
64.60
—
52.94
37.02
—
137.83
129.20d
128.60e
126.55
—
3
3-Me
27.20
19.41k
17.73m
30.35i
4
NMe
MeVal3
1
2
170.46g
58.22j
27.20
5.17, d (11)
2.33
0.86 (overlapped)
3
3-Me
19.37k
NH
a
Recorded at 600 MHz.
Recorded at 150 MHz.
b
c–m Assignment may be interchangeable for items with identical superscript letters c–m.
0.5% aq TFA/MeCN (30:70) over 45 min (flow rate:
0.22 mL/min) and then to 0.5% aq TFA/MeCN (10:90)
over 10 min was also used to separate the L-FDLA
derivatives. The system was allowed to equilibrate for
80 min at 30% aq MeCN prior to the next analysis. YMC
AS-3C2-3 column (2.0£150 mm) was used for separation
and maintained at 30 8C.
500 MHz, d): 8.95 (1H, s), 7.16–7.07 (5H, m), 5.77 (1H,
s), 3.99 (1H, m), 3.86 (1H, m), 3.60 (1H, dd, J¼11, 4.5 Hz),
3.53 (1H, dd, J¼11, 4.5 Hz), 2.91 (1H, dd, J¼14, 6.5 Hz),
2.82 (1H, dd, J¼14, 6.5 Hz), 1.75 (3H, m), 0.95 (3H, d,
J¼5.5 Hz), 0.88 (3H, d, J¼5.5 Hz).
3.2.2. D-Phenylalaninol-L-FDLA. 1H NMR (CDCl3,
500 MHz, d): 8.99 (1H, s), 7.22–7.07 (5H, m), 5.59 (1H,
s), 3.82 (2H, m), 3.60 (1H, dd, J¼11.5, 4.5 Hz), 3.56 (1H,
dd, J¼11.5, 4.5 Hz), 2.96 (1H, dd, J¼14, 6 Hz), 2.87 (1H,
dd, J¼14, 8 Hz), 1.72 (3H, m), 0.96 (3H, d, J¼6 Hz), 0.88
(3H, d, J¼6 Hz).
Standard amino acids were derivatized as described above.
The each peak was identified by co-injection with the
respective FDLA derivative of L- or D-amino acid.
Resulting data are given in Table 2.
3.2.1. L-Phenylalaninol-L-FDLA. 1H NMR (CDCl3,
3.2.3. Synthesis of L-N-methylpyroglutamic acid (3). A

7058
S. Aoki et al. / Tetrahedron 60 (2004) 7053–7059
b-H of Glu), 1.27 (3H, t, J¼7.5 Hz, Et). 13C NMR (CDCl3,
125 MHz, dc): 175.81 (d-C of Glu), 171.26 (CO of Phe),
170.92 (CO of Glu), 135.77, 129.08, 128.56, 127.22, 63.60
(a-C of Glu), 61.71 (Et), 52.58 (a-C of Phe), 37.72 (b-C of
Phe), 29.23 (g-C of Glu), 28.91 (N-Me), 23.09 (b-C of Glu),
14.08 (Et).
Table 2. HPLC analysis of the Marfey’s derivatives of the hydrolysates of
kendarimide A (1)
Amino acid
tR of standard (min)
Amino acid found by
co-injection
Condition (a) Condition (b)
L-MeAla
D-MeAla
L-MeVal
D-MeVal
L-Phe
30.13
32.65
35.53
44.69
35.28
37.63
20.43
24.47
39.68
49.73
42.97
44.25
51.16
57.28
52.29
59.79
22.87
25.20
54.89
60.94
53.16
59.71
U
U
U
U
U
U
3.3. Bioassay
Human epidermoid carcinoma KB cells (KB-3-1) were used
as the parental cell line for the present study. KB-3-1 cells
were cultured in RPMI 1640 medium with 0.44 mg/mL of
glutamine, 50 mg/mL of kanamycin sulfate, supplemented
with 10% newborn calf serum. MDR cell line, KB-C2,10
was selected from KB-3-1 cells and maintained in the
medium containing 2 mg/mL of colchicine. Reversing
activity and cytotoxicity were measured by means of
MTT colorimetric assay performed in 96-well plates.
Equal numbers of cells (10,000) were inoculated into each
well with 100 mL of the culture medium. After 24 h
preincubation (37 8C, 5% CO2), a 50 mL solution of
colchicine and testing sample were added to each well and
the whole were further incubated for 48 h. Thereafter,
25 mL of MTT solution (2 mg/mL in PBS) was added to
each well and incubated for further 3 h. After removing the
medium by aspiration, the resulting formazan was dissolved
in 200 mL of dimethylsulfoxide. The percentage of cell
growth inhibition was calculated from the absorbance at
540 nm. The cytotoxic activity of the testing sample was
also examined by MTT assay using parental KB 3-1 cells.
D-Phe
L-MeGlu
D-MeGlu
L-MeIle
D-MeIle
L-Phenylalaninol 54.55
D-Phenylalaninol 64.15
H2O solution of L-N-methylglutamic acid (50 mg/100 mL)
was autoclaved at 132 8C for 60 min. Water was evaporated
under reduced pressure to give L-N-methylpyroglutamic
acid (3). L-N-Methylpyroglutamic acid (3): FAB MS: m/z
144 (MþH)þ.
3.2.4. Synthesis12 of N-Boc-L-Phe-OEt (4). To a suspen-
sion of N-Boc-L-Phe-OH (3) (265 mg, 1 mmol) and sodium
hydrogen carbonate (168 mg, 2 mmol) in 5 mL of DMF,
ethyl iodide (0.4 mL, 5 mmol) was added at room
temperature. The reaction mixture was stirred for 24 h,
and then partitioned into H2O–EtOAc mixture. The organic
phase was washed with H2O, dried over sodium sulphate,
evaporated under reduced pressure, and the crude product
was purified by silica gel column chromatography [hexane–
EtOAc (8:2)] to furnish N-Boc-L-Phe-OEt (4) (286 mg,
98%). Compound 4: FAB MS, m/z 294 (MþH)þ. 1H NMR
(CDCl3, 500 MHz, d): 7.21 (5H, m), 7.09 (2H, m), 4.95 (1H,
brd), 4.51 (1H, m), 4.11 (2H, q, J¼7.5 Hz), 3.04 (2H, d,
J¼6 Hz), 1.37 (9H, s), 1.18 (3H, t, J¼7.5 Hz).
Acknowledgements
The authors are grateful to Prof. R.W.M. Soest, Zoologisch
Museum, University of Amsterdam, for identification of the
sponge. The authors are also grateful to the Takeda Science
Foundation, the Houansha Foundation, the Tokyo Bio-
chemical Research Foundation, the Uehara Memorial
Foundation and the Ministry of Education, Culture, Sports,
Science, and Technology of Japan for financial support.
3.2.5. Synthesis of L-pyroMeGlu-L-Phe-OEt (5). To a
solution of 4 (50 mg, 0.17 mmol) in CH2Cl2 (1.5 mL) at
0 8C, TFA (1.5 mL) was added dropwise. The mixture was
stirred at 0 8C for 2 h, and then concentrated in reduced
pressure. The solution of the resulting amine salt and L-N-
methylpyroglutamic acid (2) (25 mg, 0.17 mmol) in DMF
(1 mL) was treated with diethyl phosphorocyanidate13
(DEPC) (34 mL, 0.23 mmol) at 0 8C. After 15 min,
triethylamine (TEA) (49 mL, 0.36 mmol) was added, and
the mixture was stirred at 0 8C for 16 h. The reaction
mixture was partitioned into EtOAc/benzene–H2O mixture,
and the organic layer was washed with ice-cold 1 M aq
KHSO4 solution, sat. NaHCO3 solution, and brine, dried
over NaSO4, and concentrated in reduced pressure.
The crude product was purified by silica gel column
chromatography (hexane/EtOAc¼1:2 then EtOAc) to give
L-pyroMeGlu-L-Phe-OEt (5) as a white solid (38 mg, 81%).
Compound 5: [a]2D5¼211.68 (MeOH, c¼0.253). HR-FAB
MS: obsd; m/z 319.1673. Calcd for C17H23N2O4; m/z
319.1658 (MþH)þ. 1H NMR (CDCl3, 500 MHz, d):
7.26–7.23 (3H, m), 7.12 (2H, m), 6.41 (1H, d, J¼8 Hz,
NH), 4.91 (1H, dd-like, a-H of Phe), 4.20 (2H, q, J¼7.5 Hz,
Et), 3.88 (1H, brs, a-H of Glu), 3.24 (1H, dd, J¼14, 5 Hz,
b-H of Phe), 3.01 (1H, dd, J¼14, 8 Hz, b-H of Phe), 2.76
(3H, s, N-Me), 2.27 (3H, m, b,g-H of Glu), 1.73 (1H, t-like,
References and notes
1. Horio, M.; Gottesman, M. M.; Pastan, I. Prod. Natl. Acad. Sci.
U.S.A. 1988, 85, 3580–3584.
2. Krishna, R.; Louis, St., M.; Mayer, L. D. Int. J. Cancer 2000,
85, 131–141.
3. (a) Aoki, S.; Yoshioka, Y.; Miyamoto, Y.; Higuchi, K.;
Setiawan, A.; Murakami, N.; Chen, Z. S.; Sumizawa, T.;
Akiyama, S.; Kobayashi, M. Tetrahedron Lett. 1998, 39,
6303–6306. (b) Aoki, S.; Setiawan, A.; Yoshioka, Y.;
Higuchi, K.; Fudetani, R.; Chen, Z. S.; Sumizawa, T.;
Akiyama, S.; Kobayashi, M. Tetrahedron 1999, 55,
13965–13972. (c) Aoki, S.; Chen, Z. S.; Higashiyama, K.;
Setiawan, A.; Akiyama, S.; Kobayashi, M. Jpn. J. Cancer Res.
2001, 92, 886–895.
4. Aoki, S.; Okano, M.; Matsui, K.; Itoh, T.; Satari, R.; Akiyama,
S.; Kobayashi, M. Tetrahedron 2001, 57, 8951–8957.
5. Boulanger, A.; Abou-Mansour, E.; Badre, A.; Banaigs, B.;

S. Aoki et al. / Tetrahedron 60 (2004) 7053–7059
7059
Combaut, G.; Francisco, C. Tetrahedron Lett. 1994, 35,
4345–4348.
10. Akiyama, S.; Fojo, J. A.; Hanover, J. A.; Pastan, I. H.;
Gottesman, M. M. Somat. Cell Mol. Genet. 1985, 11,
117–126.
6. Bodanszky, M.; Stahl, G. L. Prod. Natl. Acad. Sci. U.S.A.
1974, 71, 2791–2794.
11. Hait, W. N.; Stein, J. M.; Koletsky, A. J.; Harding, M. W.;
Handschumacher, R. E. Cancer Commun. 1989, 01, 35–43.
12. Bocchi, V.; Casnati, G.; Dossena, A.; Marchelli, R. Synthesis
1979, 961–962.
7. Schiering, N.; Kabsch, W.; Moore, M. J.; Distefano, M. D.;
Walsh, C. T.; Pai, E. F. Nature 1991, 352, 168–172.
8. Creighton, C. J.; Reynolds, C. H.; Lee, D. H. S.; Leo, G. C.;
Reitz, A. B. J. Am. Chem. Soc. 2001, 123, 12664–12669.
9. (a) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
(b) Fujii, K.; Ikai, Y.; Oka, H.; Susuki, M.; Harada, K. Anal.
Chem. 1997, 69, 5146–5151.
13. Takuma, S.; Hamada, Y.; Shioiri, T. Chem. Pharm. Bull. 1982,
30, 3147–3153.