New analogue of arenastatin A, a potent cytotoxic spongean depsipeptide, with anti-tumor activity

Article abstract of DOI:10.1016/j.bmcl.2004.02.080

Two analogues possessing steric hindered substituents on C-15 of arenastatin A (1), a potent cytotoxic spongean depsipeptide, were synthesized and shown to enhance stability in mouse serum. Notably, 15-tert-butylanalogue (6) with higher cytotoxicity exhib

Full text of DOI:10.1016/j.bmcl.2004.02.080

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2597–2601
New analogue of arenastatin A, a potent cytotoxic spongean
depsipeptide, with anti-tumor activity
Nobutoshi Murakami,a Satoru Tamura,a Kouhei Koyama,a Masanori Sugimoto,a
Ryuji Maekawab and Motomasa Kobayashia,*
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
bShionogi Research Laboratories, Shionogi & Co., Ltd, 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
Received 21 January 2004; accepted 18 February 2004
Abstract—Two analogues possessing steric hindered substituents on C-15 of arenastatin A (1), a potent cytotoxic spongean dep-
sipeptide, were synthesized and shown to enhance stability in mouse serum. Notably, 15-tert-butylanalogue (6) with higher cyto-
toxicity exhibited in vivo anti-tumor activity through iv administration different from 1. Additionally, conformation analysis among
the two analogues and arenastatin A (1) indicated that the torsion angle from C-14 to C-20 is a conclusive factor for the potent
cytotoxicity of 1.
ꢀ 2004 Elsevier Ltd. All rights reserved.
In the course of our search for new biologically active
principles from marine metabolites, arenastatin A (1)
was isolated from the marine sponge Dysidea arenaria as
a minute amount of a substance with extremely potent
cytotoxicity against a tumor cell line (IC50: 5 pg/mL, KB
cells).1 Thereafter, we achieved the total synthesis of 12
and elucidated the cytotoxicity of 1 to be ascribable to
inhibition of microtubule assembly.3 However, arenast-
atin A (1) displayed little in vivo anti-tumor activity
through intravenous (iv) administration because of its
lability in mouse serum. Furthermore, synthesis of the
three amide analogues revealed the 15,20-ester linkage in
1 to be cleaved in mouse serum,4 indicative of the sig-
nificant reduction of biological potency in vivo. On the
basis of this metabolic behavior, we next synthesized the
carbaanalogue (3) and the deoxoanalogue (4) in antici-
pation of potent cytotoxicity as well as robustness in
serum. In spite of acquisition of nearly complete sta-
bility in serum for 3 and 4, their cytotoxic activities were
fairly weaker than that of 1.5;6 This finding discloses that
the 15,20-ester group in 1 must be essential for the
potent cytotoxicity of 1. In this context, we considered
that steric hindered substituents on C-15 would prevent
metabolism of the ester function. This report describes
15-tert-butylanalogue (6) of arenastatin A (1) not only
shows enhanced stability in serum but also displays in
vivo anti-tumor activity (Chart 1).
In order to protect the 15–20 ester function of arenast-
atin A (1) from metabolism in blood, one way would be
to add steric hindrance in the neighborhood of this
function. In our previous study7 of the structure–activity
relationship of 1, each of the synthetic epimers of 1 at
the position of 7,8-epoxide, 6-methyl, and OMe-tyrosine
did not show any cytotoxicity at concentration below
0.1 lg/mL. On the other hand, 15-epi-arenastatin A,8
which has been synthesized by the method7 using
D
-leucine, was shown to exhibit moderate cytotoxicity
(IC50 ¼ 20 ng/mL). Therefore, exchange of the isobutyl
group at the C-15 position for bulky substituents was
assumed to keep the reduction of activity to a minimum.
According to this working hypothesis, we designed the
15-i-propylanalogue (5) and the 15-tert-butylanalogue
(6). The synthetic protocol for 5 and 6 was the same as
for the synthesis of 1, as shown in Scheme 1. Namely,
introduction of the 7,8-epoxy portion was carried out in
the final stage and a precursory cyclic depsipeptide was
constructed from the four segments A–D. As the start-
ing material for the synthesis of segment C, (S)-leucinic
acid for 1 was replaced with 2-(S)-hydroxy-3-meth-
ylbutylic acid (7) for 5 and 3,3-dimethyl-2-(S)-hydroxy-
butylic acid (8) for 6, respectively.
After protecting the carboxyl group in the segment C (7
or 8) as benzyl esters (9 or 10), condensation of 9 or 10
* Corresponding author. Tel.: +81-6-6879-8215; fax: +81-6-6879-8219;
e-mail: kobayasi@phs.osaka-u.ac.jp
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.080

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2597–2601
13
8
1
5
O
O
7
25
O
O
O
O
O
HN
HN
O
O
15
HN
24
O
OMe
X
HN
O
OMe
R
22
O
Y
20
arenastatin A (1) : X = Y = O
15-i-propylanalogue (5) : R = CH(CH3)2
15-t-butylanalogue (6) : R = C(CH3)3
triamideanalogue (2) : X = NH, Y = O
carbaanalogue (3) : X = CH2, Y = O
deoxoanalogue (4) : X = O, Y = H2
Chart 1.
O
R
OH
O
R
OH
O
O
OBn
OH
b, c
a
O
OH
NHBoc
R
segment C
HO
NHBoc
O
9 : R = CH(CH3)2
10 : R = C(CH3)3
7 : R = CH(CH3)2
8 : R = C(CH3)3
segment D (11)
segment C-D
12 : R = CH(CH3)2
13 : R = C(CH3)3
O
d
e, f, g
O
R
O
O
HN
O
O
O
OMe
OH
HN
O
O
TMS
TMS
O
OMe
NHBoc
15 : R = CH(CH3)2
16 : R = C(CH3)3
segment A-B (14)
O
O
O
O
O
O
HN
HN
O
R
O
HN
HN
h
R
O
OMe
O
O
OMe
O
O
17 : R = CH(CH3)2
18 : R = C(CH3)3
15-i-propylanalogue (5) : R = CH(CH3)2
15-t-butylanalogue (6) : R = C(CH3)3
Scheme 1. Synthesis of 15-i-propylanalogue (5) and 15-tert-butylanalogue (6). Reagents and conditions: (a) BnBr, NaHCO3, TBAI, CH2Cl2–H2O;
(b) 11, EDCIÆHCl, DMAP, CH2Cl2; (c) H2, Pd–C, MeOH, 66% (12, three steps), 89% (13, three steps); (d) 14, EDCIÆHCl, DMAP, CH2Cl2, quant.
(15), 69% (16); (e) TFA, CH2Cl2, 0 ꢁC; (f) HCl–Et2O; (g) DPPA, NaHCO3, DMF, 0 ꢁC, 64% (17), 48% (18); (h) dimethyldioxirane, CH2Cl2, 0 ꢁC,
65% (5), 52% (6).
and the segment D (11) in the presence of 4-dimethyl-
aminopyridine (DMAP) and 3-(3-dimethylaminopro-
pyl)-1-ethylcarbodiimide hydrochloride (EDCIÆHCl)
followed by deprotection of the benzyl ester by use of
Pd–C under hydrogen atmosphere gave a segment C–D
(12 or 13) in 66% or 89% yield for three steps. Subse-
quently, the segments A–B (14) and C–D (12 or 13) were
connected by using the same method to afford a coupled
product (15 or 16) quantitatively or in 69% yield,
respectively. Cleavage of the two protective groups of 15
or 16 followed by HCl treatment gave a seco amino acid
as an HCl salt, which was further subjected to intra-
molecular macrolactamization using diphenylphospho-
rus azide (DPPA) and NaHCO3 to furnish a cyclic
depsipeptide (17 or 18) in 64% or 48% yield, respec-
tively. Finally, epoxidation of 17 was successfully
accomplished by using dimethyldioxirane to afford a
15-i-propylanalogue (5)9 in 65% yield as a major
product. Similarly, the 15-tert-butylanalogue (6)10 was
also prepared from 18 in 52% yield.
Assessment of stability of the two synthesized analogues
(5, 6) in mouse serum was carried out in the same
manner as that in our previous report4 and the result is
shown in Figure 1. Although both 5 and 6 were grad-
ually metabolized, they were shown to be more stable
than 1. Notably, 5 and 6 displayed moderate cytotox-
icities against KB-3-1 cells (IC50; 5: 30 ng/mL, 6: 10 ng/
mL), respectively.11
Next, the 15-tert-butylanalogue (6) with both higher
stability and stronger cytotoxicity was assessed for anti-
tumor activity in vivo against Lewis lung carcinoma
subcutaneously (sc) implanted in mice.12 As shown in

N. Murakami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2597–2601
2599
examined by using SYBYL 6.5 (Tripos Ass., St. Louis,
MO) in the same manner as our previous report.6 NOE
cross peaks in the 1H NMR spectra of 5 or 6 were
classified into three classes depending on their intensities
as strong, medium, or weak and then translated into
distance restraints (upper bound of distance: 2.8, 3.5,
ꢀ
and 5.0 A), respectively. Consequently, an initial struc-
ture was built with the aid of the NOE data with a
complete random array of atoms and refined by energy
minimization using the BFGS method.13 After this
refined structure was subjected to 50 cycles of molecular
dynamic calculation, the 50 structures obtained were
refined by energy minimization once again. Among the
resulting conformers, acceptable conformers were
selected on the basis of criteria that torsion angles of the
amide linkages and enone moieties were within 15ꢁ.
The average value of pairwise root-mean-square dis-
tance deviation (rmsd) for the backbone (16-membered
ring) heavy atoms and the restraint violations of are-
nastatin A (1) and the two analogues (5, 6) are sum-
marized in Table 2. Each compound exhibited not only
good convergence on the 16-membered ring portions but
also adequate compatibility with NOE information.
Because of the crucial participation of the two ester
linkages in 1 for the potent cytotoxicity, we compared
the conformation of the 16-membered ring portion of
each compound focusing on the two torsion angles (C4–
C5–O–C14 and C14–C15–O–C20).
Figure 1. Stability of 15-i-propylanalogue (5) and 15-tert-butylana-
logue (6). Each sample (10 lL of 0.1 mg/mL solution) was treated with
fresh mouse serum (100 lL) and incubated at 37 ꢁC for 0, 10, 30, 60,
and 300 min, respectively. After extraction of the reaction mixture with
EtOAc, each extract was analyzed by reversed-phase HPLC to deter-
mine the remaining amounts of 1, 5, and 6, respectively.
In each compound, a characteristic cluster consisting of
predominant conformers appeared as depicted in Figure
2. Furthermore, the percentage abundance of conform-
ers belonging to each cluster was estimated by Boltz-
mann distribution. Apparently, the two analogues (5, 6)
adopted the torsion-1 angle around ꢀ70ꢁ different from
arenastatin A (1). This conformational feature can ac-
count for the difference in the cytotoxic scores of 1, 5,
and 6. On the other hand, the lower ratio of the con-
former similar to 1 and the presence of a second domi-
nant cluster with respect to 5 may give rise to weaker
cytotoxicity than that of 6.
Table 1, 15-tert-butylanalogue (6) reduced tumor vol-
ume in a dose-dependent manner through iv adminis-
tration. It is noteworthy that 6 significantly inhibited the
growth of tumor volume at the dose of 10 mg/kg without
any signs of acute toxicity such as body weight loss,
diarrhea, and mortality.
Previously, conformation analysis of the four com-
pounds (1–4) by molecular dynamic calculation with
distance restraints revealed both the spatial orientation
of the C-20 carbonyl group and the conformation of the
16-membered ring of 1 to be significantly associated to
potent cytotoxicity.6 The foregoing large difference in
cytotoxic outcome among arenastatin A (1) and the two
analogues (5, 6) was presumed to be attributable to the
difference in conformations of the 16-membered ring
portion. Thus, conformation analysis of 5 and 6 was
In conclusion, substitution of the steric hindered tert-
butyl group for the isobutyl group on C-15 of arenast-
atin A (1) provided anti-tumor efficacy in vivo through
iv administration as well as enhancement of robustness
in serum. Conformational analysis by molecular
Table 1. In vivo anti-tumor activity of 15-tert-butylanalogue (6)
Table 2. Structural statistics and distance restraint violations
Arenastatin A 15-Isopropyl 15-tert-Butyl
T/Cc
Dosea
(mg/kg)
Tumor volumeb
(cm3, day 14)
Treatment
(1)
(5)
(6)
Number of
accepted conformers
ꢀ
Rmsd (A) for
43
45
46
6
2.5
5
6.6 1.1
7.1 1.0
4.9 1.5*
4.2 0.9*
3.3 0.7*
6.9 1.3
0.96
1.03
0.71
0.62
0.48
1.00
0.62 0.22
0.18 0.27
0.58 0.19
1.63 0.38
0.63 0.22
1.99 0.46
10
backbone heavy
atoma
Mitomycin C
Vehicle
1.65
3.3
Restraint violation
(kcal/mol)a;b
a The given numbers are the mean SD. The backbone means 16-
membered ring.
b When the calculated distance is greater than the distance restraint, the
energy incurred is 200ðdmeasure ꢀ drestrainÞ.2
*p < 0:05 against vehicle control (Dunnett method).
a Iv administration · 3 (days 3, 7, 11).
b Mean SD.
c Treated/control.

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2597–2601
Figure 2. Distribution of conformers of 1, 5, and 6 obtained by molecular dynamic calculation.
(1H, m, 15-H), 4.73 (1H, q-like, J ¼ ca: 8 Hz, 24-H), 3.86
dynamic calculation of arenastatin A (1) and the two
analogues 5, 6 established the torsion angle from C-14 to
C-20 as a conclusive factor for the potent cytotoxicity of
1.
(1H, m, 22-Ha), 3.76 (3H, s, 29-OMe), 3.76 (1H, m, 22-
Hb), 3.65 (1H, d, J ¼ 1:8 Hz, 8-H), 3.13 (2H, q-like,
J ¼ ca: 10 Hz, 25-H), 2.84 (1H, dd, J ¼ 1:8, 7.3 Hz, 7-H),
2.70 (1H, m, 4-Ha), 2.47 (3H, m, 4-Hb, 21-H), 1.85 (1H, q-
like, J ¼ ca: 6:5 Hz, 6-H), 1.54 (2H, m, 16-Ha, 17-H), 1.41
(1H, m, 16-Hb), 1.14 (3H, d, J ¼ 7:3 Hz, 13-H), 0.79, 0.81
(both, 3H, d, J ¼ 6:7 Hz, 18, 19-H). FABMS m=z: 607
[M+H]þ. FAB-HRMS m=z: calcd for C34H42O8N2+H:
Acknowledgements
607.3019. Found: 607.3033.
22
D
The authors are grateful to Messrs Naomi Uchida and
Tohru Wada, Shionogi Research Laboratories, for in
vivo experiment. This study was financially supported
by Grants-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports, and Culture of
Japan. The authors are also grateful to the Naito
Foundation and the Hoh-ansha Foundation for finan-
cial support.
9. 5: white powder, ½aꢁ +35.2 (c 0.66, CHCl3). IR (KBr):
3297, 2924, 1744, 1674, 1514 cmꢀ1
.
1H NMR (500 MHz,
CDCl3) d: 7.24–7.38 (5H, m, Ph), 7.11 (2H, d, J ¼ 8:5 Hz,
27-H), 6.93 (1H, br, 22-NH), 6.81 (2H, d, J ¼ 8:5 Hz, 28-
H), 6.68 (1H, ddd, J ¼ 4:3, 9.8, 15.3 Hz, 3-H), 5.69 (1H, d,
J ¼ 15:3 Hz, 2-H), 5.66 (1H, d, J ¼ 8:5 Hz, 24-NH), 5.19
(1H, ddd, J ¼ 1:8, 4.9, 11.0 Hz, 5-H), 4.75 (1H, ddd,
J ¼ 5:5, 7.3, 8.5 Hz, 24-H), 4.73 (1H, d, J ¼ 4:3 Hz, 15-H),
3.77 (3H, s, 30-H), 3.68 (1H, d, J ¼ 1:8 Hz, 8-H), 3.54 (1H,
m, 22a-H), 3.40 (1H, m, 22b-H), 3.15 (1H, dd, J ¼ 5:5,
14.0 Hz, 25a-H), 3.00 (1H, dd, J ¼ 7:3, 14.0 Hz, 25b-H),
2.88 (1H, dd, J ¼ 1:8, 7.3 Hz, 7-H), 2.55 (3H, m, 4a, 21-
H), 2.40 (1H, ddd, J ¼ 9:8, 11.0, 14.0 Hz, 4b-H), 1.90 (1H,
m, 6-H), 1.79 (1H, m, J ¼ 6:7 Hz, CH(CH3)2), 1.15 (3H, d,
J ¼ 6:7 Hz, 13-H), 0.88, 0.74 (both 3H, d, J ¼ 6:7 Hz,
CH(CH3)2). FABMS m=z: 593 (M+H)þ. FAB-HRMS
m=z: calcd for C33H4212O8N2: 593.2863. Found: 593.2861.
References and notes
1. Kobayashi, M.; Aoki, S.; Ohyabu, N.; Kurosu, M.; Wang,
W.; Kitagawa, I. Tetrahedron Lett. 1994, 35, 7969.
2. Kobayashi, M.; Kurosu, M.; Wang, W.; Kitagawa, I.
Chem. Pharm. Bull. 1994, 42, 2394.
3. Koiso, Y.; Morita, K.; Kobayashi, M.; Wang, W.;
Ohyabu, N.; Iwasaki, S. Chem. Biol. Interact. 1996, 102,
183.
4. Murakami, N.; Wang, W.; Ohyabu, N.; Ito, T.; Tamura,
S.; Aoki, S.; Kobayashi, M.; Kitagawa, I. Tetrahedron
2000, 56, 9121.
5. Murakami, N.; Wang, W.; Tamura, S.; Kobayashi, M.
Bioorg. Med. Chem. Lett. 2000, 10, 1823.
10. 6: white powder, ½aꢁ +55.1 (c 0.48, CHCl3). IR (KBr):
D
3279, 2926, 1740, 1678, 1514 cmꢀ1
.
1H NMR (500 MHz,
CDCl3) d: 7.24–7.38 (5H, m, Ph), 7.11 (2H, d, J ¼ 8:5 Hz,
27-H), 7.03 (1H, m, 22-NH), 6.82 (2H, d, J ¼ 8:5 Hz, 28-
H), 6.68 (1H, ddd, J ¼ 6:9, 7.9, 15.3 Hz, 3-H), 5.71 (1H, d,
J ¼ 15:3 Hz, 2-H), 5.65 (1H, d, J ¼ 7:9 Hz, 24-NH), 5.39
(1H, dt, J ¼ 9:2, 4.3 Hz, 5-H), 4.70 (1H, ddd, J ¼ 5:5, 7.9,
7.9 Hz, 24-H), 4.64 (1H, s, 15-H), 3.78 (3H, s, 30-H), 3.71
(1H, d, J ¼ 2:4 Hz, 8-H), 3.56 (1H, m, 22a-H), 3.39 (1H,
m, 22b-H), 3.14 (1H, dd, J ¼ 5:5, 14.6 Hz, 25a-H), 3.02
(1H, dd, J ¼ 7:9, 14.6 Hz, 25b-H), 2.91 (1H, dd, J ¼ 2:4,
6.7 Hz, 7-H), 2.57 (3H, m, 4a, 21-H), 2.49 (1H, m, 4b-H),
1.88 (1H, m, 6-H), 1.15 (3H, d, J ¼ 6:7 Hz, 13-H), 0.98
(9H, s, C(CH3)3). FABMS m=z: 607 (M+H)þ. FAB-
HRMS m=z: calcd for C34H43O8N2: 607.3019. Found:
607.3041.
6. Murakami, N.; Tamura, S.; Wang, W.; Takagi, T.;
Kobayashi, M. Tetrahedron 2001, 57, 4323.
7. Kobayashi, M.; Wang, W.; Ohyabu, N.; Kurosu, M.;
Kitagawa, I. Chem. Pharm. Bull. 1995, 43, 1598.
21
D
CHCl3). IR (KBr): 3271, 2930, 1741, 1676, 1591,
8. 15-Epiarenastatin A: white powder, ½aꢁ +16.3 (c 0.22,
1514 cmꢀ1 1H NMR (500 MHz, CDCl3) d: 7.20–7.34
.
(5H, m, Ph), 7.14 (2H, d, J ¼ 8:5 Hz, 27-H), 6.96 (1H, br,
22-NH), 6.82 (2H, d, J ¼ 8:5 Hz, 28-H), 6.50 (1H, ddd,
J ¼ 7:0, 8.5, 15.3 Hz, 3-H), 5.75 (1H, d, J ¼ 15:3 Hz, 2-H),
5.50 (1H, d, J ¼ 8:5 Hz, 24-NH), 5.15 (1H, m, 5-H), 5.03
11. Murakami, N.; Tamura, S.; Iwata, E.; Aoki, S.; Akiyama,
S.; Kobayashi, M. Bioorg. Med. Chem. Lett. 2002, 12, 3267.
12. On day 0, a tumor fragment (1 mm3) of Lewis lung
carcinoma was implanted through sc administration into

N. Murakami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2597–2601
2601
the back of BDF1 mice. The analogue 6 or mitomycin C
was administered through iv to the tumor-bearing mice at
various doses on days 3, 7, and 11. Each group consisted
of six mice. On day 14, the tumor volumes were
determined as 0:5a2b, where a and b are the minor and
major tumor axes. In this study, the statistical significance
in comparison with the nontreated group and the treated
groups was evaluated using Dunnettꢀs test.
13. Press, W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling,
W. T. Numerical Recipes in C. In The Art of Scientific
Computing; Cambridge University Press: Cambridge,
1988; pp 307–312.