Synthesis and biological property of carba and 20-deoxo analogues of arenastatin A

Article abstract of DOI:10.1016/S0960-894X(00)00356-5

The carba analogue, in which a methylene group is substituted for the oxygen atom linked to C-15, and 20-deoxo analogue of arenastatin A, a potent cytotoxic spongean depsipeptide, were synthesized. Both analogues lacking the 15,20-ester function, which was easily metabolized in serum, showed good stability in serum as well as moderate cytotoxic activity against KB cells and better solubility. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00356-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1823±1826
Synthesis and Biological Property of Carba and 20-Deoxo
Analogues of Arenastatin A
Nobutoshi Murakami, Weiqi Wang, Satoru Tamura and Motomasa Kobayashi*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
Received 15 May 2000; accepted 19 June 2000
AbstractÐThe carba analogue, in which a methylene group is substituted for the oxygen atom linked to C-15, and 20-deoxo ana-
logue of arenastatin A, a potent cytotoxic spongean depsipeptide, were synthesized. Both analogues lacking the 15,20-ester func-
tion, which was easily metabolized in serum, showed good stability in serum as well as moderate cytotoxic activity against KB cells
and better solubility. # 2000 Elsevier Science Ltd. All rights reserved.
In the course of our search for new, biologically active
principles from marine organisms, we isolated and char-
acterized the extremely cytotoxic (IC50 5 pg/mL against
KB cells) depsipeptide named arenastatin A (1), from
the Okinawan marine sponge of Dysidea arenaria as a
minute constituent.1,2 Later, we achieved the total
synthesis of 13,4 and elucidated that 1 inhibited micro-
tubule assembly5,6 by binding to the rhizoxin/maytansine
site on tubulin. A closely similar depsipeptide named
cryptophycin 1 (3) has been found from a terrestrial cya-
nobacterium of Nostoc sp.7 Cryptophycin 1 (3) showed
potent cytotoxicity and excellent in vivo anti-tumor
activity. On the other hand, a signi®cant reduction of
the in vivo anti-tumor activity of arenastatin A (1) was
found. Furthermore, the metabolism of the 15,20-ester
function in 1 was disclosed through the synthesis of
three analogues containing an amide function in place of
the ester linkage of 1 and the evaluation of their stability
in serum. Among them, only triamide analogue-II (2)
with a 15,20-amide function showed sucient stability
as well as moderate cytotoxicity (IC50 6 ng/mL). How-
ever, 2 showed very poor solubility in polar solvents
(such as MeOH, DMSO, and water) applicable to in
vivo anti-tumor testing.
been reported.8 11 Then, we have undertaken to syn-
thesize carba analogue (4) having a 15,20-methylene
bridge and 20-deoxo analogue (5) lacking the 20-car-
bonyl group in 1. This paper describes the synthesis
and biological assessment of the two analogues (4, 5)
(Chart 1).
Carba analogue (4), in which a methylene group is sub-
stituted for the oxygen atom linked to C-15 of 1, was
synthesized through condensation between segment A-B
(13) and segment C-D (12) as illustrated in Scheme 1. The
key segment 12 was prepared by the following procedure.
Carbonyldiimidazole induced the coupling of N-Boc-b-
alanine (7) and lithium enolate generated from tert-
butyl acetate (6) to provide a ketoester (8) in 83% yield.
SN2 Nucleophilic substitution of the carbanion of 8 with
a tri¯ate 10, which was converted from (R)-leucinic acid
(9) through treatment with trimethylsilyldiazomethane
followed by tri¯uoromethanesulfonylation, a€orded a
diastereomeric mixture 11 in 71% yield. Removal of the
tert-butyl group in 11 and subsequent re-introduction of
the Boc group gave a half-ester, which was subjected to
decarboxylation under re¯ux in benzene to yield a keto-
ester. Saponi®cation of the resulting ketoester by LiOH
furnished the desired segment C-D (12) with high
enantioselectivity in a ratio of 30:1 in 84% overall yield
through four steps.
This background prompted us to search for new analogues
of 1 possessing good stability in serum as well as potent
cytotoxicity and sucient solubility applicable to in
vivo anti-tumor testing. So far, more than 15 synthetic
studies of cryptophycin 1 (3) and arenastatin A (1) have
The absolute con®guration of 12 was con®rmed by
Kusumi's method.12 The proton signals due to the iso-
butyl residue of (S)-PGME amide (12a) appeared at
lower ®eld than those of (R)-PGME amide (12b), while
the proton signals assignable to the b-alanine portion of
12a were observed at higher ®eld than those of 12b as
*Corresponding author. Fax:+81-6-6879-8219; e-mail: kobayasi@
phs.osaka-u.ac.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00356-5

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1823±1826
Chart 1.
Scheme 1. Synthesis of carba analogue (4). Reagents and conditions: (a) LDA, THF, 78 ꢀC; (b) carbonyldiimidazole, THF, 2 steps 83%; (c)
CHN2TMS, CH2Cl2; (d) Tf2O, 2,6-lutidine, 20 ꢀC, 2 steps 95%; (e) NaH, THF, 71%; (f) TFA; (g) (Boc)2O, Et3N, CH2Cl2; (h) benzene, re¯ux; (i)
LiOH, THF:H2O (3:1), 4 steps 84%; (j) 2,4,6-trichlorobenzoyl chloride, Et3N, THF; (k) Et3N, DMAP, toluene, 2 steps 97%; (l) TFA, CH2Cl2; (m)
HCl±Et2O; (n) DPPA, NaHCO3, DMF, 5 ꢀC, 3 steps 66%; (o) dimethyldioxirane, CH2Cl2, 66%.
shown in Figure 1. Consequently, the absolute con®g-
uration at the C-15 position in 12 was con®rmed to be
the desired R con®guration.
dimethyldioxirane to furnish the desired carba analogue
(4)14 predominantly in a ratio of ca. 2:1.
Next, 20-deoxo analogue (5) lacking the 20-carbonyl
group of 1 was synthesized as illustrated in Scheme 2.
An alkoxy carboxylic acid 16 was prepared from d-leu-
cine according to TenBrink's procedure.15 The resulting
As a result of examining various activating reagents for
the condensation of 12 and 13, esteri®cation was
achieved by using 2,4,6-trichlorobenzoyl chloride in the
presence of Et3N to furnish the desired diester 14 in
97% yield.13 Simultaneous cleavage of two protecting
groups in 14 followed by HCl treatment a€orded a seco
amino acid as a hydrochloride salt, which was subjected to
intramolecular macrolactamization by use of diphenyl-
phosphoryl azide (DPPA) and NaHCO3 to give a cyclic
depsipeptide 15 in 66% overall yield through three steps.
Finally, epoxidation of 15 was successfully executed with
.
carboxylic acid 16 was coupled with 13 using EDCI HCl
in the presence of DMAP in CH2Cl2 to give a con-
jugated product 17 quantitatively. After removal of two
protecting groups followed by conversion to a hydro-
chloride salt, the resulting seco acid was also cyclized
intramolecularly to give a cyclic depsipeptide 18 in
moderate overall yield (74%). Treatment of 18 with
dimethyldioxirane furnished the 20-deoxo analogue

N. Murakami et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1823±1826
1825
.
Scheme 2. Synthesis of 20-deoxo analogue (5). Reagents and conditions: (a) EDCI HCl, DMAP, CH2Cl2, quant; (b) TFA, CH2Cl2; (c) HCl±Et2O;
(d) DPPA, NaHCO3, DMF, 5 ꢀC, 3 steps 74%; (e) dimethyldioxirane, CH2Cl2, 65%.
anti-tumor testing unlike the triamide analogue-II (2).18
The signi®cant decrease (10,000-fold less) of the cyto-
toxicity for 4 and 5 compared with that of arenastatin A
(1) might be caused by the conformational di€erence in
the 16-membered ring system between 1 and 4, 5, inas-
much as we have already clari®ed that the synthesized
stereoisomers relative to epoxy, 6-methyl, and OMe-tyr-
osine did not show any potent cytotoxicity at concentra-
tions below 0.1 mg/mL.4 Conformational comparison of
the ring structure between arenastatin A (1) and its
analogues is currently under way.
Figure 1. Con®rmation of absolute con®guration in 12.
Acknowledgements
The authors are grateful to the Naito Foundation, the
Houansha Foundation, and the Ministry of Education,
Sciences, Sports, and Culture of Japan for ®nancial
support.
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.; Ohyabu, N.; Wang, W.; Fujii,
S.; Kitagawa, I. Chem. Pharm. Bull. 1994, 42, 2196.
3. Kobayashi, M.; Kurosu, M.; Wang, W.; Kitagawa, I.
Chem. Pharm. Bull. 1994, 42, 2394.
4. Kobayashi, M.; Wang, W.; Ohyabu, N.; Kurosu, M.;
Kitagawa, I. Chem. Pharm. Bull. 1995, 43, 1598.
5. Koiso, Y.; Morita, K.; Kobayashi, M.; Wang, W.; Ohyabu,
N.; Iwasaki, S. Chem.-Biol. Interact. 1996, 102, 183.
6. Morita, K.; Koiso, Y.; Hashimoto, Y.; Kobayashi, M.; Wang,
W.; Ohyabu, N.; Iwasaki, S. Biol. Pharm. Bull. 1997, 20, 171.
Figure 2. Stability of arenastatin A (1) and its analogues (4, 5) in
mouse serum. Each sample (10 mL of 0.1 mg/ml solution) was treated
with fresh mouse serum (100 mL) and incubated at 37 ꢀC for 0, 10, 30,
60, 300 min, respectively. After extraction of the reaction mixture with
7. Barrow, R. A.; Hemscheidt, T.; Liang, J.; Paik, S.; Moore,
R. E.; Tius, M. A. J. Am. Chem. Soc. 1995, 117, 2479.
8. Varie, D. L.; Shih, C.; Hay, D. A.; Andis, S. L.; Corbett, T.
H.; Gossett, L. S.; Janisse, S. K.; Martinelli, M. J.; Moher, E.
D.; Schultz, R. M.; Toth, J. E. Bioorg. Med. Chem. Lett. 1999,
9, 369.
9. Liang, J.; Hoard, D. W.; Khau, V. V.; Martinelli, M. J.;
Moher, E. D.; Moore, R. E.; Tius, M. A. J. Org. Chem. 1999,
64, 1459.
10. Patel, V. F.; Andis, S. L.; Kennedy, J. H.; Ray, J. E.;
Schultz, R. M. J. Med. Chem. 1999, 42, 2588.
11. White, J. D.; Hong, J.; Robarge, L. A. J. Org. Chem.
1999, 64, 6206.
EtOAc, each extract was analyzed by reversed phase HPLC to
determine the remain of 1, 4 and 5.
(5)16,17 as a major oxygenated product (a-epoxide:b-
epoxide=1:2).
Carba (4) and 20-deoxo (5) analogues exhibited moderate
cytotoxicity against KB cells with IC50 values of 0.07 and
0.04 mg/mL, respectively. In addition, both analogues (4,
5) also showed nearly complete stability in mouse serum
as we expected (Fig. 2). Notably, the two analogues
possessed better solubility for application to in vivo
12. Nagai, Y.; Kusumi, T. Tetrahedron Lett. 1995, 36, 1853.

1826
N. Murakami et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1823±1826
13. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yama-
guchi, M. Bull. Chem. Soc. Jpn. 1979, 52, 1989.
15. TenBrink, R. E. J. Org. Chem. 1987, 52, 418.
16. 5: [a]1D8+44.1ꢀ (c=0.11, CHCl3). IR (KBr) cm 1: 2928,
1745, 1660, 1535, 1514. H NMR (500 MHz, CDCl3) d 7.36±
14. 4: [a]1D8+43.9ꢀ (c=0.33, MeOH). IR (KBr) cm 1: 2930,
1
1
2860, 1728, 1668, 1514. H NMR (500 MHz, CDCl3) d 7.40±
7.23 (5H, m, Ph), 7.12 (2H, d, J=8.5 Hz, 27-H), 6.84 (1H,
ddd, J=3.7, 11.0, 14.6, 3-H), 6.81 (2H, d, J=8.5, 28-H), 6.52
(1H, brd, J=6.7, 22-NH), 5.67 (1H, d, J=14.6, 2-H), 5.66 (1H, d,
J=8.5, 24-NH), 5.33 (1H, ddd, J=2.4, 5.5, 9.2, 5-H), 4.66 (1H, q-
like, J=ca. 8.0, 24-H), 3.77 (3H, s, 30-H), 3.76 (1H, m, 22-Ha),
3.67 (1H, d, J=1.8, 8-H), 3.60 (1H, dd, J=3.1, 10.4, 15-H), 3.26
(1H, m, 22-Hb), 3.18 (1H, dd, J=7.3, 14.6, 25-Ha), 3.08 (1H, m,
20-Ha), 2.92 (1H, dd, J=1.8, 7.9, 7-H), 2.89 (1H, m, 25-Hb), 2.84
(1H, m, 20-Hb), 2.59 (1H, m, 4-Ha), 2.37 (1H, m, 4-Hb), 1.78
(3H, m, 6, 21-H), 1.64 (1H, m, 17-H), 1.49 (1H, m, 16-Ha), 1.23
(1H, m, 16-Hb), 1.14 (3H, d, J=7.3, 6-CH3), 0.84, 0.83 (both, 3H,
d, J=6.7, 18, 19-H). FABHRMS: Obsd; m/z 593.3217. Calcd for
C34H44N2O7; m/z 593.3227 (M+H)+.
17. Murakami, N.; Ito, T.; Tamura, S.; Aoki, S.; Kobayashi,
M. Abstract Papers, Part 2, P-160, 118th Annual Meeting of
the Japanese Society of Pharmaceutical Sciences, Kyoto, 31
March±2 April, 1998.
18. For example, the solubility of arenastatin A (1) and its
analogues (2, 4, 5) in MeOH was 2.8, 0.20, 2.9 and 1.6 mg/mL,
respectively.
7.25 (5H, m, Ph), 7.18 (1H, m, 22-NH), 7.09 (2H, d, J=8.5 Hz,
27-H), 6.82 (2H, d, J=8.5, 28-H), 6.71 (1H, ddd, J=8.0, 14.7,
15.3, 3-H), 5.73 (1H, d, J=15.3, 2-H), 5.50 (1H, d, J=7.3, 24-
NH), 5.25 (1H, m, 5-H), 4.65 (1H, ddd, J=5.5, 7.3, 7.4, 24-H),
3.79 (3H, s, 30-H), 3.69 (1H, d, J=1.9, 8-H), 3.63 (1H, m, 22-
Ha), 3.14 (1H, m, 22-Hb), 3.10 (1H, dd, J=5.5, 14.0, 25-Ha),
3.05 (1H, dd, J=7.4, 14.0, 25-Hb), 2.94 (1H, dd, J=1.9, 7.3,
7-H), 2.90 (1H, dd, J=8.5, 16.2, 150-Ha), 2.87 (1H, m, 15-H),
2.61 (1H, m, 21-Ha), 2.53 (1H, m, 21-Hb), 2.33 (1H, brd,
J=16.2, 150-Hb), 1.65±1.40 (3H, m, 16, 17-H), 1.40 (1H, m, 6-
H), 1.13 (3H, d, J=7.4, 6-CH3), 0.86, 0.83 (both, 3H, d,
J=6.8, 18, 19-H). FABHRMS: Obsd; m/z 605.3224. Calcd for
C35H45N2O7; m/z 605.3227 (M+H)+. The stereochemistry of
the epoxy moiety was established by comparison of the che-
mical shifts due to the 8-H and 6-Me groups with those of
arenastatin A (1). The practical data of 8-H and 6-Me were as
follows: 1: d 3.68, 1.14; a-epoxy isomer of 1: d 3.59, 1.05; a-
epoxy isomer of 4: d 3.59, 1.04; a-epoxy isomer of 5: d 3.59,
1.03.