Synthesis and anticancer activity of polyhydroxylated 18-membered analogue of antimycin A3

Article abstract of DOI:10.1016/j.tet.2012.01.080

A novel polyhydroxylated 18-membered analogue of antimycin A₃ was synthesized. Our synthesis commenced with Boc-l-threonine and was achieved by way of one-pot homocoupling/ring-closing olefin metathesis reaction cascade and Sharpless asymmetric

Full text of DOI:10.1016/j.tet.2012.01.080

Tetrahedron 68 (2012) 2884e2891
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and anticancer activity of polyhydroxylated 18-membered analogue
of antimycin A3
,
c
,
a
a
b,
Ade Arsianti a , Hiroki Tanimoto , Tsumoru Morimoto , Anton Bahtiar d, Tatsuo Takeya b,
*
Kiyomi Kakiuchi a
a Graduate School of Materials Science, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
b Graduate School of Biological Science, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
c Department of Chemistry, Faculty of Medicine, University of Indonesia, Jl. Salemba Raya No. 4, Jakarta, Indonesia
d Faculty of Pharmacy, University of Indonesia, Kampus UI Depok, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel polyhydroxylated 18-membered analogue of antimycin A3 was synthesized. Our synthesis
Received 8 December 2011
Received in revised form 26 January 2012
Accepted 28 January 2012
Available online 2 February 2012
commenced with Boc-L-threonine and was achieved by way of one-pot homocoupling/ring-closing olefin
metathesis reaction cascade and Sharpless asymmetric dihydroxylation. The analogue exhibited a greater
anticancer activity against HeLa cells, breast MDA-MB-231 cells, and prostate PC-3 cells compared to the
original antimycin A3.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Anticancer
Polyhydroxylated 18-membered ring
Antimycin A3
Analogue
1. Introduction
Antimycin A3 was isolated from Streptomyces sp. in 1949 and in
4
later, this was revealed as mixture of two molecules, A3a and A3b
.
The search for new anticancer agents has become a major
challenge for current research in medicinal chemistry, especially
with an increase in the incidence of cancer drug resistance
exhibited by tumor cells that seriously reduce the effectiveness of
anticancer therapy.1,2 The recent drug discovery process shows
that useful anticancer drugs were derived not only from natural
sources but also came from synthetic chemistry processes.3 Con-
sidering that research and development of new synthetic anti-
cancer drugs with complex structures is difficult, expensive, and
time consuming, analogue synthesis of anticancer drugs has
emerged as a promising strategy to seek new anticancer agents
that are more effective and responsive in anticancer therapy. An-
alogue synthesis can have two advantages. First, it has short du-
ration and high probability of yielding more active compounds by
modifying the structure. Second, it gives information about the
structureeactivity relationship between the analogue and the
original compound.
Antimycin A3 is one of the most active agents that inhibits the
electron transfer activity of ubiquinol-cytochrome c oxidoreductase
and prevents the growth of human cancer cells. Antimycin A3 was
also found to induce apoptosis of cancer cells by selectively killing
cancer cells that expressed high levels of anti-apoptotic Bcl-2 and
5e8
Bcl-XL
.
The structure of antimycin A3 consisting of a nine-
membered dilactone is similar to the antifungal antibiotic UK-3A
that was also isolated from Streptomyces sp. in 1997.9,10 We re-
cently reported the synthesis of novel 2-hydroxynicotinoyl-serine-
butyl esters related to antibiotic UK-3A, which demonstrated sig-
nificant activity as a growth inhibitor of Bacillus subtilis and
Staphylococcus aureus.11 In this research, we focused on the syn-
thesis of antimycin A3 analogue (1) and examined its anticancer
activity (Fig. 1). The total and formal syntheses of antimycin A3 have
been accomplished by some researchers,4,12 however, studies on its
analogue synthesis by specifically modifying the dilactone core are
still limited. Therefore, in this work, we carried out the synthesis of
antimycin A3 analogue, which are more focus on the modification
of the nine-membered dilactone core. Studies on the structur-
eeactivity relationship of antimycin A3 by Miyoshi et al. in 1995
revealed that the hydroxyl group and 3-formamide group in the
salicylyl moiety, as well as the amide bond were necessary for
* Corresponding author. Tel./fax: þ81 743 72 6084; e-mail addresses: a-arsianti@
ms.naist.jp, ade.arsianti@ui.ac.id (A. Arsianti).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.01.080

A. Arsianti et al. / Tetrahedron 68 (2012) 2884e2891
2885
Fig. 1. Structure of antimycin A3, UK-3A, respirantin, and desired antimycin A3 analogue (1).
anticancer activity. On the other hand, the nine-membered dilac-
tone core in antimycin A3 was found less effective for anticancer
activity than 3-formamidosalicylyl moiety.13 These results in-
dicated that, it is quite possible to carry out the synthesis of anti-
mycin A3 analogue by replacing the nine-membered dilactone core
with another active core that contributes to the improvement of its
anticancer activity. Therefore in this work, the nine-membered
introduction of the 3-formamidosalicylyl group into this core, is
expected to synergistically improve its anticancer activity com-
pared to that of the original compound antimycin A3. The retro-
synthetic analysis of the antimycin A3 analogue is outlined in
Scheme 1. As shown, analogue 1 can be built by amidation of tetraol
3 with 3-formamidobenzoic acid moiety (2), followed by hydro-
genolysis of the benzyl group. Tetraol 3 can be prepared from Boc
deprotection of 4, whereby 4 can be derived from 18-membered
tetralactone 5 by way of Sharpless dihydroxylation.16,17 Tetra-
lactone 5 can be constructed from one-pot homocoupling ring-
closing olefin metathesis18,19 of diester 6, and 6 can be synthe-
dilactone core of antimycin A3 was replaced by
a poly-
hydroxylated 18-membered tetralactone core in our analogue. It
has been reported that the 18-membered ring of polylactone, such
as respirantin, which has a similar structure with antimycin A3
showed potent antitumor activity against human cancer cell lines
sized from two-step esterification of commercially available Boc-
threonine (7).
L-
with IC50 range 0.00018e0.47 mg/mL. Furthermore, respirantin with
Scheme 1. Retrosynthetic analysis of antimycin A3 analogue (1).
IC50: 0.0037
A3 (IC50: 0.015
m
g/mL, showed a stronger cytotoxicity than antimycin
m
2. Results and discussion
2.1. Synthesis of tetraol 3
g/mL) against mouse Leukemia P-388 cells.14 It is
also known that the presence of hydroxyl groups in biologically
active compound significantly increases their biological activity
due to the enhancement of solubility in water, which is the one of
important factors influencing the efficacy of drugs.3,15 Thus, the
polyhydroxylated 18-membered tetralactone core in our analogue
is expected to be active as an anticancer agent. Moreover,
Scheme 2 outlines the synthesis of 18-membered tetraol 3.
Starting from esterification of Boc- -threonine (7) with allyl bro-
mide under basic condition, the synthesis was conducted according
to Wu et al.,20 and afforded Boc-
-threonine allyl ester (8) in 96%
L
L

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A. Arsianti et al. / Tetrahedron 68 (2012) 2884e2891
Scheme 2. Synthesis of 18-membered tetraol 3. Reagents and conditions: (a) Allyl bromide, Na2CO3, DMF, rt (96%); (b) Acrylic acid, DIC (diisopropyl carbodiimide), DMAP, CH2Cl2,
ꢀC to rt (92%); (c) 4 mol % Zhan catalyst 1B, CH2Cl2, 45 ꢀC (73%); (d) OsO4, K3Fe(CN)6, K2CO3, NaHCO3, (DHQ)2PHAL, MeSO2NH2, t-BuOH/H2O, 0 ꢀC (61%); (e) concd HCl, EtOAc, rt
0
(81%); (f) 2, DIC/DMAP or EDCI/DMAP or EDCI/HOBt or EDCI/HOSu, NMM.
yield. Condensation of ester 8 with acrylic acid employing the
combination of DIC and DMAP occurred easily to afford 92% yield of
diester 6. In the next step, Ru-catalyzed homocoupling ring-closing
metathesis (RCM)18,19 was used to transform diester 6 into an 18-
membered tetralactone 5. Three kinds of Ru-catalyst, Grubbs first
generation, Grubbs second generation, and Zhan catalyst 1B,21 were
tested in this metathesis reaction (Fig. 2). Homocoupling RCM of
diester 6 with the less reactive Grubbs first catalyst failed to form
tetralactone 5, while the use of the more reactive Grubbs second
catalyst provided 5 in only 32% yield. The best result, 73% yield of 5
was achieved by performing the reaction with 4 mol % of Zhan
catalyst 1B in concentration 0.1 M at 45 ꢀC. Subsequently, Sharpless
asymmetric dihydroxylation of 5 with OsO4, (DHQ)2PHAL ligand,
and K3Fe(CN)6 as a co-oxidant gave 67% yield of a 12:1 di-
astereomeric mixture of tetraol products. This mixture could be
separated by medium pressure liquid chromatography to afford
61% yield of pure major tetraol 4, which was then treated with with
concd HCl in EtOAc to afford the polyhydroxylated 18-membered
tetralactone 3 in 81% yield as a white solid. The X-ray crystallo-
graphic analysis of 322 clarified its stereochemistry (Fig. 3A).
EDCI/DMAP, EDCI/HOBt, and EDCI/HOSu under many reaction
conditions, the desired tetraol diamide 9 was not obtained (step f in
Scheme 2). We postulated that not only the amine groups but also
four hydroxyl groups of 3 can react with carboxylic acid 2, and
might cause byproducts instead of the desired tetraol diamide 9. To
solve this problem, we abandoned this strategy, and proposed
a new one.
2.2. Synthesis of tetralactone 11 and analogue 1
The revised synthetic strategy for the synthesis of antimycin A3
analogue 1 is summarized in Scheme 3. We planned to carry out the
amidation of non-hydroxylated tetralactone 11 with acid 2 to give
diamide 10 in the initial stage, and then introduce the hydroxyl
groups into diamide 10 by Sharpless asymmetric dihydroxylation
followed by deprotection of benzyl groups in the late stage, to
generate desired the analogue 1. With this method, the amidation
of non-hydroxylated tetralactone 11 with acid 2 was expected to
proceed well without the formation of byproducts, which might be
obtained from the esterification of a polyhydroxylated tetralactone
with acid 2. The non-hydroxylated tetralactone 11 derived from Boc
deprotection of 18-membered tetralactone 5, which we had already
prepared in the former strategy.
As shown in Scheme 4, tetralactone 11 as diammonium
dichloride salts was formed from Boc deprotection of 5 with concd
HCl. Amidation of 11 with acid 2 could be performed using the base
NMM and the combination of EDCI/HOBt in THF as a solvent
afforded diamide 10 in 45% yield. In the next step, Sharpless dihy-
droxylation of 10 in the presence of 6 equiv of NMO with 50 mol %
of both OsO4 and (DHQ)2PHAL proceeded smoothly to give tetraol
diamide products in 66% yield as a 4:1 inseparable diastereomeric
mixture, with 9 as a major diastereomer. As the final step, hydro-
genolysis of this diastereomeric mixture with 10% Pd/C resulted in
simultaneous cleavage of both Bn group, and afforded a 4:1 di-
astereomeric mixture of the corresponding Bn deprotected tetraol
diamide products, which was successfully separated in this step by
medium pressure liquid chromatography to give a pure major
Fig. 2. Structure of Zhan catalyst 1B.
With key intermediate 3 in hand, our subsequent plan was to
conduct the amidation of tetraol
3 with benzylated 3-
formamidobenzoic acid 2, which was prepared according to Pettit
et al.,14 to give tetraol diamide 9. Unfortunately, after extensive
attempts with a variety of amidation reagents, such as DIC/DMAP,

A. Arsianti et al. / Tetrahedron 68 (2012) 2884e2891
2887
analogue 1 in 51% yield. Subsequently, the analogue 1 could be
recrystallized readily by methanol. The X-ray crystallographic
analysis of 123 has its stereochemistry assigned, as shown in Fig. 3B.
2.3. Cytotoxicity of tetralactone 11, tetraol 3, and analogue 1
After completion of the synthesis, cytotoxicities of the analogue
1 and two intermediate products, 18-membered ring tetraol 3 and
18-membered ring tetralactone 11 were evaluated as inhibitors of
cancer cell growth versus HeLa cells, breast cancer MDA-MB-231
cells, and prostate cancer PC-3 cells (Table 1). Tetralactone 11
with IC50 over 4000
cancer cells. In contrast to tetralactone 11, tetraol 3, which has four
hydroxyl groups showed the cytotoxicity in concentration 250 M,
112 M, and 205 M against HeLa cells, breast MDA-MB-231 cells,
mM showed no cytotoxicity against all tested
m
m
m
and prostate PC-3 cells, respectively. The cytotoxicity of tetraol 3
was greatly improved by the presence of the hydroxyl groups
compared to that of tetralactone 11. This suggested that the hy-
droxyl groups are very important for the anticancer activity. Com-
pared to analogue 1, tetraol 3, which does not possess 3-
formamidosalicylyl group, showed less cytotoxicity, suggesting
that the introduction of 3-formamidosalicylyl group on 18-
membered ring tetraol moiety in analogue 1 was potentially re-
sponsible for the increase in its anticancer activity. Moreover, an-
alogue 1, which contains a polyhydroxylated 18-membered ring
core and two parts of 3-formamidosalicylyl groups exhibited
greater anticancer activity than that of the original antimycin A3 on
all cancer cells tested, with IC50: 39, 40, and 43 mM against HeLa
cells, breast MDA-MB-231 cells, and prostate PC-3 cells, re-
spectively. These results indicated that introducing two parts of
formamidosalicylyl groups and replacing the nine-membered
dilactone ring of antimycin A3 with a polyhydroxylated 18-
membered ring in analogue 1 successfully improved its antican-
cer activity. Thus, analogue 1, which strongly inhibited the growth
of HeLa cells, breast MDA-MB-231 cells, and prostate PC-3 cells,
should be considered as a promising candidate for the treatment of
human cervical, breast, and prostate cancers.
3. Conclusion
In conclusion, we synthesized a novel analogue of antimycin A3
(1) from Boc-L-threonine through olefin metathesis cascade. Ana-
Fig. 3. (A) ORTEP drawing of 3$2H2O, (B) ORTEP drawing of the analogue 1.
logue 1 showed greater anticancer activity against HeLa cells, breast
MDA-MB-231 cells, and prostate PC-3 cells compared to the origi-
nal antimycin A3.
Scheme 3. The revised retrosynthetic analysis of antimycin A3 analogue (1).

2888
A. Arsianti et al. / Tetrahedron 68 (2012) 2884e2891
Scheme 4. Completion of the synthesis of antimycin A3 analogue (1). Reagents and conditions: (a) concd HCl, EtOAc, rt (90%); (b) 2, EDCI, HOBt, NMM, THF, rt (45%); (c) OsO4, NMO,
(DHQ)2PHAL, t-BuOH/THF/H2O, rt (66%, 4:1 dr); (d) 10% Pd/C, H2, MeOH, rt (51%).
Table 1
Cytotoxicities (IC50
against HeLa cells, breast MDA-MB-231 cells, and prostate PC-3 cells
internal standard. Mass spectra were recorded on Shimadzu
GCeMS QP-5000 or JEOL JMS-AX 700 spectrometers. X-ray crys-
tallography was performed on Rigaku RAXIS-RAPID imaging plate
diffractometer. Specific rotation, [a]D, were measured on JASCO
, mM) of tetralactone 11, tetraol 3, analogue 1, and antimycin A3
Compound
Cytotoxicity (IC50, mM)
DIP-1000 digital polarimeter. Cytotoxicity evaluations of the syn-
thesized products were carried out in Molecular Oncology Labo-
ratory of the Graduate School of Biological Sciences, Nara Institute
of Science and Technology (NAIST). The tested cancer cells: HeLa
cells, breast MDA-MB-231 cells, and prostate PC-3 cells are the
culture collections of Molecular Oncology Laboratory, Graduate
School of Biological Sciences, Nara Institute of Science and Tech-
nology (NAIST).
HeLa
Breast MDA-MB-231
Prostate PC-3
Tetralactone 11
Tetraol 3
Analogue 1
Antimycin A3
4514
250
39
4289
112
40
4002
205
43
60
79
50
The values in bold represent the best results for the analogue compound.
4. Experimental section
4.2. Boc-
L
-threonine-allyl ester 8
-Threonine (2.0 g, 9.12 mmol) in
4.1. General experimental methods
To a stirred solution of Boc-
L
Unless otherwise noted, all reactions were performed in an
oven-dried glassware, sealed with a rubber septum under nitrogen
atmosphere. Anhydrous THF, CH2Cl2, and toluene were purchased
from Kanto Chemical Co., DMF and tert-butyl alcohol were distilled
prior to use. Methanol, ethyl acetate, n-hexane, chloroform, Et2O,
and dimethyl sulfoxide (DMSO) were purchased from Wako Pure
Chemical Industries. Grubbs catalysts and Zhan catalyst 1B were
DMF (50 mL) was added Na2CO3 (1.93 g, 4.56 mmol), followed by
allyl bromide (0.93 mL, 10.94 mmol) and water (1.6 mL). The
resulting mixture was stirred for 40 h at rt. The solvent was re-
moved from the reaction mixture in vacuo and water (50 mL) was
added. The aqueous layer was extracted with EtOAc (3ꢁ50 mL) and
the combined organic layers were dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo, followed by purification by
column chromatography on silica gel (gradient elution 20:1 to 18:2,
hexane/EtOAc) to give ester 8 (2.09 g, 96%) as colorless liquid. Rf
0.63 (1:1 hexane/EtOAc); IR (neat) 3440, 2979, 2935, 2362, 1718,
purchased from Stream Chemicals. Boc-L-Threonine, allyl alcohol,
allyl bromide, acrylic acid, p-toluenesulfonic acid monohydrate,
NaHCO3, K2CO3, K3Fe(CN)6, DMAP (N,N-4-dimethylaminopyridine),
NMM (N-methylmorpholine), HOBt (1-hydroxybenzo-triazole),
HOSu (N-hydroxysuccinimide), EDCI (1-ethyl-3-(3-diaminopropyl)
carbodiimide hydrochloride), MeI, Benzylbromide, formamide, and
OsO4 (osmium (VIII) oxide) were obtained from Wako Pure
Chemical industries. 3-Aminosalicylic acid was obtained from
Tokyo Chemical Industries Co. Ltd. DIC (diisopropyl carbodiimide),
(DHQ)2PHAL, (DHQD)2PHAL, and antimycin A3 were purchased
from SigmaeAldrich Chemical Company. Flash column chroma-
tography was carried out using Merck silica gel 60 (spherical/
1507, 1367, 1165, 1067, 989 cmꢂ1
5.92e5.85 (m, CH]CH2, 1H), 5.34e5.22 (m, Thr-NHþCH]CH2,
3H), 4.65e4.63 (m, OCH2CH], 2H), 4.29e4.24 (m, Thr-H
þThr-H
2H), 2.13 (br s, Thr-OH, 1H), 1.43 (s, t-Bu, 9H), 1.23 (d, J¼6.2 Hz, CH3,
;
1H NMR (500 MHz, CDCl3):
d
a
b,
3H); 13C NMR (125 MHz, CDCl3):
d 171.3, 156.3, 131.6, 118.7, 80.0,
68.0, 66.0, 59.0, 28.3, 19.9. HRMS FABþ calcd for C12H22NO5
[MþH]þ: 260.1498, found: 260.1506.
40e63
mm). Reactions and chromatography fractions were ana-
4.3. Diester 6
lyzed employing precoated silica gel 60 F254 plates (Merck). Com-
pounds were visualized using an ultraviolet lamp (254 nm) and/or
by staining with ninhydrin (in EtOH), p-anisaldehyde (in EtOH), and
ammonium molybdate (in 10%H2SO4). All melting points were
determined on Yanaco micro melting point apparatus and un-
corrected. IR spectra were measured on a JASCO FT/IR-420 spec-
trophotometer. 1H NMR and 13C NMR spectra were recorded on
JEOL JNM-ECP500 (500 MHz) spectrometers with tetramethylsi-
To a solution of ester 7 (0.2 g, 0.77 mmol) in dry CH2Cl2 (15 mL)
was added acrylic acid (0.16 mL, 2.31 mmol) and DIC (0.36 mL,
2.31 mmol) stirred at 0 ꢀC. The mixture was stirred at the same
temperature 0 ꢀC for 1 h before DMAP (9.4 mg, 0.077 mmol) was
introduced. The stirring mixture was then continued at 0 ꢀC for 6 h
and then allowed to warm to rt until TLC showed disappearance of
the starting material Boc-
L-Threonine allyl ester (24 h). The reaction
lane (d 0), CHCl3 (d 7.26), CH3OH (d 3.30) or DMSO (d 2.49) as an
mixture was diluted with Et2O (20 mL), filtered and evaporated in

A. Arsianti et al. / Tetrahedron 68 (2012) 2884e2891
2889
vacuo. The crude product was purified by flash column chroma-
tography on silica gel with hexane/EtOAc 20:1 to afford diester 6
(222.7 mg, 92%) as colorless oil. Rf 0.63 (2:1 hexane/EtOAc); IR
(neat) 3359, 2980, 2938, 1725, 1637, 1507, 1406, 1367, 1267, 1168,
chromatography on silica gel (gradient elution 10:1 to 6:4, CHCl3/
CH3OH) gave an ammonium chloride salt of 3 (101 mg, 81%) as
a white solid. Subsequently, the white crystalline solid of 3 was
obtained as dihydrate after recrystallization from methanol/EtOAc.
Rf 0.12 (3:1 CHCl3/CH3OH); mp 126e130 ꢀC; IR (KBr disc) 3432,
2979, 1750, 1509, 1455, 1392,1369, 1278, 1209, 1165, 1131, 1091,
1063, 987 cmꢂ1
;
1H NMR (500 MHz, CDCl3):
d
6.38 (d, J¼17.7 Hz,
1H), 6.06 (dd, J¼17.1 and 10.4 Hz, 1H), 5.87e5.83 (m, 2H), 5.49 (m,
1H), 5.31 (d, J¼17.1 Hz, 1H), 5.23 (d, J¼10.4 Hz, 2H), 4.61 (m, 2H),
4.50 (dd, J¼9.8 and 2.4 Hz, 1H), 1.47 (s, 9H), 1.35 (d, J¼6.1 Hz, 3H);
1063 cmꢂ1
;
1H NMR (500 MHz, CD3OD):
d
5.38 (d, J¼6.1 Hz, 1H),
4.54 (d, J¼11.0 Hz, 1H), 4.43 (s, 1H), 4.31e4.25 (m, 3H), 4.16e4.12
13C NMR (125 MHz, CDCl3):
d
169.8 (s), 164.8 (s), 155.8 (s), 131.5 (t),
(m, 2H), 1.45 (d, J¼6.1 Hz, 3H); 13C NMR (125 MHz, CD3OD):
d 171.9
131.4 (d), 127.9 (d), 119.1 (t), 80.3 (s), 70.9 (d), 66.3 (t), 57.3 (d), 28.3
(q), 16.9 (q); HRMS FABþ calcd for C15H24NO6 [MþH]þ: 314.1604,
found: 314.1606.
(s), 168.4 (s), 72.0 (d), 70.6 (d), 69.0 (t), 57.3 (d), 49.8 (d), 16.2 (q);
HRMS FABþ calcd for C16H26N2O12Na [(Mꢂ72)þNa]þ: 461.1383,
found: 461.1375; ½a D30
ꢂ9 (c 1.0, CH3OH).
ꢃ
4.4. 18-Membered tetralactone 5
4.7. Tetralactone 11
A round-bottomed flask was charged with diester 6 (0.25 g,
0.80 mmol) dissolved in CH2Cl2 (8 mL). To this solution Zhan cat-
alyst 1B (23.4 mg, 4 mol %) was added. The mixture was refluxed
under nitrogen atmosphere for 21 h. After reaction completed, the
solvent was evaporated and the crude was purified by column
chromatography on silica gel (gradient elution 20:1 to 15:5, hex-
ane/EtOAc) gave tetralactone 5 (0.17 mg, 73%) as colorless liquid. Rf
0.35 (2:1 hexane/EtOAc); IR (neat) 3383, 2980, 2937, 1729, 1665,
To a stirred solution of tetralactone 5 (0.53 g, 0.92 mmol) in
EtOAc (25 mL) was added 35% (w/v) of HCl (1.44 mL, 13.8 mmol)
and the mixture was stirred at rt for 10 h. After reaction was
complete, the solvent was evaporated and the crude was purified
by column chromatography on silica gel (gradient elution 6:1 to 3:1,
CHCl3/CH3OH) gave diammonium chloride salts of 11 (0.37 g, 90%)
as a white solid. Rf 0.38 (5:1 CHCl3/CH3OH); mp 187e190 ꢀC; IR (KBr
disc) 3440, 2926, 1755, 1664, 1631, 1517, 1438, 1391, 1311, 1179,
1504, 1453, 1368, 1308, 1278, 1165, 1065, 987 cmꢂ1
(500 MHz, CDCl3):
;
1H NMR
1031 cmꢂ1 1H NMR (500 MHz, CD3OD):
; d 6.95e6.88 (m, 1H), 6.15
d
6.88 (dt, J¼15.9 and 7.3 Hz, 1H), 5.90 (d,
(d, J¼15.9 Hz, 1H), 5.63 (dq, J¼9.8 and 2.4 Hz, 1H), 5.13e4.80 (m,
J¼15.9 Hz, 1H), 5.52 (m, 1H), 5.23 (d, J¼9.8 Hz, 1H), 4.83 (s, 2H), 4.59
2H), 4.52 (d, J¼2.4 Hz, 1H), 1.43 (d, J¼7.3, 3H); 13C NMR (125 MHz,
(d, J¼9.8 Hz, 1H), 1.50 (s, 9H), 1.34 (d, J¼6.7 Hz, 3H); 13C NMR
CD3OD):
d 167.4 (s), 165.1 (s), 143.0 (d), 124.2 (d), 69.3 (d), 65.1 (t),
(125 MHz, CDCl3):
d
169.1 (s), 164.1 (s), 155.8 (s), 141.8 (d), 122.2 (d),
57.7 (d), 16.3 (q); HRMS FABþ calcd for C16H23N2O8 [(Mꢂ72)þH]þ:
80.6 (s), 71.4 (d), 62.9 (t), 57.3 (d), 28.3 (q), 16.3 (q); HRMS FABþ
371.1454, found: 371.1453; ½a D28
ꢂ12 (c 1.0, CH3OH).
ꢃ
calcd for C26H38N2O12Na [MþNa]þ: 593.2322, found: 593.2327.
4.8. Diamide 10
4.5. Tetraol 4
NMM (1.0 mL, 9.0 mmol) was added to a solution of 11 (0.2 g,
0.45 mmol) in THF (20 mL). The mixture was stirred at rt for 1.5 h
before 3-formamidobenzoic acid 2 (0.25 g, 0.9 mmol), EDCI (0.17 g,
0.9 mmol) and HOBt (0.18 g, 1.35 mmol) were successively in-
troduced. Stirring of mixture continued at rt for 10 h. The reaction
was terminated by addition of saturated KHSO4 (20 mL) and
extracted with EtOAc (3ꢁ15 mL). The extract was dried and evap-
orated, the residue was flash chromatographed on silica gel (gra-
dient elution 100:0 to 100:0.3, CHCl3/CH3OH) to give diamide 10
(0.18 g, 45%) as a pale yellow oil. Rf 0.78 (9:1 CHCl3/CH3OH); IR (neat)
3389, 3035, 2937, 1730, 1667, 1520,1427, 1381, 1309,1277, 1179,1067,
To a stirred solution of tetralactone 5 (0.1 g, 0.18 mmol) in t-
BuOH/H2O (1:1) was added K3Fe(CN)6 (0.28 g, 0.90 mmol), K2CO3
(0.12 g, 0.90 mmol), NaHCO3 (0.074 g, 0.90 mmol), MeSO2NH2
(25 mg, 0.27 mmol), and (DHQ)2PHAL (54.5 mg, 40 mol %). The
resulting mixture was stirred at rt for 5e10 min and then this so-
lution was cooled to 0 ꢀC. To this solution was added OsO4 (18 mg,
40 mol %), the resulting solution was stirred at 0 ꢀC and monitoring
by TLC until disappearance of starting material 5 (6 h). The reaction
was quenched by addition Na2SO3 and H2O. The resulting mixture
was extracted by CH2Cl2. The combined CH2Cl2 layers were dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The crude residue was then purified by column chroma-
tography on silica gel (gradient elution 100:0 to 97:3, CHCl3/
CH3OH) and gave a diastereomeric mixture of tetraol products
(75.1 mg, 67%), which were separated by medium pressure liquid
chromatography and afforded major diastereomer 4 (68.3 mg, 61%)
as a white solid. Rf 0.61 (5:1 CHCl3/CH3OH); mp 112e115 ꢀC; IR (KBr
disc) 3845, 2989, 1731, 1668, 1493, 1365, 1302, 1278, 1091,
984, 914 cmꢂ1 1H NMR (500 MHz, DMSO-d6):
; d 9.77 (s, 2H),
8.92e8.83 (m, 2H), 8.30 (s, 2H), 8.18 (d, J¼7.9 Hz, 2H), 7.43 (d,
J¼7.9 Hz, 2H), 7.33e7.28 (m, 6H), 7.24e7.14 (m, 4H), 6.89 (dt, J¼11.0
and 5.5 Hz, 2H), 5.98 (d, J¼15.3 Hz, 2H), 5.37e5.31 (m, 2H), 5.06 (dd,
J¼8.6 and 4.3 Hz, 2H), 4.97 (s, 4H), 4.86 (d, J¼15.9 Hz, 2H), 4.63 (d,
J¼15.9 Hz, 2H),1.18 (d, J¼6.7 Hz, 6H); 13C NMR (125 MHz, DMSO-d6):
d
168.7 (s), 167.1 (s), 164.5 (s), 160.5 (d), 145.6 (s), 143.7 (d), 136.5 (s),
131.6 (s), 130.3 (s), 128.4 (d), 128.2 (d), 128.1 (d), 124.4 (d), 124.1 (d),
123.6 (d),119.9 (d), 75.8 (t), 70.3 (d), 62.9 (t), 55.3 (d),15.7 (q); HRMS
ESIþ calcd for C46H44N4O14Na [MþNa]þ: 899.2752, found: 899.2751.
1065 cmꢂ1; 1H NMR (500 MHz, CDCl3):
d
5.39 (dq, J¼6.1 and 1.8 Hz,
1H), 5.33 (d, J¼9.8 Hz, 1H), 4.58 (dd, J¼9.8 and 1.8 Hz, 1H), 4.48 (m,
2H), 4.31 (s, 1H), 4.14 (t, J¼12.2 Hz, 1H), 4.05 (m, 2H), 3.80 (d,
J¼8.6 Hz, 1H), 1.48 (s, 9H), 1.41 (d, J¼7.3 Hz, 3H); 13C NMR (125 MHz,
4.9. Tetraol diamide 9
CDCl3): d 171.6 (s), 170.1 (s), 155.9 (s), 80.6 (s), 74.6 (d), 70.8 (d), 69.2
(d), 66.5 (t), 56.7 (d), 28.3 (q), 16.4 (q); HRMS FABþ calcd for
To a solution of diamide 10 (0.37 g, 0.42 mmol), (DHQ)2PHAL
(164 mg, 50 mol %), and NMO (297 mg, 2.52 mmol) in t-BuOH/THF/
H2O (7.5:7.5:1.5) was added OsO4 (53.6 mg, 50 mol %). The resulting
mixture was stirred at rt and monitored by TLC until disappearance
of starting material 10. The reaction was quenched with addition of
Na2SO3 (0.63 g). The resulting mixture was extracted by CH2Cl2
(3ꢁ15 mL). The combined CH2Cl2 layers were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude resi-
due was purified by column chromatography on silica gel (gradient
elution 99:1 to 96:4, CHCl3/CH3OH) to give a white solid of 4:1 dr of
C26H42N2O16Na [MþNa]þ: 661.2432, found: 661.2437.
4.6. Tetraol 3
A round-bottomed flask was charged with tetraol 4 (0.16 g,
0.24 mmol) dissolved in EtOAc (10 mL). 35% (w/v) of HCl (0.63 mL,
6.0 mmol) was added into this solution and the mixture was stirred
at rt for 12 h. After reaction was complete, the solvent was evap-
orated in vacuo, and the crude was purified by flash column

2890
A. Arsianti et al. / Tetrahedron 68 (2012) 2884e2891
tetraol diamide products (0.26 g, 66%), with 9 as major diastereomer.
This inseparable diastereomeric mixture was then used for the next
step. Rf 0.43 (8:1 CHCl3/CH3OH); mp 135e138 ꢀC; IR (KBr disc) 3370,
2934,1752,1684,1605,1583,1524,1427,1396,1268,1205,1123,1081,
MDA-MB-231 cells, and prostate PC-3 cells was determined by CCK-
8 (Cell Counting Kit-8), which is a very convenient assay utilizing
Dojindo’s highly water soluble tetrazolium salt, WST-8 [2-(2-
methoxy-4-nitrophenyl)-2H-tetrazolium, monosodium salt]. WST-
8 is reduced by dehydrogenase in cells to give a yellow-colored
WST-8 formazan. The amount of WST-8 formazan dye generated
by the activity of dehydrogenase in cells was directlyproportional
to the number of living cells.
973, 915 cmꢂ1 1H NMR (500 MHz, CDCl3), based on 9:
; d 8.37 (d,
J¼7.9 Hz, 2H), 8.22 (d, J¼8.6 Hz, 2H), 7.92 (s, 2H), 7.77 (d, J¼7.9 Hz,
2H), 7.39e7.18 (m, 12H), 5.49e5.47 (m, 2H), 5.29 (d, J¼12.2 Hz, 2H),
5.20 (dd, J¼8.6 and 4.3 Hz, 2H), 4.85e4.76 (m, 4H), 4.41e4.32 (m,
2H), 4.17e4.06 (m, 4H), 3.97 (br s, 2H), 3.84 (d, J¼7.3 Hz, 2H), 1.94 (br
s, 2H), 1.39 (d, J¼6.7 Hz, 6H); 13C NMR (125 MHz, CDCl3), based on 9:
4.11.4. Procedure of cytotoxicity evaluation of the analogue. Cell
suspension (100 mL) (5000 cells/well) of HeLa cells, breast MDA-MB-
d
171.5 (s), 169.7 (s), 165.8 (s), 158.6 (d), 146.2 (s), 135.4 (s), 131.5 (s),
129.3 (d), 129.1 (d), 128.9 (d), 126.4 (d), 125.9 (s), 125.2 (d), 125.1 (d),
78.6 (t), 74.1 (d), 70.8 (d), 69.2 (d), 66.9 (t), 55.5 (d), 16.7 (q); HRMS
ESIþ of 4:1 dr of tetraol diamide products:calcd for C46H48N4O18Na
[MþNa]þ: 967.2861, found: 967.2860.
231 cells, and prostate PC-3 cells, respectively, was dispensed to
a 96-well plate. The plate was pre-incubated for 24 h in a humidified
incubator containing 5% CO2 at 37 ꢀC. The various concentration of
tested samples and antimycin A3 (0e100 mM) were added into the
culture media in the plate. The plate was incubated for an appro-
4.10. Analogue 1
priate length of time (72 h) in the incubator. 10 mL of CCK-8 solution
was then added to each well of the plate, and the plate was sub-
sequently incubated for 1e4 h in the incubator. This mixture was
then measured for absorbance at 450 nm using a microplate reader.
Each plate contained multiple wells at a given experimental condi-
tion and multiple control wells. This procedure was replicated for
2e4 plates/condition. The half maximal inhibitory (IC50) was de-
termined using the probit method by plotting the concentration of
the sample against percentage (%) of living cells.
Mixture of 4:1 dr of tetraol diamide products (90 mg) and 10% Pd/
C (135 mg) in methanol (12 mL) were stirred under H2 atmosphere
(balloon-pressure) at room temperature for 3 h. After reaction was
complete, the solution phase was filtered through Celite and the
solid phase washed with 1:1 EtOAc/MeOH (50 mL). The combined
solvent filtrate and washings was evaporated and the residue was
flash chromatographed on silica gel (gradient elution 99:1 to 90:10,
CHCl3/CH3OH) to give a mixture of 4:1 dr of the corresponding Bn-
deprotected products. This mixture was separated by medium
pressure liquid chromatography afforded major diastereomer 1
(37.3 mg, 51%) as a white solid. The pure product 1 was then obtained
as white crystalline solid after recrystallization from methanol. Rf
0.31 (5:1 CHCl3/CH3OH); mp 142e145 ꢀC; IR (KBr disc) 3350, 2924,
2852,1745,1679,1642,1540,1434,1364,1259,1210,1128,1086,1014,
Acknowledgements
This research was supported partially by grant-in-aid for sci-
entific research from Nara Institute of Science and Technology
(NAIST) for Advanced Research Partnership Project, which is
gratefully appreciated. We also thank to Ms.Yoshiko Nishikawa for
High Resolution Mass Spectrometry measurement, and Mr. Shou-
hei Katao for X-ray Crystallographic analysis.
752; 1H NMR (500 MHz, CD3OD),
d
8.33 (s, 2H), 8.28 (d, J¼7.9 Hz, 2H),
7.75 (d, J¼7.9 Hz, 2H), 6.87 (t, J¼8.6 Hz, 2H), 5.50e5.45 (m, 2H), 5.17
(d, J¼2.4 Hz, 2H), 4.55e4.49 (m, 2H), 4.25 (s, 2H), 4.19e4.16 (m, 4H),
1.40 (d, J¼6.7 Hz, 6H); 13C NMR (125 MHz, CD3OD),
d 172.5 (s), 172.1
Supplementary data
(s), 170.8 (s), 162.1 (d), 142.9 (s), 128.2 (s), 126.4 (d), 124.1 (d), 119.3
(d), 115.8 (s), 73.9 (d), 72.3 (d), 71.1(d), 68.3 (t), 56.9 (d), 16.7 (q);
HRMS ESIþ calcd for C32H36N4O18Na [MþNa]þ: 787.1922, found:
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2012.01.080.
787.1923; ½a 3D1
þ8 (c 0.25, CH3OH).
ꢃ
References and notes
4.11. Cytotoxicity evaluation of antimycin A3 analogues
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4.11.1. Cell culture. The human cervical adenocarcinoma HeLa cell,
human breast MDA-MB-231 cells and prostate PC-3 cells were
maintained in humidified room air containing 5% CO2 at 37 ꢀC. HeLa
cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM),
breast MDA-MB-231 cells were in DMEM (low glucose), and Pros-
tate PC-3 cells were in RPMI-1640. These cells were supplemented
with 10% fetal bovineserum (FBS) and 1% penicillin-streptomycin
(GIBCO BRL, Grand Island, NY). Cells were routinely grown in
100 mm plastic tissue culture dishes and harvested with a solution
of trypsin-EDTA when they reached 80e90% of confluence. Cells
were maintained with these culture conditions for all experiment.
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22. Crystallographic data for tetraol 3 is deposited with the Cambridge Crystallo-
graphic Data Centre (Supplementary Publication No: CCDC 813908) and can be
obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK (email: deposit@ccdc.cam.ac.uk).
4.11.2. Reagent and tested sample. Antimycin A3 from Sigma-
eAldrich Chemical Co. was dissolved in ethanol at 2ꢁ10ꢂ2 M as
a stock solution. The stock solution was wrapped in foil and kept at
4
ꢀC or ꢂ20 ꢀC. The tested samples, tetralactone 11, tetraol 3, and
analogue 1 were prepared as previously described in this chapter.
Tetralactone 11 and tetraol 3 were dissolved in water at the variety
of concentration 0e100 mM, whereas analogue 1 was dissolved in
DMSO at the variety of concentration 0e100 mM.
4.11.3. Cytotoxicity assay.24,25 The effect of the tested compounds
and antimycin A3 on the cell proliferation of HeLa cells, breast

A. Arsianti et al. / Tetrahedron 68 (2012) 2884e2891
2891
23. Crystallographic data for analogue 1 is deposited with the Cambridge Crys-
tallographic Data Centre (Supplementary Publication No: CCDC 813907) and
can be obtained, free of charge, on application to CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK (email: deposit@ccdc.cam.ac.uk).
24. Nakagawa, T.; Takahashi, M.; Ozaki, T.; Watanabe, K.; Todo, S.; Mizuguchi, H.;
Hayakawa, T.; Nakagawara, A. Mol. Cell. Biol. 2002, 22, 2575.
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