Synthesis and biological activity of new quinoxaline antibiotics of echinomycin analogues

Article abstract of DOI:10.1016/j.bmcl.2003.09.086

Novel quinoxaline antibiotics having the methylenedithioether bridge as an analogue of echinomycin have been synthesized by insertion of methylene moiety between -S-S- bond. The compound 1a shows remarkable cytotoxicities against human tumor various cell

Full text of DOI:10.1016/j.bmcl.2003.09.086

Bioorganic & Medicinal Chemistry Letters 14 (2004) 541–544
Synthesis and biological activity of new quinoxaline antibiotics of
echinomycin analogues§
Yun Bong Kim,a Yong Hae Kim,a,* Ju Youn Parkb and Soo Kie Kimb
aCenter for Molecular Design and Synthesis, Department of Chemistry, Korea Advanced Institute of Science and Technology,
Taejon 305-701, South Korea
bDepartment of Microbiology, Wonju College of Medicine, Institute of Basic Medical Science, IFBB, Yonsei University,
Wonju 220-701, South Korea
Received 27 August 2003;revised 24 September 2003;accepted 24 September 2003
Abstract—Novel quinoxaline antibiotics having the methylenedithioether bridge as an analogue of echinomycin have been synthe-
sized by insertion of methylene moiety between –S–S– bond. The compound 1a shows remarkable cytotoxicities against human
tumor various cell lines, and is active VRE (vancomycin-resistant enterococci) within MIC range 0.5–8 mg/mL. According to the
eukaryotic or prokaryotic data, 1a might be a first analogue to replace echinomycin.
# 2003 Elsevier Ltd. All rights reserved.
The quinoxaline antibiotics of bicyclic octadepsipep-
tide1 show activity against gram-positive bacteria2 and
certain animal tumors,3 and also are potent inhibitors of
RNA synthesis.4 The mechanism of action apparently
occurs by binding to DNA in which they function as
bifunctional intercalating agents.5 Two antibiotic famil-
ies of the antibiotic echinomycin,4,5 and the triostins,6
are well known. Both series are similar in composition,
consist of two quinoxaline-2-carboxylic acid moieties
attached to a cyclic octadepsipeptide containing a sulfur
cross-linkage. Echinomycin contain a thioacetal cross
bridge. Few reports7 have appeared in the synthetic
studies on the quinoxaline antibiotics. In our earlier work
on echinomycin, sulfonium salts of echinomycin showed
stronger antitumor activity than that of echinomycin.8
bridge (1a,b). Djenkolic acid-[3,30-(methylenedithio)dia-
lanine], isolated from the djenkol bean, has a unique
methylene dithioether structure corresponding a mono-
carba analogue of the biscystein trisulfide.9
The 6a and 6b have been achieved basically according to
the method for preparing triostins, which involves the
preparation of tetradepsipeptides (5a,b) as a key inter-
mediate. Tetradepsipeptides represents one-half of the
symmetrical octadepsipeptides portions of 1a and 1b.
Fragment coupling of free C-terminal and free N-terminal
tetradepsipeptide each prepared from 5aand 5bby removal
of appropriate protecting group, gave linear octadepsipep-
tide possessing the complete amino acid sequence of 1a and
1b. Further transformations involve disulfide formation,
cyclization, methylene insertion and introduction of qui-
noxaline chromophore provided 1a and 1b (Fig. 1).
However echinomycin or such an analogue of echinomy-
cin having a methylenedithioether moiety has never been
reported yet. In order to study the biological activity of
echinomycin analogues, a series of new quinoxaline anti-
biotics (1–4) containing thioether, sulfoxide and sulfone
moiety have been prepared. We wish to describe herein
an efficient synthesis of a quinoxaline antitumor anti-
biotic having a djenkolic acid moiety as a sulfur cross
The Cbz-d-Ser-Opa10 was coupled with Boc-l-MeVal-
OH11 by using DCC-HOBt (N-hydroxy benzotriazole)
in pyridine to give didepsipeptide [Cbz-d-Ser-Opa-Boc-
l-Val], in 82% yield. After removal of the Boc group
with CF3CO2H (TFA) in CH2Cl2, resulting didepsi-
peptides were coupled with Boc-l-MeCys(Bam)-OH12 or
Boc-l-Cys(Bam)-OH using DCC-HOBt to give tridepsi-
peptide [Cbz-d-Ser-Opa-l-MeVal-Boc-l-MeCys (Bam)],
in 74% yield. The Boc group was removed from tridepsi-
peptide and then reacted with (Boc-l-Ala)2O to give tetra-
depsipeptide [Cbz-d-Ser-Opa-l-MeVal-l-MeCys(Bam)-
Boc-l-Ala] (5a). The protected tetradepsipeptides (5a)
Keywords: Echinomycin;Anticancer;Active-VRE;Apoptosis.
§Supplementary data associated with this article can be found at
doi:10.1016/j.bmcl.2003.09.086
* Corresponding author. Tel.: +82-42-869-2818;fax: +82-42-869-
5818;e-mail: kimyh@kaist.ac.kr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.086

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Y. B. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 541–544
Figure 1. Structure of echinomycin and new quinoxaline antibiotics.
Scheme 1. (a) NaSeH, EtOH, 0 ꢀC;(b) TBAF– xH2O, CH2Cl2;(c) (i) 30% HBr in AcOH;(ii) Q xCl, Et3N, DMF.
was treated with Zn in 90% aqueous acetic acid effected
reductive cleavage of the phenylacyl ester function to
provide tetradepsipeptide[Cbz-d-Ser-l-MeVal-l-MeCys
(Bam)-Boc-l-Ala], having a free C-terminal carboxyl
group. Tetradepsipeptide [Cbz-d-Ser-Opa-l-Val-l-
MeCys(Bam)-l-Ala] was prepared by treatment of 5a
with TFA-CH2Cl2 as the trifluoroacetate salt. The cou-
pling of free C-terminal and free N-terminal tetra-
depsipeptide with DCC-HOBt in THF afforded the
linear octadepsipeptide [Cbz-d-Ser-Opa-l-MeVal-l-Me
Cys(Bam)-Boc-l-Ala-Cbz-d-Ser-l-MeVal-l-MeCys
(Bam)-Boc-l-Ala] in 65% yield. Cyclization of linear
octadepsipeptide to provide the 26-membered cyclic
octadepsipeptides (6a) with ring closure was accom-
plished through four steps sequential Pa ester deprotec-
tion (Zn, 90% aqueous AcOH, 0 ꢀC, 4 h),13 disulfide
bond formation of free C-terminal linear octadepsipep-
tide (disulfide-linkage octadepsipeptide, I2, CH2Cl2–
MeOH, 25 ꢀC),14 Boc deprotection, and cyclization was
followed by treatment with [Cbz-d-Ser-l-MeVal-l-
MeCys(Bam)-Boc-l-Ala-Cbz-d-Ser-l-Me-Val-l-Me-
Cys(Bam)-l-Ala] [10.0 equiv of 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (EDCI),
HOBt, CH2Cl2, 0 ꢀC, 24 h].
The synthesis of novel quinoxaline antibiotic was ach-
ieved through four steps from 6a. Reductive cleavage of
the disulfide gave dithiol (7a) by ethanolic NaSeH.15
A
methylene insertion to construct an S–CH2–S bridge
between two N-Me cysteine residue was performed by
using tetrabutylammonium fluoride hydrate in CH2Cl2
(8a).16 Removal of the benzyloxycarbonyl group (HBr
in acetic acid) and acylation with 2-quinoxalyl chlo-
ride17 gave 1a. The 1b, 7b, and 8b were prepared by the
same method described for 1a and their yields are
shown in Scheme 1.
Oxidation of 1a and 1b with m-CPBA or dimethyl-
dioxirane provided the corresponding monosulfoxide
(2a), disulfoxide (3a) and disulfone (4a,b) (Scheme 2).
Anticelluar activities of new compounds were evaluated
in vitro against various cell lines.18 The results are
summarized in Table 1.

Y. B. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 541–544
543
Table 2. Apoptosis induced in HT-29 by various stimuli using
FACScan
Stimuli
Percentage of
apoptotic cells
Control
Echinomycin
1a
9.6ꢁ1.1
47.5ꢁ0.9
28.9ꢁ0.8
HT-29 cells were treated with echinomycin (2 mg/mL) and 1a (10 mg/
mL) for 24 h (each concentration is the lowest one to initiate the
apoptosis of HT-29 cell).
The percentage of apoptotic cells was assessed by flow cytometry.
Results are expressed as meanꢁSEM of at least three separate
experiments.
In summary, the synthesis of new compounds 1a, 1b, 2a,
3a, 4a and 4b were successfully achieved. According to
the eukaryotic or prokaryotic data, the novel compound
1a might be a first analogue to replace echinomycin.
Acknowledgements
Scheme 2. Oxidation of 1a by m-CPBA and 1a,b by dimethyldioxir-
ane.
This work was supported by Grant No. R02-2002-000-
00097-0 from Korea Science & Engineering Foundation.
a
Table 1. MTT assay for IC50 values of novel antibiotics on various
cell lines
References and notes
1a 1b 2a
3a 4a
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PANC-1 (pancreas)
BeWO (placenta)
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4.0 >20 >20 >20 >20 >20
2.7 >20 >20 >20 >20 >20
2.2
1.8
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enabled to induce apoptosis of HT-29 cells (Table 2).
The signaling mechanism exerted by echinomycin or 1a
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cytotoxicity against solid cancer cells compared to
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Moreover, 1a is active VRE (vancomycin-resistant ent-
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mycin: 0.25 mg/mL). Echinomycin and related com-
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overcome by 1a, analogues of echinomycin.

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Y. B. Kim et al. / Bioorg. Med. Chem. Lett. 14 (2004) 541–544
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