Synthesis of tetrocarcin derivatives with specific inhibitory activity towards Bcl-2 functions

Article abstract of DOI:10.1016/S0960-894X(01)00094-4

Tetrocarcin A was recently identified as an inhibitor of the anti-apoptotic function of Bcl-2. We synthesized novel tetrocarcin derivatives in order to increase their selective inhibitory activity against Bcl-2. It was found that 21-acetoxy-9-glycosyloxy derivatives had potent Bcl-2 inhibitory activity without significant antimicrobial activity.

Full text of DOI:10.1016/S0960-894X(01)00094-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 887–890
Synthesis of Tetrocarcin Derivatives with Specific Inhibitory
Activity Towards Bcl-2 Functions
Masami Kaneko,* Takayuki Nakashima, Yuko Uosaki, Mitsunobu Hara,
Shun-ichi Ikeda and Yutaka Kanda
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, 3-6-6 Asahi-machi, Machida, Tokyo 194-8533, Japan
Received 16November 2000; accepted 31 January 2001
Abstract—Tetrocarcin A was recently identified as an inhibitor of the anti-apoptotic function of Bcl-2. We synthesized novel
tetrocarcin derivatives in order to increase their selective inhibitory activity against Bcl-2. It was found that 21-acetoxy-9-glycosyl-
oxy derivatives had potent Bcl-2 inhibitory activity without significant antimicrobial activity. # 2001 Elsevier Science Ltd. All
rights reserved.
Introduction
Bcl-2 is a membrane protein that inhibits apoptosis
induced by various stimuli.1 Elevated expression of Bcl-
2 is often found in human cancers. Consequently, those
cancer cells resist apoptosis.2 In many cases, cancers
that overexpress Bcl-2, such as follicular lymphoma and
hormone-refractory prostate cancer, are also resistant to
Chemistry
chemotherapeutic agents. Thus, inhibitors of anti-
apoptotic function of Bcl-23 are potentially useful in
treating human malignancies, particularly those that
overexpress Bcl-2. We have recently reported tetro-
carcin A (1) as the first small molecule inhibitor of the
anti-apoptotic function of Bcl-2.4 Compound 1 induced
apoptosis in cell lines that overexpressed Bcl-2 and were
resistant to cell death stimuli, such as those induced by
anti-Fas antibody (aFas), tumor necrosis factor a or
staurosporine. Although 1 was originally discovered as
an antibiotic that was active against Gram-positive
bacteria,5 the absence of Bcl-2 family proteins in bac-
teria suggests that its antibacterial activities are not
related to its activity against Bcl-2. We synthesized
tetrocarcin derivatives in order to achieve potent Bcl-2
inhibitory activity without antibacterial activities. Here
we report the synthesis and biological evaluation of
tetrocarcin derivatives.
Monomethyl ether of the tetronic acid moiety 2 was
synthesized by treatment of 1 with trimethylsilyl (TMS)
diazomethane.6 In order to introduce various sub-
stituents to the 9 position of 1, the original sugar chain
was removed by hydrolysis under acidic conditions.5d
Compound 3, which has hydroxy groups at the 9 and 21
positions, was converted to disilyloxy derivative 4 by
treatment of 10 equiv of tert-butyldimethylsilyl chloride
(TBDMSCl) for 6days and only the silyl group at
the 21 position was removed selectively with tetra-
butylammonium fluoride (TBAF). On the other hand,
21-monosilyloxy derivative 6 was obtained by treatment
with 5 equiv of TBDMSCl for 2 days in 77% yield.
Different kinds of modifications at the 9 and 21 posi-
tions can be achieved by using 5 and 6 as starting
materials. For example, 6 was condensed with carboxylic
acids in the presence of 1-(3-dimethylamino)propyl-3-
ethylcarbodiimide (EDCI) and 4-dimethylaminopyridine
(DMAP), followed by removal of the silyl group to give
9-acyloxy derivatives, such as 8 (Scheme 1). We also
synthesized derivatives with substituents at the 9 and
21 positions from 9-hydroxy-21-acetoxy derivative 9 as
*Corresponding author. Fax:+81-42-726-8330; e-mail: masami.kaneko@
kyowa.co.jp
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00094-4

888
M. Kaneko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 887–890
Scheme 1.
shown in Scheme 2. Compound 9 was synthesized from
the peracetate of 1 by hydrolysis under the same condi-
tions as synthesis of 3.5d Compound 10 was synthesized
from 9 and 4-methoxybenzyl 2,2,2-trichloroacetimidate
in the presence of trifluoromethanesulfonic acid
(TfOH).7 By the reaction of 9 with tetra-O-acetyl-
glucosyl 2,2,2-trichloroacetimidate and trimethylsilyl
trifluoromethanesulfonate (TMSOTf), orthoester 11
was obtained in 45% yield. Tetrahydropyranyl (THP)
ether 12a, the simplest analogue of the original sugar,
and deoxyglycosides 12b and 12c, could be prepared
from 9 and 3,4-dihydro-2H-pyran (DHP) or corre-
sponding glucals. THP ether 12a was a mixture of
diastereomers. Compound 12b and 12c were obtained
(Table 1). Addition of aFas to HeLa/bcl-2 cells showed
no caspase activation, but co-treatment of aFas-resis-
tant HeLa/bcl-2 cells with 1 and aFas caused DEVDase
activation. The concentration of 1 needed for aFas-
dependent 3-fold activation of DEVDase (AC3) was
2.2 mM. In the absence of aFas, 1 weakly activated
DEVDase in an aFas-independent manner with the
AC3 of 7.7 mM, indicating that 1 itself retained weak
activity as an apoptotic stimulus. Although methyl ether
2 showed more potent Fas-dependent DEVDase activity
than 1, its Fas-independent DEVDase activity was also
increased with AC3 of 2.5 mM. One class of non-sugar
type derivatives, such as 3, 4, 6 and 8, lost the DEVDase
activity both in the presence and absence of aFas. The
other non-sugar type of derivatives, such as 5 and 10,
showed aFas-dependent DEVDase activity but their
aFas-independent DEVDase activities were increased.
Deoxy sugar derivatives 12b–e showed no aFas-
independent DEVDase activity but still retained aFas-
dependent DEVDase activity, indicating that they
did not induce apoptosis by themselves but instead
inhibited anti-apoptotic function of Bcl-2. aFas-depen-
dent DEVDase activity of 12b (KF67544) was almost
comparable to 1. The SAR obtained for DEVDase
activation was very similar to that obtained from XTT
cell viability assay. It is interesting to note that these
derivatives showed very weak antibacterial activities.
Thus, the introduction of deoxy sugar at the 9 position
was effective for selective Bcl-2 inhibitory activity, but
had no effect on other Bcl-2-unrelated properties,
such as antimicrobial activities and apoptosis inducing
activities.
.
stereoselectively using Ph3P HBr without Ferrier
rearrangement.8 Ferrier rearrangement product 12d was
.
synthesized by using BF3 OEt2 in good yield. Compound
12d was converted to 12e by treatment with TBAF.
Biological Evaluations
Bcl-2 overexpressing HeLa cells (HeLa/bcl-2) were used
for studying the Bcl-2 inhibitory activities of tetrocarin
derivatives.4 We have previously determined that acti-
vation of caspases is a quantitative indicator for apop-
tosis induction associated with Bcl-2 inhibition by 1.
Caspase assay (DEVDase assay) using a tetrapeptide
substrate, Ac-DEVD-MCA [acetyl-l-aspartyl-l-gluta-
myl-l-valyl-l-aspartic acid a-(4-methyl-coumaryl-7-
amide)], was used for SAR analysis of Bcl-2 inhibition

M. Kaneko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 887–890
889
Scheme 2.
Table 1. Biological activities of tetrocarcin derivatives
Compds
DEVDase assaya
XTT assayb
BSc
AC3 (mM)
IC50 (mM)
MIC (mg/mL)
R1
R9
R21
Fas+
FasÀ
Fas+
FasÀ
1
2
3
4
5
6
7
8
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A
A
H
H
H
2.2
1
7.7
2.5
>10
>10
1.8
>10
>10
>10
>10
4.8
>10
4.9
1.4
0.8
4.0
1.5
<0.049
5.2
H
TBDMS
TBDMS
>10
>10
1.3
>10
9.5
>10
8.4
>10
>10
2.0
>10
3.9
>10
>10
2.0
>10
6.4
>50
>50
>50
>50
>50
>50
>50
TBDMS
H
TBDMS
TBDMS
H
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
MeO(CH2CH2O) 2CH2CO
MeO(CH2CH2O) 2CH2CO
>10
>10
>10
>10
9
H
o-MeOC6H5CH2
10
11
12a
12b
12c
12d
12e
3.8
5.7
3.9
2.1
3.0
4.9
3.6>10
7.65.8
>50
>50
B
THP
C
D
E
1.8
3.9
1.1
2.0
4.3
>10
>10
>50
6.3
16.7
>50
>50
4.8
>10
>10
>10
>10
>10
>10
F
3.7
aThe effect of compds on DEVD cleavage with (+) or without (-) anti-Fas antibody treatment. (AC3=concentration of 3-fold activation).
bXTT assay is for evaluation of cell viability with (+) or without (-) anti-Fas antibody treatment (ref. 4).
cAntimicrobial activity against Bacillus subtilis.

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M. Kaneko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 887–890
3. (a) Jansen, B.; Schlagbauer-Wadl, H.; Brown, B. D.; Bryan,
Conclusion
R. N.; van Elsas, A.; Muller, M.; Wolff, K.; Eichler, G. H.;
Pehamberger, H. Nat. Med. 1998, 4, 232. (b) Wang, J.-L.; Liu,
D.; Zhang, Z.-J.; Shan, S.; Han, X.; Srinivasula, S. M.; Croce,
C. M.; Alnemri, E. S.; Haung, Z. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 7124.
From a SAR study of tetrocarcin derivatives, it was
clear that the sugar moiety at the 9 position played an
important role in Bcl-2 inhibitory activity. Among the
sugar derivatives of 1, a series of deoxy sugar analogues
was found to retain Bcl-2 inhibitory activity but lost
both apoptosis inducing activity and antimicrobial
activity. Combined use of this class of tetrocarcin deri-
vatives with clinically useful anticancer agents might be
beneficial for treating chemotherapy-resistant human
tumors that overexpress Bcl-2.
4. Nakashima, T.; Miura, M.; Hara, M. Cancer Res. 2000, 60,
1229.
5. (a) Tomita, F.; Tamaoki, T.; Shirahata, K.; Kasai, M.;
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Acknowledgements
The authors thank K. Hasegawa and Y. Akasaka for
XTT and DEVDase assay; M. Kamigaki and M.
Kusunoki for antimicrobial assay; S. Kobayashi,
M. Nogi and Dr. M. Yoshida for MS and NMR
measurement; Dr. H. Nakano and Dr. S. V. Sharma
for helpful suggestions.
6. Aoyama, T.; Terasawa, S.; Sudo, K.; Shioiri, T. Chem.
Pharm. Bull. 1984, 32, 3759.
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