Novel non-peptide inhibitors targeting death receptor-mediated apoptosis

Article abstract of DOI:10.1016/j.bmcl.2003.08.003

We have previously reported that ECH, (2R, 3R, 4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one inhibits Fas-mediated apoptosis by blocking self-activation of pro-caspase-8 in the death-inducing signaling complex (DISC). A series of ECH derivatives were asymmetrically synthesized via key synthetic intermediates obtained from lipase-catalyzed kinetic resolution. Inhibitory activities of the derivatives towards death receptor-mediated apoptosis both in type I and type II cells were investigated, revealing that novel non-peptide inhibitors, RKTS-33 and RKTS-34, are effective as ECH.

Full text of DOI:10.1016/j.bmcl.2003.08.003

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3743–3746
Novel Non-peptide Inhibitors Targeting Death
Receptor-Mediated Apoptosis
Hideaki Kakeya,a,* Yasunobu Miyake,a,c Mitsuru Shoji,b Satoshi Kishida,b
Yujiro Hayashi,b Takao Kataokac and Hiroyuki Osadaa,*
aAntibiotics Laboratory, RIKEN Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
bDepartment of Industrial Chemistry, Faculty of Engineering, Tokyo University of Science,
Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
cDivision of Bioinformatics, Center for Biological Resources and Informatics, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
Received 14 July 2003; revised 2 August 2003; accepted 3 August 2003
Abstract—We have previously reported that ECH, (2R, 3R, 4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-
5-en-1-one inhibits Fas-mediated apoptosis by blocking self-activation of pro-caspase-8 in the death-inducing signaling complex
(DISC). A series of ECH derivatives were asymmetrically synthesized via key synthetic intermediates obtained from lipase-cata-
lyzed kinetic resolution. Inhibitory activities of the derivatives towards death receptor-mediated apoptosis both in type I and type II
cells were investigated, revealing that novel non-peptide inhibitors, RKTS-33 and RKTS-34, are effective as ECH.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
The resulting assemblage of proteins is known as the
death-inducing signaling complex (DISC), formation of
which is a trigger for downstream activation.7,8 In Fas-
mediated apoptosis, two types of cells are proposed to
transmit distinct death signals.9,10 In type I cells, such as
human Burkitt’s lymphoma SKW6.4 cells, sufficient
activation of pro-caspase-8 is initiated at the DISC fol-
lowed by direct activation of pro-caspase-3. However,
in type II cells, such as human T lymphoma Jurkat cells,
pro-caspase-8 is inadequate for direct activation of pro-
caspase-3, and cleaves the Bcl-2family member Bid
instead, activating the mitochondrial pathway with the
release of cytochrome c, which leads to the activation of
caspase-9/-3 cascade.
Apoptosis, or programmed cell death, plays a vital role
in the development and homeostasis of multicellular
organisms.1À3 Depending on death stimuli, one of two
signaling pathways is activated: a death receptor-
dependent pathway and a death receptor-independent
pathway via the mitochondria.2,3 In both cases, stimu-
lation of the death pathway leads to processing and acti-
vation of initiator caspases, that is pro-caspase-8 or -9,
which subsequently transmit the signal to downstream
effector caspases, for example caspase-3, -6, or-7.4,5
Fas antigen (CD95/Apo-1) is a cell surface death recep-
tor molecule, a member of the tumor necrosis factor
(TNF), introducing apoptosis-inducing signals into Fas-
bearing cells by stimulation with Fas ligand (FasL) or
agonistic anti-Fas monoclonal antibodies.2,3 Following
stimulation by FasL, the cytosolic tail of the Fas recep-
tor associates with the death domain (DD) of the adap-
tor molecule FADD,6 which then recruits caspase-8 via
a homophilic death effector domain (DED) interaction.
Since some pathological conditions such as rheumatoid
arthritis, liver inflammation, autoimmune diseases, and
neurodegenerative disorders are characterized by excess
receptor-mediated apoptosis, we screened anti-apopto-
sis agents among microbial metabolites, and reported
identification of an inhibitor of Fas-mediated apoptosis,
ECH [(2R, 3R, 4S)-2,3-epoxy-4-hydroxy-5-hydroxy-
methyl-6-(1E)-propenyl-cyclohex-5-en-1-one], which is
produced by a fungus.11À14 ECH blocks the self-activa-
tion of pro-caspase-8 in the DISC and selectively inhi-
bits death receptor-mediated apoptosis.14 Herein, we
*Corresponding author. Tel.: +81-48-467-9542; fax: +81-48-462-
4669; e-mail: hkakeya@riken.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.003

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H. Kakeya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3743–3746
Scheme 1. Synthesis of RKTS-33 and RKTS-34.
describe design, synthesis, and structure–activity rela-
tionships (SAR) of ECH-related molecules, shown in
Figure 1, in receptor-mediated apoptosis.
furan and acryloylchloride proceeded smoothly without
a Lewis acid catalyst, affording the addition product.
Hydrolysis, followed by the iodolactonization reaction,
provided iodolactone, (Æ)-2-iodo-4,8-dioxa-tricyclo
[4.2.1.03,7]nonan-5-one, in large quantity without col-
umn chromatography or distillation. After conversion
of the iodolactone to cyclohexenol (Æ)-1, the kinetic
resolution by Pseudomonas stutzeri lipase (Meito TL)
proceeded with high efficiency, affording (+)-1 and
acetate (À)-2 in 49% with 99% ee and in 48% with 96%
ee, respectively.17 Hydroxy group-directed Sharpless
epoxidation18 of (+)-1 proceeded diastereoselectively to
provide (+)-3. The reduction of ester (+)-3 with
NaBH4 and protection of the generating primary alco-
hol with tert-butyldimethylsilylchloride (TBSCl) affor-
ded silyl ether (+)-5 in good yield. Oxidation of (+)-5
with Dess–Martin periodinane (DMP),19 followed by
isomerization with silica gel, produced (+)-6. Depro-
tection of the alcohol gave RKTS-33 in 71% yield.20
Protection of the diol RKTS-33 with acetonide and
iodonation afforded the key intermediate (+)-7. As the
key intermediate (+)-7 was in hand, the next task was
the introduction of the side chain. The Suzuki coupling
reaction with dihydroxy(1-methyl-1-propenyl)borane
was examined in detail and it was found that coupling
reaction proceeded smoothly under Johnson’s condi-
tions,21 affording (+)-8. Final deprotection under acidic
conditions gave RKTS-34 in 57% yield over two
steps.22
Design and Synthesis of ECH-related Molecules
In order to prepare several C-6 side-chain derivatives of
ECH efficiently, we designed 6-iodocyclohexenone deri-
vative (+)-7 as a key synthetic intermediate, because the
Suzuki coupling reaction of (+)-7 with several alke-
nylboranes would afford the corresponding side-chain
analogues (Scheme 1).
The synthesis starts from the Diels–Alder reaction
between furan and an acrylate derivative. Though we
have already reported the HfCl4-mediated highly dia-
stereoselective Diels–Alder reaction of furan and chiral
acrylate with Corey’s auxiliary,15,16 a more practical
method for the synthesis of the key intermediate has
been developed as follows. The Diels–Alder reaction of
RKTS-31, RKTS-35, and RKTS-17 were also synthe-
sized from the key intermediate (+)-7 by the same
Suzuki coupling reaction with dihydroxyvinylborane,
dihydroxystyrylborane, and dihydroxy(1-heptenyl)bor-
ane, respectively, followed by deprotection of the silyl
group. RKTS-14 was prepared from ECH in three
steps: The protection of the primary alcohol with
TBSCl, protection of the secondary alcohol with methyl
ether (MeI, Ag2O),23 and deprotection of the silyl ether
with tetrabutylammonium fluoride (TBAF) afforded
RKTS-14 in 54% yield. The enantiomer of ECH, ent-
ECH, was prepared from (À)-1, which was synthesized
Figure 1. Structures of ECH and ECH-related compounds. The geo-
metry of the double bond at the C-7 position is an E configuration.

H. Kakeya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3743–3746
3745
Table 1. Effect of novel death receptor-mediated apoptosis inhibitors
on FasL- and staurosporine-induced apoptosis in SKW6.4 and Jurkat
cells
SKW 6.4 cells (Type I)a
Jurkat cells (Type II)a
b
c
d
b
c
d
ED50
ED50
IC50
ED50
ED50
IC50
ECH
5
20
20
10
4
10
nee
10
>80
>80
>80
>80
>80
>80
ne
>80
80
6
25
25
18
6
10
ne
ne
>80
>80
>80
>80
>80
>80
ne
>80
60
ent-ECH
RKTS-14
RKTS-33
RKTS-31
RKTS-34
RKTS-35
RKTS-17
RKTS-32ne
RKTS-36
>80
>80
>80
>80
30
>80
>80
>80
>80
20
ne
35
ne
15
ne
9
ne
ne
8
ne
ne
7
ne
ne
10
aSee text.
b50% effective dose (mM) of FasL-induced apoptosis inhibition.
c50% effective dose (mM) of staurosporine-induced apoptosis inhibi-
tion.
dConcentration (mM) of 50% inhibitory activity on cell growth.
eNo effect due to its cytotoxicity.
with the ED50 value of 10 mM selectively inhibited
apoptosis induced by FasL as almost effectively as
ECH, while RKTS-17 exhibited cytotoxicity at con-
centrations over 20 mM (Figure 2b, c and e). Moreover,
RKTS-35, which possesses a bulky phenyl group, did
not inhibit FasL-induced apoptosis but rather showed
cytotoxicity at an IC50 value of 30 mM (Fig. 2d). In
striking contrast to RKTS-33 and RKTS-34, RKTS-32
exhibited cell toxicity without any inhibitory action on
FasL-induced apoptosis (Fig. 2f), suggesting that, intri-
guingly, the apoptosis-inhibitory activity is due to the
length of the side chain at C-6 as well as the secondary
hydroxy group at C-4. A summary of all the compounds
tested in type I and type II cells is shown in Table 1.
Compounds ent-ECH, the enantiomer of ECH, and
RKTS-14, with a methoxy group at C-4 of ECH,
inhibited Fas-induced apoptosis with ED50 values of
20ꢀ25 mM in both types of cells, indicating that the
nature of stereochemistry in addition to the secondary
hydroxy group of ECH is of importance. RKTS-31,
RKTS-33, and RKTS-34 selectively blocked Fas-
induced, but not staurosporine-induced apoptosis,
without remarkable cell toxicity, suggesting that these
inhibitors are effective in blocking death receptor-medi-
ated apoptosis in both types of cells. Moreover, these
three inhibitors also inhibited apoptosis induced by
agonistic anti-Fas antibody CH-11 (data not shown).
On the contrary, an epoxyquinoid derivative, RKTS-36,
with a ketone group at C-4, did not inhibit FasL-
induced apoptosis at all; that is consistent with the case
of RKTS-32.
Figure 2. Effect of novel receptor-mediated apoptosis inhibitors on
FasL-induced apoptosis in SKW6.4 cells. *: none, *: FasL, &:
staurosporine.
by the hydrolysis of ester (À)-2 in good yield.17 Epoxy-
quinone derivatives RKTS-32and RKTS-36 were syn-
thesized in three steps from ECH and RKTS-17,
respectively: Protection of the primary alcohol with
TBSCl, followed by oxidation with DMP and depro-
tection of the silyl ether, gave epoxyquinone derivatives.
Structure–Activity Relationships (SAR) in Blocking
FasL-induced Apoptosis
In both type I and type II cells, once pro-caspase-3 is
efficiently activated, it cleaves a variety of cellular sub-
strates including DNA repair enzymes, structural pro-
teins, and endonucleases to produce the changes
associated with apoptosis. We therefore investigated the
biological activities of the above synthetic compounds
both in SKW6.4 (type I) and Jurkat (type II) cells.
Apoptosis-inhibitory activities of test compounds were
assessed by the measurement of cell viability.24 Cross-
linked FasL and staurosporine (a broad-specificity pro-
tein kinase inhibitor) are able to induce apoptosis in
both types of cells primarily via the Fas/FasL-system or
the mitochondria pathway, respectively.14 Figure 2
shows the dose-dependent effect of selected compounds
on FasL- and staurosporine-induced apoptosis in
SKW6.4 cells. ECH inhibited FasL-induced apoptosis
without decreasing cell viability at the ED50 value of 5
mM (Fig. 2a). We found that RKTS-33 and RKTS-34
Conclusions
Novel apoptosis inhibitors, RKTS-33 and RKTS-34,
which were asymmetrically synthesized via a key syn-
thetic intermediate resulting from lipase-catalyzed
kinetic resolution, exhibited selective inhibitory activity
toward Fas-mediated, but not staurosporine-induced,
apoptosis. Although the class of substrate-mimicking

3746
H. Kakeya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3743–3746
peptide inhibitors has been previously reported, the
important aspect of ECH derivatives described here is
that these are membrane-permeable inhibitors as well as
non-peptide inhibitors.
14. Miyake, Y.; Kakeya, H.; Kataoka, T.; Osada, H. J. Biol.
Chem. 2003, 278, 11213.
15. Shoji, M.; Yamaguchi, J.; Kakeya, H.; Osada, H.; Haya-
shi, Y. Angew. Chem. Int. Ed. 2002, 41, 3192.
16. Hayashi, Y.; Nakamura, M.; Nakao, S.; Inoue, T.; Shoji,
M. Angew. Chem. Int. Ed. 2002, 41, 4079.
17. Shoji, M.; Kishida, S.; Takeda, M.; Kakeya, H.; Osada,
H.; Hayashi, Y. Tetrahedron Lett. 2002, 43, 9155.
18. Sharpless, K. B.; Michaelson, R. C. J. Am. Chem. Soc.
1973, 95, 6136.
19. (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48,
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Acknowledgements
This work was supported in part by a Special Project
Funding for Basic Science (Chemical Biology Research)
from RIKEN, and a grant from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan.
20. 1H NMR (400 MHz, CD3OD) d 3.39–3.42(1H, m), 3.75–
3.77 (1H, m), 4.18 (1H, d, J=17.2Hz), 4.42(1H, d, J=17.2
Hz), 4.49 (1H, s), 6.03 (1H, brs); 13C NMR (100 MHz, CDCl3)
d 53.89, 58.19, 62.85, 63.85, 120.52, 161.94, 195.76; IR (neat) n
3352, 2920, 2852, 1676, 1132, 1041, 856, 806 cmÀ1; MS (FAB-
MS) m/z 155 (MÀH)À.
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