Apoptosis-Inducing activity of synthetic intermediates of Halichlorine

Article abstract of DOI:10.1016/S0960-894X(02)00373-6

Synthetic intermediates of alkaloid halichlorine with the azaspiro core structure have been found to induce apoptosis of cultured human cells including an acute monocytic leukemia cell line (THP-1) at micromolar concentrations. The novel biological activi

Full text of DOI:10.1016/S0960-894X(02)00373-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2069–2072
Apoptosis-Inducing Activity of Synthetic Intermediates of
Halichlorine
Midori Itoh, Jun Kuwahara, Kohji Itoh,* Yu-ichi Fukuda, Mikiko Kohya,
Mitsuru Shindo and Kozo Shishido
Institute for Medicinal Resources, University of Tokushima, Sho-machi 1-78, Tokushima 770-8505, Japan
Received 12 April 2002; accepted 18 May 2002
Abstract—Synthetic intermediates of alkaloid halichlorine with the azaspiro core structure have been found to induce apoptosis of
cultured human cells including an acute monocytic leukemia cell line (THP-1) at micromolar concentrations. The novel biological
activity of the intermediates was suggested to depend on the skeletal structure and silyloxymethyl functionality on the five-mem-
bered ring.# 2002 Elsevier Science Ltd. All rights reserved.
Halichlorine1 is an alkaloid isolated from the sponge
Halichondria okadai Kadota in 1996 that possesses an
azaspiro-[4,5]decane substructure (Fig. 1).
used as a key intermediate in our previous synthetic
studies4h on halichlorine, as shown in Scheme 1.
This substance was shown to inhibit the induced
expression of VCAM-1 (vascular cell adhesion mole-
cule-1)2 in cultured human umbilical vein endothelial
cells, and might be a potential target for the treatment
of allergic inflammation, arteriosclerosis and cancer.3
The unique structure and biological activity of the
compound has promoted a variety of synthetic approa-
ches4 including total synthesis.5 In this study, we ana-
lyzed the biological effects on human cultured cells of
several compounds with a spirocyclic core subunit pre-
pared in the course of total synthetic studies on hali-
chlorine. Some of these compounds were unexpectedly
found to exhibit apoptosis-inducing activity as a novel
biological function.
Cytotoxic Activities of Azaspirobicyclic Intermediates
The cytotoxic activities of synthesized key intermediates
1–17 were assayed by the XTT method5 with a cultured
human monocytic leukemia cell line (THP-1).6 Cell via-
bility was colorimetrically estimated by conversion of
the XTT reagent to its formazan derivative with the
succinate tetrazolium reductase in the living cell culture
system in RPMI1640 medium containing 10% fetal calf
serum in the presence of 5% CO2 at 37 ꢀC Figure 2 shows
the dose-dependency of the cytotoxic effects of the syn-
thetic intermediates with the azaspirobicyclic structure
on THP-1 cells when ethanol solutions of them were
added to the culture medium. Compounds 6, 9, 15 and 16
induced dose-dependent decreases in cell viability,
whereas 7, 10 and 12 had lower effects in the same range.
Syntheses of Compounds with the Spirocyclic Core
Structure of Halichlorine
Table 1 summarizes the concentrations necessary to
decrease cell viability by 50% (IC50). The values for 16,
For biological activity evaluation, five bicyclic com-
pounds (2–5, 9) and eleven tricyclic compounds (6–8,
10–17) were prepared from tricyclic ester 1, which was
*Corresponding author. Tel.: +81-88-633-7290; fax: +81-88-633-
7290; e-mail: kitoh@ph2.tokushima-u.ac.jp
Figure 1. Halichlorine.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00373-6

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M. Itoh et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2069–2072
5, 9, 6, 15, 4, 14 and 10 were 5.0, 8.9, 25, 32, 32, 40, 63
and 71 mM, respectively. The other agents had lower
cytotoxic effects at concentrations below 100 mM. From
the aspect of the structure–activity relationship, the
spirobicyclic core substructure, the protecting t-butyldi-
phenylsilyl (TBDPS) group at C14 and the distinct
functional groups were considered to be responsible for
the expression of the cytotoxic activity. Spirotricyclic 16
exhibited the most potent effect among the compounds,
having a substructure similar to that of the natural
product. Interestingly, 12, a derivative lacking a double
bond (C2–C3), exhibited little cytotoxic activity.
remarkable condensation (data not shown). In contrast,
non-toxic intermediates including 3 and 12 did not
induce chromatin condensation at all. In the case of
potent cytotoxic 5, chromatin condensation was hardly
observed, although considerable cell debris was detected
under the experimental conditions (data not shown).
Partial induction by the other cytotoxic compounds was
observed when they were added at concentrations above
50 mM.
Activation of Caspase by the Cytotoxic Intermediates
We also examined whether or not intracellular activa-
tion of caspase 38 and/or 7,9 as apoptotic executioners,
could be induced by the addition of the cytotoxic inter-
mediates. After treatment of THP-1 cells with the com-
pounds for a definite period, cell extracts were prepared,
and then caspase activity was measured fluorometrically
with acetyl-DEVD-AMC10,11 as a substrate. As shown
in Figure 4, cytotoxic 16, 15 and 4, but not 5, induced
the activation of caspase time-dependently while 3 and
12, which exhibit little cytotoxicity at the indicated
concentrations, did not induce caspase 3 activation.
These results strongly suggest that some of the key
intermediates of halichlorine cause apoptosis of THP-1
cells. In contrast, such activation was not observed
when cells were treated with potent cytotoxic 5. One of
the reasons for this is that the cell extract was hard to
prepare because 5 induced the destruction of a large
number of the cells immediately on treatment at 5 mM.
It is also possible that the cytotoxic effect of 5 may
be induced by a different mechanism from that of
apoptosis.
Induction of Chromatin Condensation by the Cytotoxic
Intermediates
We also examined the effects of the cytotoxic inter-
mediates on induction of chromatin condensation as
one of the apoptotic phenomena in THP-1 cells. Vital
staining with Hoechst 332587 for 2 h was performed
after treatment with the compounds for 4 h. From the
fluorescent microscopic images shown in Figure 3,
cytotoxic 16 and 4 were found to induce chromatin
condensation at the concentrations of 5 and 40 mM,
respectively. Twenty micromolar 6 and 15 also caused
t
Scheme 1. (a) Bu(Ph)2SiCl, imidazole, 4-DMAP, CH2Cl2, rt, quant.;
(b) H2, PtO2, EtOH, rt, quant.; (c) LiAlH4, THF, rt, quant.; (d) tri-
.
phosgene, Et3N, CH2Cl2, rt, quant.; (e) HF pyridine, THF, rt, 81%
for 7, 63% for 17; (f) nBu4NF, THF, 50 ꢀC, ꢀ76% for 8, 89% for 11,
.
68% for 13; (g) MeLi, BF3 OEt2, Et2O,À78 C, quant.; (h) I2, Ph3P,
Figure 2. Cytotoxic activity of 6 (&), 9 (~), 15 (^), and 16 (*)
towards the THP-1 cell line treated with the indicated concentrations
for 4 h at 37 ꢀC. Each value is the mean of two independent measure-
ments with the XTT method. Compounds 7 (&), 10 (~), and 12 (*)
did not exhibit the cytotoxic activity.
imidazole, benzene, rt, 95%; (I) LDA, THF, À78 ꢀC, 95%; (j) LDA,
ClCO2Me, THF, À78 ꢀC, quant.; (k) LiBH4, Et2O, MeOH, reflux,
.
82%; (l) BH3 THF, THF, rt, 87%; (m) LDA, PhSeCl, THF, rt; (n)
mCPBA, CH2Cl2, 58% (two steps).

M. Itoh et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2069–2072
2071
Figure 3. Induction of chromatin condensation in THP-1 cells by spirocyclic intermediates: (A) control (no agent); (B) 5 mM 16; (C) 40 mM 4; (D) 50 mM
3. The cells were stained with Hoechst 33258 (0.2 mM) in culture after incubation with the compounds for 4 h at 37 ꢀC. Original magnification, Â400.
Table 1. Cytotoxic activity of key intermediates 1–17 towards the
THP-1 cell line in culture
Compd
IC50, mMa
1
2
3
4
>100
>100
>100
40
5
8.9
6
32
7
8
9
>100
>100
25
10
11
12
13
14
15
16
17
71
>100
>100
>100
63
32
5.0
>100
aValues represent the concentrations of the compounds in the culture
medium necessary to decrease the cell viability by 50%, that were
determined from dose–response curves like those shown in Figure 2.
Figure 4. Time-dependent activation of caspase 3 and/or 7 in THP-1
cells by 5 mM 16 (–*–), 20 mM 16 (*), 20 mM 15 (^) and 20 mM 4
(&) at 37 ꢀC. 20 mM 3 (!), 20 mM 12 (*) and 20 mM 5 (~) did not
induce caspase activation at all.
References and Notes
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This work was supported by a Grant-in-Aid for Scien-
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Sports and Culture, Japan. Financial support for K.S.
was also provided by the Uehara Memorial Founda-
tion. One of the authors (K.S.) is grateful for the gen-
erous financial support by the Uehara Memorial
Foundation.

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