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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).