M. Horikawa et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2130–2133
2133
Table 1 (continued)
Compound
Viability (%)a
O
O
O
O
20
21
109.6 2.1
96.2 2.8
N
H
OH
OH
N
H
a Viability values of P388D1 cells incubated with 50 lM of acyl-HSL analogs are shown as means SD from three determinations compared to that
of the nontreated cells.
In our previous study,7 the apoptosis activity of a limit-
ed number of compounds, 2–7, was tested and com-
pared with that of 1 using the U937 cell lines. The test
with this cell line later turned out to give a poor repro-
ducibility. In the present study, therefore, the apoptosis-
inducing activities of all compounds (1–21) were
measured on the macrophage P388D1 cell line, which
provided reproducible and thus more reliable results.
In summary, we demonstrated the synthesis of a series
of acyl-HSL analogs and their apoptosis-inducing activ-
ity. The present results revealed the structural character-
istics of the acyl-HSL analogs necessary for the
apoptosis-inducing activity in macrophages, that is, the
P388D1 cell lines, suggesting the presence of a putative
receptor in eukaryotic cells. Further investigation to dis-
cover the molecular target of 3-oxo-C12-HSL for the
induction of apoptosis in macrophages is under way.
The apoptotic activity of 1 was reconfirmed, whereas
compounds 2–7 showed no activity in agreement with
the previous result using the U937 cell lines. This again
clearly proved the importance of the presence and suf-
ficient chain length of the 3-oxo acyl group (analogs 5
and 4) and of the homoserine lactone moiety (analog
7) including the (S)-configuration at its C-2 position
(analog 6). The role of the homoserine lactone moiety
was further confirmed by the lack of activity in the
analogs 20 and 21, which are a Pseudomonas quo-
rum-sensing agonist and an antagonist,10 respectively.
Thus, we focused on compounds having comparable
or longer 3-oxo-acyl chains, and found that com-
pounds 9, 11, 13, 14, and 15 retained potent apopto-
sis-inducing activities. According to the method
previously described for 1,7 these active analogs were
also confirmed to induce active caspase-3 as one of
the apoptosis markers (data were not shown). Their
activities would be equal to that of 1 because of the
structural similarities between 1 and them. On the
other hand, 8, 10, and 12 possessing shorter chains lost
the apoptosis activity. The change in the 3-oxo group
to the 4- or 5-oxo position reduced the activity as
exemplified by compounds 16 and 17. Furthermore,
the introduction of hydrophilic functional groups, such
as hydroxy or carboxy groups, at the x-position re-
duced the apoptosis inducing ability as exemplified
by compounds 18 and 19 (Table 1).
Acknowledgments
This research was supported in part by a grant from the
Japan Society for the Promotion of Science (JSPS) and
the Ministry of Education, Culture, Sports, Science
and Technology of Japan to promote multi-disciplinary
research projects.
References and notes
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Iglewski, B. H. Science 1993, 260, 1127.
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J. Immunol. 2002, 169, 2636.
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S. W.; Daykin, M.; Williams, P.; Telford, G.; Pritchard,
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Miyairi, S.; Pechere, J. C.; Standiford, T. J.; Ishiguro, M.;
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8. Compounds 8 and 9 were synthesized in moderate yields,
respectively.
The present structure–activity relationship study dem-
onstrated that the 3-oxo group in the acyl side chain
and the homoserine lactone moiety of the L-form are
crucial for the apoptosis-inducing activity. Thus, a plau-
sible target receptor is expected to have a specific hydro-
philic pocket. Furthermore, the acyl side chains
possessing the polar groups in the end eliminated the
activity. In addition, the hydrophobic acyl side chains
longer and bulkier than that of the natural counterpart
1 kept the activity, whereas the shorter ones lost the
activity. Thus, the target receptor is also expected to
have a large and flexible hydrophobic pocket.
1. LiHMDS
then crushed dry ice
O
O
O
O
R
N
R
2. EDCI, HOBt
O
H
O
R = nC7H15
or
H2N
O
8: nC7H15 (45%)
9: nC11H23 (53%)
R = nC11H23
9. Blackwell, H. E.; O’Leary, D. J.; Chatterjee, A. K.;
Washenfelder, R. A.; Bussmann, D. A.; Grubbs, R. H.
J. Am. Chem. Soc. 2000, 122, 58.
10. Smith, K. M.; Bu, Y.; Suga, H. Chem. Biol. 2003, 10, 563.