N. Ishimata et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
the activity of scavenging degranulation factor was related to elec-
A
30 μM
100 μM
300 μM
2
3
tron density on the benzene ring. Therefore, this result indicates
that the ester structure itself of VA esters, an oxycarbonyl group
directly connected to the benzene ring, is a critical factor for inhi-
bition of the degranulation in mast cells.
4
3
2
1
0
0
0
0
0
In design 2, structure–activity relationships of functional
groups on the benzene ring were investigated (Fig. 3B). When the
positions of the hydroxyl and methoxy groups on the benzene ring
of derivative 5 were changed, the degranulation inhibitory activity
of 9 showed the same tendency as that of 5. However, removal of
all functional groups (14) resulted in loss of the inhibitory activity.
These results suggested that functional groups on the benzene ring
were essential for the degranulation inhibitory activity. When only
the hydroxyl group on the benzene ring was in the para or meta
position, analogs 10 and 11 showed greater degranulation inhibi-
tory activities than those of 5 and 9 with the methoxy group and
hydroxyl group on the benzene ring. On the other hand, degranu-
lation inhibitory activity of analogs 12 and 13 methylated to the
hydroxyl group of the analogs 10 and 11 was abolished, suggesting
that the methoxy group on the benzene ring of VA esters inhibited
their activity. From these results, it was found that a simple struc-
ture with a hydroxyl benzoic acid ester such as paraben or m-
hydroxy benzoate showed potent degranulation inhibitory activity.
Parabens have been reported to cause allergic contact dermati-
tis.24 Indeed, parabens with a C7–10 side chain showed significant
histamine release in vitro and heptylparaben elicited a strong skin
reaction in vivo, but parabens with a C1–4 side chain caused no sig-
nificant degranulation and butylparaben did not induced a strong
skin reaction. It was also reported that methyl paraben possibly
*
*
*
*
Cont.
Ox.
BVA
PPA
EPA
Control Oxatomide
75 μM)
5
7
8
(
B
30 μM
100 μM
300 μM
#
#
4
3
2
1
0
0
0
0
0
#
#
*
*
*
*
*
*
**
*
*
*
*
2
5
has some inhibitory effects on histamine release. Although para-
bens have been reported to cause allergic contact dermatitis,
degranulation might be inhibited by a paraben with adequate
lipophilicity of the alkyl side chain. Our data agree with data in
those previous reports. The VA ester with the longest chain, deriva-
tive 6, showed cytotoxicity and increased degranulation as result,
although VA esters with a C1–4 chain inhibited degranulation. It
is thought that allergic contact dermatitis induced by paraben with
a heavy side chain is indirectly caused by cytotoxicity. Therefore,
these results of the structure–activity relationship study revealed
that directly connecting the butyl ester moiety on the benzene ring
was required for potent degranulation inhibitory activity and that
at least one hydroxyl group on the benzene ring was required for
the activity.
�
BVA
�BꢀVA
�B4A
�B3A
�BPA
�BMA
�BA
5
9
10
11
12
13
14
�
Cont.
�Ox.
Figure 3. Inhibitory effects of 5 analogs on antigen-induced degranulation in RBL-
H3 cells. (A) Degranulation inhibitory activity of analogs 7 and 8 in design 1. (B)
Degranulation inhibitory activity of analogs 9–14 in design 2. DNP-IgE-sensitized
RBL-2H3 cells were incubated with 75 M oxatomide for a positive control or with
indicated concentrations of these samples for 20 min and stimulated with DNP-HSA
for 1 h. Release of b-hexosaminidase was measured. Each value is the mean of three
2
l
**
independent cultures, and the bars show SD. p < 0.01 (Dunnett’s test) as compared
with the control. #p < 0.05 and ##p < 0.01 (Student’s t-test) as compared with 5 or 9.
inhibitory activity and whether the type of functional group on the
benzene ring was important for exhibiting the activity. We propose
two kinds of design for developing structure–activity relationships
Table 1). Synthetic methods are shown in Supplementary data. In
design 1, VA ester analogs 7 and 8, in which the position of the
4
In conclusion, derivative 5 with a C straight-chain ester had the
most potent degranulation inhibitory activity among the VA ester
derivatives, and it exhibited approximately three-fold greater inhi-
bitory activity than that of original compound 1. Moreover, analog
(
1
0 demethoxylated from derivative 5 and its isomer (11) had sig-
ester group in the lipophilic chain varied, were designed. In design
, VA ester analogs 9–14, in which the types of functional groups at
nificantly potent activity in comparison with that of derivative 5.
From the results of study on structure–activity relationships of
VA esters, we found that the minimum structure for potent degran-
ulation inhibitory activity required direct connection of butyl ester
moiety on the benzene ring and at least one hydroxyl group on the
benzene ring. The results of this study are expected to contribute to
the development of novel anti-allergic drugs with simple
structures.
2
the para and/or meta positions on the benzene ring were changed,
were designed. These VA ester analogs were designed on the basis
of derivative 5, which showed the most potent degranulation inhi-
bitory activity among VA ester derivatives 1–6.
In design 1, the position of the ester group was changed from
the benzoic acid group to phenylacetic acid and phenylpropionic
acid groups (7 and 8) in order to investigate whether only the
lipophilicity of the ester group was necessary for the role in mem-
brane permeability and distribution or whether the structure of the
ester group was involved in the degranulation inhibitory activity.
When the ester moiety was not directly connected to the benzene
ring (7 and 8), the inhibitory degranulation activity was abolished
Acknowledgments
The authors are grateful to the SC-NMR Laboratory of Okayama
University and the MS Laboratory of Faculty of Agriculture,
Okayama University. We thank Dr. Kenichi Harada and Prof.
Yoshiyasu Fukuyama at Faculty of Pharmaceutical Sciences,
Tokushima Bunri University for the measurement of NMR spectra.
(
Fig. 3A). This result suggested that the resonance structure
between the benzene ring and oxycarbonyl group interacted with
the factor of degranulation. Actually, it has been reported that
Ishimata, Nao
