2770
K. Kubota et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2766–2771
ITR
LTR
0.200
0.160
0.120
0.080
0.040
0.000
0.100
0.080
*
**
***
***
30
0.060
0.040
0.020
0.000
***
30
###
###
###
###
OVA(-) Vehicle
1
3
10
10
OVA(-) Vehicle
1
3
10
38
10
39
38
39
27
mg/kg
27
mg/kg
Figure 2. Effect of 27 on OVA-induced biphasic reaction model. Each column with vertical bar represents the mean SE of 6-7 mice. *: P <0.05, **: P <0.01 and ***: P <0.001
when compared with the vehicle (0.5% MC)—treated group.[Dunnett’s test] . ###: P <0.001 when compared with the vehicle (0.5% MC)—treated group.[t-test]. 38 is Ketotifen
fumarate. 39 is Prednisolone.
proved PB affinity (94.9%) compared to 22, and had good in vivo
anti-histaminic activity. Homopiperazine compound 28 showed
more potent H1 receptor binding affinity than piperazine com-
pound 27, and this result showed good correlation with the result
of benzoate (21 and 22) and propionate (29 and 30).
Our efforts were next directed towards modification of the
phenylacetate part. The propionate 29 with piperazine unit
showed high PB (99.1%), although corresponding homopiperazine
compound 30 showed low PB (98.8%) with proper H1 binding affin-
ity, so further modification was carried out with homopiperazine
anti-inflammatory potential in mice OVA-induced biphasic cutane-
ous reaction model. Among the compounds tested, compound 27
showed potent histamine H1-receptor antagonistic activity and
remarkable anti-inflammatory activity in vivo model. It is therefore
believed that compound 27 is a promising next generation anti-
histamine.
Acknowledgments
Authors thank Mr. Kido, Ms. Negishi, and Dr. Goto for their bio-
logical evaluation of the synthesized compounds and helpful
suggestions.
compounds. Introduction of an alkyl group onto the a-position of
the carboxylate was effective in improving Caco-2 cell permeabil-
ity with a slight loss in H1 binding affinity. However, the improve-
ment in Caco-2 cell permeability did not translate into
improvement of oral bioavailability (31, 32, and 33). The pheno-
xyacetate 34 had a quite poor Caco-2 cell permeability with low
PB (95.3%), and the acrylate 35 showed weak H1 binding affinity.
The C-junction derivative 36 was also prepared for comparison
with the N-junction derivatives 21. Compound 36 exhibited in gen-
eral good properties, including favorable oral bioavailability.
Next we evaluated the anti-inflammatory effects of the phenyl-
acetate derivatives in OVA-induced biphasic reaction model. As ex-
pected, compound 27 showed inhibitory activity on both ITR and
LTR. Homopiperazine compound 30 also showed inhibitory activity
on both ITR and LTR, even though its bioavailability was low. How-
ever, compound 28 suppressed neither ITR nor LTR. This result did
not correlate with the result obtained with compound 22. It is dif-
ficult to explain the reason clearly, but one reason might be the dif-
ference of distribution to the skin. To explain this discrepancy,
clarification of the anti-inflammatory mechanism is required. The
C-junction derivative 36 showed no inhibitory activity on both of
ITR and LTR.
Finally we selected compound 27, which met all criteria, and
evaluated its inhibition of ITR and LTR in OVA-induced biphasic
reaction model. As shown in Figure 2, 27 inhibited both ITR and
LTR in a dose dependent manner. Ketotifen (38), a well known
launched anti-histamine, inhibited only ITR, while Prednisolon
(39) potently inhibited LTR. The biological properties of 27 are dif-
ferent from those of known anti-histamines, and its potential to in-
duce sedation is minimal due to the presence of a carboxylate. It is
therefore believed that compound 27 could be a next generation
anti-histamine.
References and notes
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11. Membrane of stable recombinant CHO-K1 cells expressing the human
histamine H1 receptor (Euroscreen) was incubated with various
concentrations of compounds and 1.25 nM [3H]-pyrilamine in
a
total of
l of binding buffer (50 mM Tris–HCl, pH 7.5). Nonspecific binding was
determined in the presence of 1.25 M triprolidine (Sigma). The assay mixture
200
l
l
was incubated for 3 h at room temperature and then filtered through GF/B
filters. These were washed and counted by a scintillation counter. Specific
binding was calculated as the difference between total and nonspecific
bindings.
In future studies, we will continue further structural optimiza-
tion of compound 27 and will try to clarify the mechanism of its
anti-inflammatory effect.
We synthesized a series of phenothiazine carboxylic acid deriv-
atives, having 6-amino-pyrimidine-2,4(1H,3H)-dione moiety and
evaluated their affinity toward human histamine H1 receptor and
Caco-2 cell permeability. Selected compounds were further evalu-
ated for their oral anti-histaminic activity in mice and bioavailabil-
ity in rats. Finally, promising compounds were examined for their
12. The apical side of Caco-2 cell line monolayer was incubated with the
compounds at 37 °C for 120 min. The compound concentration of an apical
side and a basal side was measured. The permeability coefficient (Papp) was
calculated by following scheme.
Papp = dC/dt ꢁ VR/(A ꢁ C0); C0: initial donor concentration (
reserver well (mL), A: membrane surface area (cm2), dC/dt: slope of the
cumulative reserver concentration ( M/s).
lM), VR: volume of
l
13. Eight-week old female mice (ddY, SLC) was administered orally with various
doses of compounds suspended in 0.5% methylcellulose 1 h before the