Antiallergic activity profile in vitro RBL-2H3 and in vivo passive cutaneous anaphylaxis mouse model of new sila-substituted 1,3,4-oxadiazoles

Article abstract of DOI:10.1021/jm300421h

A new class of sila-substituted 1,3,4-oxadiazoles was synthesized and evaluated for antiallergic activity using RBL-2H3 as the in vitro model and the in vivo anaphylactic mouse model. We observed that compound 5c effectively suppressed DNP-HSA-induced mast cell degranulation, compared to carbon analogue 9, and also suppressed the expression of TNF-α mRNA and Akt phosphorylation in antigen-stimulated RBL-2H3 cells. We also studied the effect of 5c in an in vivo passive cutaneous anaphylaxis (PCA) mouse model. The suppression by 5c was more effective than that by diphenylhydramine (DPH), a typical anti-histamine drug.

Full text of DOI:10.1021/jm300421h

Article
pubs.acs.org/jmc
Antiallergic Activity Profile in Vitro RBL-2H3 and in Vivo Passive
Cutaneous Anaphylaxis Mouse Model of New Sila-Substituted 1,3,4-
Oxadiazoles
Guda Dinneswara Reddy,†,§ Sae-Jin Park,‡,§ Hyeon Mo Cho,† Tack-Joong Kim,*,‡ and Myong Euy Lee*,†
†Department of Chemistry and Medical Chemistry, College of Science and Technology, Research and Education Center for
Advanced Silicon Materials, Yonsei University, Wonju, Gangwon-do 220-710, South Korea
‡Division of Biological Science and Technology, College of Science and Technology, Yonsei University, Wonju, Gangwon-do
220-710, South Korea
S
* Supporting Information
ABSTRACT: A new class of sila-substituted 1,3,4-oxadiazoles
was synthesized and evaluated for antiallergic activity using
RBL-2H3 as the in vitro model and the in vivo anaphylactic
mouse model. We observed that compound 5c effectively
suppressed DNP-HSA-induced mast cell degranulation,
compared to carbon analogue 9, and also suppressed the
expression of TNF-α mRNA and Akt phosphorylation in
antigen-stimulated RBL-2H3 cells. We also studied the effect
of 5c in an in vivo passive cutaneous anaphylaxis (PCA) mouse
model. The suppression by 5c was more effective than that by
diphenylhydramine (DPH), a typical anti-histamine drug.
extracellular matrix degradation is an important event during
the remodeling of inflamed tissue.10
INTRODUCTION
■
Day-by-day allergy disease increases drastically throughout the
world because of host (host factors include heredity, gender,
race, and age) and environmental factors.1 Allergy is an
immunological disorder that occurs when the immune system
reacts to normally harmless substances in the environment.
Most of the allergic reactions are distinctive because of
excessive activation of certain mast cells and basophils by an
antibody immunoglobulin E (IgE).2 Mast cells are central to
the pathogenesis of immediate hypersensitivity, and they are
located in blood vessels and at epithelial surfaces such as the
lung and intestine. Such epithelial location makes mast cells one
of the first activators of the inflammatory response.3−5 Mast
cells have a high affinity for IgE because of having a specific IgE
receptor, FcεR1, on their surface. As the IgE−multivalent
allergen complex binds to FcεR1, the mast cells become
activated and release a range of preformed species such as
newly synthesized mediators and cytokines that trigger potent
allergic reactions.6 In normal conditions, the mast cells have
preformed mediators such as histamine serine proteases and β-
hexosaminidase in their cytoplasmic granules. The activated
mast cells release these mediators in response to immune
allergic reactions through IgE−FcεR1 binding.3−9
Recently we have focused on the syntheses of sila-substituted
five-membered ring heterocycles such as 1,3,4-oxadiazoles,
1,3,4-thiadiazoles, 1,2,4-triazoles, pyrazoles, isoxazoles, and
imidazoles because these compounds would display broad
spectra of biological activities. Particularly various substituted
1,3,4-oxadiazoles have been much explored for their biological
activities such as antiallergic,11 antiviral,12 anti-inflammatory,13
anticancer,14 antibacterial,15 and antifungal agents.15
Bioorganosilicon chemistry is known to be a powerful tool
for the generation of new therapeutic agents. Up to now several
silicon-containing drugs have been reported in the literature;
those are polydimethylsiloxanes(I),16 cyclohexyl(phenyl)(3-
(piperidin-1-yl)propyl)silanol(II),17 sila-derived phthalo-
cyanine(III),18 etc. (Figure 1). Recently some of the silicon-
containing drugs have entered human clinical trials; those are
silperisone,19 Tac101,20 BNP 1350,21 etc. In this context, we
have synthesized various sila-substituted 1,3,4-oxadiazoles and
examined their antiallergic effect using RBL-2H3 as the in vitro
model and the in vivo anaphylactic mouse model. Our results
indicate that silicon-containing compound 5c not only
effectively inhibits the degranulation and expression of
cytokines involved in type 1 hypersensitivity in mast cells but
β-Hexosaminidase, a granule-associated exoglycosidase, has
been used to monitor mast cell degranulation just as histamine
has been used. β-Hexosaminidase could act with tryptases and
chymases to induce degradation of the extracellular matrix. This
Received: March 26, 2012
Published: July 6, 2012
© 2012 American Chemical Society
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Figure 1. Biologically active organosilicon compounds.
also suppresses hypersensitive reactions in the PCA mouse
model.
Scheme 2. Synthesis of 2-(4-Chlorophenyl)-5-
(neopentylsulfonylmethyl)-1,3,4-oxadiazole 9
a
RESULTS AND DISCUSSION
■
Chemistry. The sila-substituted thio/sulfone linked 1,3,4-
oxadiazoles were synthesized in high yields. The synthetic
intermediate 2-((trimethylsilyl)methylthio)acetic acid (2) was
prepared by the reaction of (chloromethyl)trimethylsilane (1)
with mercaptoacetic acid in the presence NaOH/MeOH. The
oxidation of 2 in the presence of H2O2/CH3COOH22 at 0 °C
resulted in 2-((trimethylsilyl)methylsulfonyl)acetic acid (3).
The desired heterocycles 2-aryl-5-(((trimethylsilyl)methyl-
thio)methyl)-1,3,4-oxadiazoles (4a−c) and 2-aryl-5-
(((trimethylsilyl)methylsulfonyl)methyl)-1,3,4-oxadiazoles
(5a−c) were synthesized respectively by the reaction of 2 and 3
with the corresponding benzohydrazides in the presence of
phosphorus oxychloride23 (Scheme 1).
a
Reagents and conditions: (i) HSCH2COOH, EtOH, KOH, reflux, 8
h; (ii) 30% H2O2, AcOH, 0 °C, 24 h; (iii) 4-Cl-PhCONHNH2,
POCL3, reflux, 5 h.
Effect of Silicon-Containing Compounds on β-Hex-
osaminidase Release in IgE/Ag-Sensitized RBL-2H3
Cells. The RBL-2H3 cells were activated when the IgE/Ag
complex bound their receptor, FcεR1. The aggregation of
FcεR1 by the IgE/Ag complex induced mast cell degranulation
and the release of mediators and cytokines.25−28 β-Hexosami-
nidase, one of these mediators, is a marker of mast cell
degranulation.29 To determine the antidegranulation effect of
silicon-containing compounds, we measured the level of β-
hexosaminidase release in IgE/Ag sensitized RBL-2H3 cells.
We pretreated the RBL-2H3 cells with 200 μM silicon-
containing compounds (2−5) for 30 min followed by activation
with DNP-HSA (50 ng/mL) for 15 min. Among the silicon-
containing compounds, 5c significantly inhibited IgE/Ag-
induced mast cell degranulation (Figure 2) and also effectively
suppressed IgE/Ag-induced mast cell degranulation in a dose-
dependent manner. Interestingly, 5c showed much better
suppression results than its carbon analogue 9 (Figure 3). This
result strongly indicates that the silyl group in 5c plays an
important role for the effective suppression of IgE/Ag-induced
mast cell degranulation. This would be due to the properties of
the silicon moiety having larger molecular size, lower
electronegativity, and greater lipophilicity than those of the
carbon analogue. Silicon−carbon bonds (Si−C, 1.87 Å) are
longer by 0.33 Å than carbon−carbon bonds (C−C, 1.54 Å),
which may change the molecule’s interaction with a receptor
and enhance the pharmacological selectivity and potency. In
general, silicon containing compounds show greater lip-
ophilicity than carbon analogues, which might enhance tissue
distribution.30,31 Considering those effects above, 5c is
anticipated as a better antiallergy candidate than the carbon
analogue.
Scheme 1. Synthesis of 2-Aryl-5-
(((trimethylsilyl)methylthio)methyl)-1,3,4-oxadiazoles 4
and 2-Aryl-5-(((trimethylsilyl)methylsulfonyl)methyl)-1,3,4-
a
oxadiazoles 5
a
Reagents and conditions: (i) HSCH2COOH, MeOH, NaOH, reflux,
4 h; (ii) 30% H2O2, AcOH, 0 °C, 24 h; (iii) ArCONHNH2, POCl3,
reflux, 4−6 h.
For the comparison of antiallergic activity to 5c, we
synthesized 2-(4-chlorophenyl)-5-(neopentylsulfonylmethyl)-
1,3,4-oxadiazole (9) as a carbon analogue24 of it. Compound
9 was synthesized as follows: 2-(neopentylthio)acetic acid was
synthesized from the reaction of neopentyl bromide (neopentyl
chloride did not work) with mercaptoacetic acid followed by
oxidation with H2O2/CH3COOH22 at 0 °C to give 2-
(neopentylsulfonyl)acetic acid. Finally, 2-(neopentylsulfonyl)-
acetic acid was treated with 4-chlorobenzohydrazide in the
presence of phosphorus oxychloride23 to yield target
compound 9 (Scheme 2).
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line at concentrations up to 200 μM (Figure 4). These findings
indicate that the reduction in degranulation of the activated
Figure 2. Effect of silicon-containing compounds on IgE/Ag-
stimulated mast cell degranulation. The cells were incubated overnight
in 24-well plates with 200 ng/mL DNP-specific IgE. The medium was
replaced with PIPES buffer containing 100 μM silicon-containing
compounds, and the cells were then challenged with 25 ng/mL DNP-
HSA. After 15 min, β-hexosaminidase release was determined from the
absorbance measured at 405 nm. Data are expressed as the mean SD
(n = 4). Significant differences with the IgE/Ag-stimulated controls
without silicon-containing compounds are indicated: (∗) p < 0.01 and
(∗∗) p < 0.001. Control 1 is IgE/Ag(DNP-HAS)-unstimulated control
without compounds. Control 2 is IgE/Ag(DNP-HAS)-stimulated
control without compounds.
Figure 4. Cytotoxic effects of silicon-containing compound 5c on mast
cell. To assess cytotoxicity, RBL-2H3 cells were incubated overnight in
medium containing 200 ng/mL DNP-specific IgE and then treated
with compound 5c at various concentrations for 23 h. Cell viability
was determined with the Ez-Cytox kit from the absorbance at 450 nm.
RBL-2H3 cells was a result of the properties of the silicon-
containing compound 5c and not cytotoxicity.
Effect of Silicon-Containing Compound 5c on TNF-α
mRNA Expression Level and Akt Phosphorylation in IgE/
Ag-Sensitized RBL-2H3 Cells. Aggregation of IgE-bound
FcεRI by Ag binding induces various inflammatory cytokines,
notably TNF-α, which mediate the symptoms in an allergic
reaction.32 We treated the RBL-2H3 cells with IgE (25 ng/mL)
for 24 h followed by treatment with silicon-containing
compound 5c at different concentrations (0−200 μM). The
cells were then activated with DNP-HSA (25 ng/mL) for 5
min. We observed that 5c diminished the mRNA expression
levels of TNF-α in a dose-dependent manner (Figure 5). From
Figure 5. Effect of compound 5c on the expression of TNF-α in IgE/
Ag-stimulated mast cells. The IgE/Ag-sensitized RBL-2H3 cells were
treated with various doses of silicon-containing compound 5c and then
challenged with 25 ng/mL DNP-HSA for 1 h. Total RNA was isolated
and analyzed by RT-PCR.
Figure 3. Effect of silicon-containing compounds on IgE/Ag-
stimulated mast cell degranulation. The cells were incubated overnight
in 24-well plates with 200 ng/mL DNP-specific IgE. The medium was
replaced with PIPES buffer containing the indicated concentrations of
silicon-containing compound 5c and carbon analogue compound 9,
and the cells were then challenged with 25 ng/mL DNP-HSA. After 15
min, β-hexosaminidase release was determined from the absorbance
measured at 405 nm. Data are expressed as the mean SD (n = 4).
Significant differences with the IgE/Ag-stimulated controls without
silicon-containing compound 5c or carbon analogue compound 9 are
indicated: (∗) p < 0.01 and (∗∗) p < 0.001. Control 1 is IgE/Ag-
unstimulated control without compounds 5c and 9. Control 2 is IgE/
Ag-stimulated control without compounds 5c and 9.
these findings, we inferred that the antiallergic effect of 5c was
caused by suppression of the mRNA expression of these
cytokines. We also examined the effects of 5c on
phosphatidylinositol 3-kinase32 because of their role in the
production of TNF-α. The phosphorylation of Akt, a surrogate
for the activation of phosphatidylinositol 3-kinase, was also
significantly suppressed by 5c in a dose-dependent manner
(Figure 6).
5c Reduced Anaphylactic Shock in an in Vivo Mouse
Model. On the basis of good results from the in vitro model,
we furthermore examined the effect of 5c in an in vivo allergic
mouse model.33 We induced anaphylaxis by injecting IgE
intradermally into one ear of each mouse. One day after IgE
administration, we injected a DNP-HSA-Evans blue mixture
into tail veins of each mouse to induce a systemic allergic
reaction. The IgE/Ag-sensitized mouse ears subsequently
swelled and turned blue. However, in the mice that received
silicon-containing compound 5c orally, the intensity of the blue
To confirm that the degranulation inhibition was indeed due
to the activity of 5c and its noncytotoxicity, we assessed the
cytotoxicity of 5c in the RBL-2H3 cells using an Ez-cytox assay.
We pretreated the mast cells overnight with different doses of
silicon-containing compound 5c. After 23 h, cells were treated
with the Ez-cytox solution for 1 h, and the absorbance of the
cell culture plate was measured with a microplate reader. Silicon
containing compound 5c was noncytotoxic in the RBL-2H3 cell
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vitro model. Furthermore we tested the effect of 5c on passive
cutaneous anaphylaxis in an in vivo PCA mouse model. When
we fed 5c orally to the IgE/Ag-sensitized, DNP-HSA-Evans
blue dye injected mouse, the intensity of the blue dye decreased
with increasing 5c concentration, and 5c also suppressed
anaphylaxis in the mice. The suppression by 5c was more
effective than that by DPH, an anti-histamine drug. With all the
experimental results, we believe that 5c will be a useful clinical
candidate for antiallergic drugs.
Figure 6. Effect of silicon-containing compound 5c on Akt
phosphorylation in IgE/Ag-stimulated mast cells. The IgE/Ag-
sensitized RBL-2H3 cells were treated with various doses of silicon-
containing compound 5c and then challenged with 25 ng/mL DNP-
HSA for 1 h. Phosphorylation of Akt was analyzed by Western blotting
using specific antibodies.
EXPERIMENTAL SECTION
■
Chemistry. Melting points were determined in open capillaries on
a Stuart apparatus and are uncorrected. Reagents and solvents were
obtained in the highest available purity and used without further
purification unless otherwise indicated. The purity of the compounds
dye decreased with increasing 5c concentration. 5c also
appeared to suppress anaphylaxis in the mice, as evidenced
by the results depicted in the bar graph in Figure 7. The effect
1
was checked by TLC (silica gel, ethyl acetate/hexane (1:3)). The H
NMR, 13C NMR, and 29Si NMR spectra were recorded on a Bruker
AMX 400 NMR spectrometer in CDCl3. Mass spectra were recorded
on a low-resolution (Agilent Technologies GC/MS model 6890N and
model 5973N mass selective detector) EI mass spectrometer and a
high-resolution (JEOL JMS-600W/JEOL JMS-700) instrument. The
purities of the tested compounds, which were examined by GC/MS,
were more than 95%.
2-((Trimethylsilyl)methylthio)acetic Acid (2). 2-((Tri-
methylsilyl)methylthio)acetic acid was prepared according to the
literature procedure.22 Condensation of (chloromethyl)trimethylsilane
(1) (6.1 g, 50 mmol) with mercaptoacetic acid (4.6 g, 50 mmol)
afforded the corresponding 2-((trimethylsilyl)methylthio)acetic acid
(2) in 93% yield. A colorless liquid with a boiling point of 163−165 °C
(760 Torr) was obtained. 1H NMR (CDCl3, 400 MHz): δ 0.07 (s, 9H,
Si-CH3), 1.93 (s, 2H, Si-CH2), 3.20 (s, 2H, S-CH2), 9.61 (bs, 1H,
OH). 13C NMR (CDCl3, 100 MHz): δ −1.81 (Si-CH3), 19.44 (Si-
CH2), 37.54 (S-CH2), 177.08 (CO). 29Si NMR (CDCl3, 79 MHz):
δ 1.34.
2-((Trimethylsilyl)methylsulfonyl)acetic Acid (3). 2-((Tri-
methylsilyl)methylsulfonyl)acetic acid was prepared according to the
literature procedure.22 Oxidation of 2-((trimethylsilyl)methylthio)-
acetic acid (2) (2.7 g, 15 mmol) with 30% hydrogen peroxide (6.7 mL,
66 mmol) gave 2-((trimethylsilyl)methylsulfonyl)acetic acid (3) in
84% yield. A white solid with a melting point of 61−63 °C was
Figure 7. Effect of silicon-containing compound 5c on IgE/Ag-
induced passive cutaneous anaphylaxis (PCA). Seven-week-old ICR
mice were intravenously injected with 200 μg of antigen containing 3%
Evans blue 24 h after intradermal injection of DNP-IgE (0.5 μg) into
the ear. Silicon-containing compound 5c was orally administered 1 h
before the antigen. (A) Representative pictures of the ears are shown.
(B) The dye was extracted overnight in 500 μL of formamide at 63 °C,
and the light absorbance was measured at 620 nm. Data are expressed
as the mean SD (n = 4). The asterisks indicate significant difference
from IgE/Ag-stimulated controls without silicon-containing com-
pound 5c ((∗∗) p < 0.001). Diphenylhydramine (DPH, 50 mg/kg)
was used as a typical anti-histamine reference drug.
1
obtained. H NMR (CDCl3, 400 MHz): δ 0.28 (s, 9H, Si-CH3), 2.97
(s, 2H, Si-CH2), 4.03 (s, 2H, SO2-CH2), 9.65 (bs, 1H, OH). 13C NMR
(CDCl3, 100 MHz): δ −0.65 (Si-CH3), 44.82 (Si-CH2), 61.19 (SO2-
CH2), 167.38 (CO). 29Si NMR (CDCl3, 79 MHz): δ 1.74.
General Procedure for the Preparation of 2-Aryl-5-
(((trimethylsilyl)methylthio)-methyl)-1,3,4-oxadiazole (4a−c).
A mixture of 2-((trimethylsilyl)methylthio)acetic acid (2) (1.8 g, 10
mmol), arylhydrazide (10 mmol), and POCl3 (7.0 mL, 75 mmol) was
23
heated at about 70 °C for 4−6 h. The excess POCl3 was removed
under reduced pressure, and the residue was poured into crushed ice.
The resulting precipitate was filtered, washed with saturated sodium
bicarbonate solution and then with water, dried, and recrystallized
from ethanol to afford 4a−c.
2-Phenyl-5-(((trimethylsilyl)methylthio)methyl)-1,3,4-oxa-
diazole (4a). Compound 4a was obtained from 2-((trimethylsilyl)-
methylthio)acetic acid) (2) (1.8 g, 10 mmol), benzohydrazide (1.4 g,
10 mmol), and POCl3 (7.0 mL, 75 mmol) using the general procedure
for 4a−c. A pale yellow solid with a melting point of 82−84 °C was
obtained in 91% yield. 1H NMR (CDCl3, 400 MHz): δ 0.05 (s, 9H, Si-
CH3), 1.89 (s, 2H, Si-CH2), 3.87 (s, 2H, S-CH2), 7.49−8.05 (m, 5H,
Ph). 13C NMR (CDCl3, 100 MHz): δ −1.77 (Si-CH3), 18.76 (Si-
CH2), 29.16 (S-CH2), 123.83, 126.93, 129.06, 131.78 (Ph), 164.04 (C-
2), 165.38 (C-5). 29Si NMR (CDCl3, 79 MHz): δ 1.52. MS: m/z
(relative intensity) 263 (M+ − 15, 24.2), 231 (100), 160 (57.0), 105
(23.4), 100 (14.8), 73 (46.8). HRMS: C13H19N2OSSi 279.0987 (M+ +
1, calcd), 279.0992 (found).
was comparable to that of 50 mg/kg diphenylhydramine
(DPH), an anti-histamine drug. These results confirm the
ability of 5c to inhibit passive cutaneous anaphylaxis in an in
vivo animal model, similar to that in in vitro experiments.
In conclusion, we have demonstrated antiallergy activity of
new and nontoxic sila-substituted 1,3,4-oxadiazoles using RBL-
2H3 as the in vitro and in vivo models. We observed that 2-(4-
chlorophenyl)-5-(((trimethylsilyl)methylsulfonyl)methyl)-
1,3,4-oxadiazole (5c) effectively suppressed DNP-HSA-induced
mast cell degranulation in a dose-dependent manner. Also, silyl
compound 5c effectively suppressed DNP-HSA-induced mast
cell degranulation, compared to carbon analogue 9. In addition
5c greatly suppressed the expression of TNF-α mRNA and Akt
phosphorylation in IgE/Ag-stimulated RBL-2H3 cells of the in
2-p-Tolyl-5-(((trimethylsilyl)methylthio)methyl)-1,3,4-oxa-
diazole (4b). Compound 4b was obtained from 2-((trimethylsilyl)-
methylthio)acetic acid) (2) (1.8 g, 10 mmol), 4-Me-benzohydrazide
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(1.5 g, 10 mmol), and POCl3 (7.0 mL, 75 mmol) using the general
129.61, 138.73 (Ph), 158.11 (C-2), 165.66 (C-5). 29Si NMR (CDCl3,
79 MHz): δ 2.11. MS: m/z (relative intensity) 344 (M+, 9.1), 329
(13.7), 267 (8.6), 193 (85.4), 139 (100), 111 (22.5), 73 (17.7).
HRMS: C13H17ClN2O3SSi 344.0418 (M+, calcd), 344.0423 (found).
2-(Neopentylthio)acetic Acid (7). To a solution of potassium
hydroxide (2.8 g, 50 mmol) in ethanol (30 mL) was slowly added
mercaptoacetic acid (2.3 g, 25 mmol), and then neopentyl bromide
(3.8 g, 25 mmol) was added portionwise. The mixture was refluxed for
8 h, was cooled, and was poured into ice-cold water containing
hydrochloric acid. The resultant liquid was extracted with diethyl ether
and was distilled to afford 2-(neopentylthio)acetic acid (7) in 95%
yield. A colorless liquid with a boiling point of 118−120 °C (760
procedure for 4a−c. A white solid with a melting point of 89−91 °C
1
was obtained in 89% yield. H NMR (CDCl3, 400 MHz): δ 0.04 (s,
9H, Si-CH3), 1.88 (s, 2H, Si-CH2), 2.39 (s, 3H, Ph-CH3), 3.85 (s, 2H,
S-CH2), 7.27 (d, 2H, Ph), 7.91 (d, 2H, Ph). 13C NMR (CDCl3, 100
MHz): δ −1.79 (Si-CH3), 18.71 (Si-CH2), 21.61 (Ph-CH3), 29.11 (S-
CH2), 121.04, 126.85, 129.72, 142.27 (Ph), 163.74 (C-2), 165.46 (C-
5). 29Si NMR (CDCl3, 79 MHz): δ 1.45. MS: m/z (relative intensity)
277 (M+ − 15, 24.2), 245 (100), 174 (63.2), 119 (54.4), 100 (16.1),
91 (41.1), 73 (82.3). HRMS: C14H21N2OSSi 293.1144 (M+ + 1,
calcd), 293.1134 (found).
2-(4-Chlorophenyl)-5-(((trimethylsilyl)methylthio)methyl)-
1,3,4-oxadiazole (4c). Compound 4c was obtained from 2-
((trimethylsilyl)methylthio)acetic acid) (2) (1.8 g, 10 mmol), 4-Cl-
benzohydrazide (1.7 g, 10 mmol), and POCl3 (7.0 mL, 75 mmol)
using the general procedure for 4a−c. A white solid with a melting
point of 97−99 °C was obtained in 93% yield. 1H NMR (CDCl3, 400
MHz): δ 0.05 (s, 9H, Si-CH3), 1.88 (s, 2H, Si-CH2), 3.86 (s, 2H, SO2-
CH2), 7.46 (d, 2H, Ph), 7.97 (d, 2H, Ph). 13C NMR (CDCl3, 100
MHz): −1.78 (Si-CH3), 18.80 (Si-CH2), 29.14 (S-CH2), 122.29,
128.20, 129.46, 138.07 (Ph), 164.20 (C-2), 164.58(C-5). 29Si NMR
(CDCl3, 79 MHz): δ 1.54. MS: m/z (relative intensity) 297 (M+ − 15,
25.0), 265 (100), 194 (63.2), 139 (50.7), 111 (30.4), 100 (26.5), 73
(89.0). HRMS: C13H18ClN2OSSi 313.0598 (M+ + 1, calcd), 313.0598
(found).
General Procedure for the Synthesis of 2-Aryl-5-
(((trimethylsilyl)methylsulfonyl)methyl)-1,3,4-oxadiazole (5a−
c). A mixture of arylhydrazide (10 mmol), 2-((trimethylsilyl)-
methylsulfonyl)acetic acid (3) (2.1 g, 10 mmol), and POCl3 (7.0
mL, 75 mmol) was heated at about 70 °C for 4−6 h. The excess
POCl3 was removed under reduced pressure, and the residue was
poured into crushed ice. The resulting precipitate was filtered, washed
with saturated sodium bicarbonate solution and then with water, dried,
and recrystallized from ethanol to give 5a−c.
1
Torr) was obtained. H NMR (CDCl3, 400 MHz): δ 0.91 (s, 9H, C-
CH3), 2.52 (s, 2H, C-CH2), 3.17 (s, 2H, S-CH2), 11.69 (bs, 1H, OH).
13C NMR (CDCl3, 100 MHz): δ 28.76 (-CH3), 32.26 (C-CH3) 41.32
(C-CH2), 47.70 (S-CH2), 177.36 (CO). MS: m/z (relative
intensity) 162 (M+, 97.0), 129 (11.1), 106 (97.0), 101 (39.5), 77
(14.9), 71 (18.6), 57 (100).
2-(Neopentylsulfonyl)acetic Acid (8). 2-(Neopentylsulfonyl)-
acetic acid was prepared according to the literature procedure.22
Oxidation of 2-(neopentylthio)acetic acid (7) (2.4 g, 15 mmol) with
30% hydrogen peroxide (6.0 mL, 59 mmol) gave 2-
(neopentylsulfonyl)acetic acid (8) in 81% yield. A colorless liquid
with a boiling point of 134−136 °C (760 Torr) was obtained.1H NMR
(CDCl3, 400 MHz): δ 1.17 (s, 9H, C−CH3), 3.23 (s, 2H, C-CH2),
3.99 (s, 2H, SO2-CH2), 9.89 (bs, 1H, OH). 13C NMR (CDCl3, 100
MHz): δ 29.71 (CH3), 32.37 (C-CH3), 60.29 (C-CH2) 64.50 (SO2-
CH2), 177.84 (CO).
2-(4-Chlorophenyl)-5-(neopentylsulfonylmethyl)-1,3,4-oxa-
diazole (9). 2-(4-Chlorophenyl)-5-(neopentylsulfonylmethyl)-1,3,4-
oxadiazole (9) was prepared according to the literature procedure.23 A
mixture of 4-Cl-benzohydrazide (1.7 g, 10 mmol), 2-
(neopentylsulfonyl)acetic acid (8) (2.0 g, 10 mmol), and POCl3
(7.0 mL, 75 mmol) was heated at about 70 °C for 5 h to give 9. A
white solid with a melting point of 102−104 °C was obtained in 79%
2-Phenyl-5-(((trimethylsilyl)methylsulfonyl)methyl)-1,3,4-
oxadiazole (5a). Compound 5a was obtained from 2-
((trimethylsilyl)methylsulfonyl)acetic acid (3) (2.1 g, 10 mmol),
benzohydrazide (1.4 g, 10 mmol), and POCl3 (7.0 mL, 75 mmol)
using the general procedure for 5a−c. A white solid with a melting
1
yield. H NMR (CDCl3, 400 MHz): δ 1.19 (s, 9H, C-CH3), 3.20 (s,
2H, C-CH2), 4.56 (s, 2H, SO2-CH2), 7.48 (d, 2H, Ph), 7.98 (d, 2H,
Ph). 13C NMR (CDCl3, 100 MHz): δ 29.69 (CH3), 32.45 (C-CH3),
52.36 (C−CH2), 63.49 (SO2-CH2), 121.45, 128.49, 129.63, 138.84
(Ph), 157.71 (C-2), 165.73(C-5). MS: m/z (relative intensity) 328
(M+, 5.1), 313 (5.1), 193 (63.7), 139 (100), 123 (7.4), 111 (36.2), 75
(14.8), 55 (22.9). HRMS: C14H17ClN2O3S 328.0648 (M+, calcd),
328.0650 (found).
Materials. Mouse monoclonal anti-dinitrophenol (anti-DNP) IgE
antibody, dinitrophenol-conjugated human serum albumin (DNP-
HSA), and Evans blue were obtained from Sigma-Aldrich, (St. Louis,
MO, U.S.). p-nitrophenyl-N-acetyl-β-D-glucosaminide was obtained
from MP Biomedicals, LLC (Solon, OH, U.S.).
Cell Culture. The rat mast cell line RBL-2H3 was obtained from
the American Type Culture Collection (ATCC, Rockville, MD, U.S.).
The cells were grown in Eagle’s minimal essential medium
(WelGENE, Inc., Daegu, Korea) containing 10% (v/v) fetal bovine
serum and 100 units/mL penicillin−streptomycin (Lonza Walkersville,
Inc., Walkersville, MD, U.S.) at 37 °C in a humidified 5% CO2
atmosphere. Cells were detached with trypsin-EDTA (ethylenediami-
netetraacetic acid) solution, washed with phosphate buffered saline
(PBS, pH 7.2), and resuspended in fresh medium for use in
subsequent experiments.
Measurement of β-Hexosaminidase Release. We measured
the release of β-hexosaminidase as a marker for degranulation. RBL-
2H3 cells were distributed in 24-well plates (2 × 105 cells/well) and
were sensitized overnight with anti-DNP-specific IgE at 200 ng/mL.
The IgE-sensitized cells were washed twice with PIPES buffer (25 mM
PIPES, pH 7.2, 110 mM NaCl, 4 mM KCl, 0.4 mM MgCl, 40 mM
HCl, 5.6 mM glucose, 1 mM CaCl2, and 0.1% BSA). They were then
treated for 30 min at 37 °C with the silicon-containing compounds in
PIPES buffer at the indicated concentrations. The cells were
subsequently stimulated with DNP-HSA (25 ng/mL) at 37 °C for
15 min and chilled on ice to stop the stimulation. To measure the β-
hexosaminidase release, the supernatant from the antigen (Ag)
1
point of 102−104 °C was obtained in 89% yield. H NMR (CDCl3,
400 MHz): δ 0.26 (s, 9H, Si-CH3), 2.90 (s, 2H, Si-CH2), 4.56 (s, 2H,
SO2-CH2) 7.50−8.07 (m, 5H, Ph). 13C NMR (CDCl3, 100 MHz): δ
−0.67 (Si-CH3), 43.35 (Si-CH2), 53.45 (SO2-CH2), 123.15, 127.20,
129.18, 132.33 (Ph), 157.96 (C-2), 166.45(C-5). 29Si NMR (CDCl3,
79 MHz): δ 2.05. MS: m/z (relative intensity) 310 (M+, 9.6), 296
(8.3), 231 (20.1), 159 (89.5), 105 (100), 77 (60.4). HRMS:
C13H18N2O3SSi 310.0807 (M+, calcd), 310.0807 (found).
2-p-Tolyl-5-(((trimethylsilyl)methylsulfonyl)methyl)-1,3,4-
oxadiazole (5b). Compound 5b was obtained from 2-
((trimethylsilyl)methylsulfonyl)acetic acid (3) (2.1 g, 10 mmol), 4-
Me-benzohydrazide (1.5 g, 10 mmol), and POCl3 (7.0 mL, 75 mmol)
using the general procedure for 5a−c. A white solid with a melting
1
point of 117−119 °C was obtained in 86% yield. H NMR (CDCl3,
400 MHz): δ 0.25 (s, 9H, Si-CH3), 2.40 (s, 3H, Ph-CH3), 2.89 (s, 2H,
Si-CH2), 4.55 (s, 2H, SO2-CH2), 7.29 (d, 2H, Ph), 7.92 (d, 2H, Ph).
13C NMR (CDCl3, 100 MHz): δ −0.68 (Si-CH3), 21.68 (Ph-CH3),
43.37 (Si-CH2), 53.44 (SO2−CH2), 120.36, 127.14, 129.86, 142.99
(Ph), 157.67 (C-2), 166.58(C-5). 29Si NMR (CDCl3, 79 MHz): δ
1.98. MS: m/z (relative intensity) 324 (M+, 7.2), 309 (10.9), 245
(5.1), 173 (75.9), 119 (100), 91 (31.5), 73 (24.8). HRMS:
C14H20N2O3SSi 324.0964 (calcd), 324.0961 (found).
2-(4-Chlorophenyl)-5-(((trimethylsilyl)methylsulfonyl)-
methyl)-1,3,4-oxadiazole (5c). Compound 5c was obtained from 2-
((trimethylsilyl)methylsulfonyl)acetic acid (3) (2.1 g, 10 mmol), 4-Cl-
benzohydrazide (1.7 g, 10 mmol), and POCl3 (7.0 mL 75 mmol) using
the general procedure for 5a−c. A white solid with a melting point of
126−128 °C was obtained in 91% yield. 1H NMR (CDCl3, 400 MHz):
δ 0.26 (s, 9H, Si-CH3), 2.89 (s, 2H, Si-CH2), 4.56 (s, 2H, SO2-CH2),
7.48 (d, 2H, Ph), 8.00 (d, 2H, Ph). 13C NMR (CDCl3, 100 MHz): δ
−0.67 (Si-CH3), 43.40 (Si-CH2), 53.40 (SO2-CH2), 121.60, 128.47,
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Journal of Medicinal Chemistry
Article
stimulated cells in PIPES buffer and β-hexosaminidase substrate (p-
nitrophenyl-N-acetyl-β-D-glucosaminide) in 0.1 M citrate buffer (pH
4.5) were mixed in 96-well plates and incubated at 37 °C for 1 h. This
reaction was terminated by adding 0.1 M carbonate buffer (pH 10.5),
and the absorbance was measured at 405 nm using a microplate reader.
Measurement of Cell Viability. The Ez-cytox enhanced cell
viability kit (Daeil Labservice, Seoul, Korea) was used to assess cell
viability following the manufacturer’s instructions. In brief, RBL-2H3
cells (2 × 104 cells/well) were seeded into 96-well plates and
incubated overnight with 200 ng/mL anti-DNP-specific IgE. After
incubation, the medium was replaced with serum free medium, and the
cells were treated with silicon-containing compound 5c and 25 ng/mL
DNP-HSA for 23 h. The Ez-cytox kit reagent was then added to the
medium, and the cells were incubated at 37 °C for 1 h. Cell viability
was determined based on absorbance at 450 nm measured with a
microplate reader.
cellular proteins (20 μg) were separated by 10% SDS−polyacrylamide
gel electrophoresis and transferred to a polyvinylidene fluoride
(PVDF) membrane (Bio-Rad, CA, U.S.) using a Trans-Blot SD
semidry transfer cell (Bio-Rad, CA, U.S.). Membranes were incubated
for 1 h with blocking solution (5% skim milk) at room temperature,
followed by incubation overnight at 4 °C with specific primary
antibodies (1:2000). Subsequently, membranes were washed three
times with TBS-T buffer and incubated with a secondary anti-rabbit
antibody (1:5000) for 1 h at room temperature. The membranes were
then washed three times with TBS-T, and the proteins were detected
using an enhanced chemiluminescence Western blotting detection kit
(GE Healthcare, U.K.).
Statistical Analysis. Experimental results are expressed as the
mean
SD. A one-way analysis of variance (ANOVA) with the
Dunnett’s test for multiple comparisons was performed. p < 0.01 and p
< 0.001 were considered statistically significant, as indicated.
Animals. Seven-week-old male ICR mice were purchased from
Orient Bio (Gangneung, Korea) and housed in wire cages at 20−22
°C and a relative humidity of 40−50%. All animals were given access
to standard laboratory chow and water ad libitum. The Institutional
Animal Care and Use Committee (IACUC) at Yonsei University
(Wonju, Korea) approved the protocol for this study.
ASSOCIATED CONTENT
* Supporting Information
Spectra for compounds 4−9. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
Induction of Mast-Cell-Mediated Passive Cutaneous Ana-
phylaxis in Mice. An anti-DNP-specific IgE antibody (0.5 μg) was
injected intradermally into one ear of each mouse. After 24 h, the mice
were challenged with an intravenous injection of DNP-HSA (200 μg;
antigen, Ag) in 200 μL of PBS 3% Evans blue. One hour later, the
silicon-containing compound 5c was administered orally at a dose of
50−100 mg/kg. Two hours after the Ag challenge, the mice were
euthanized and the treated ear was excised to measure the amount of
dye that had extravasated in response to the Ag. The dye was extracted
from the ear in 500 μL of formamide at 63 °C overnight and
quantified by measuring dye absorbance at 620 nm.
AUTHOR INFORMATION
Corresponding Author
*For T.-J.K.: phone, +82-33-760-2242; e-mail, ktj@yonsei.ac.kr.
For M.E.L.: phone, +82-33-760-2237; fax, +82-33-760-2182; e-
mail, melgg@yonsei.ac.kr.
■
Author Contributions
§The first two authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
RNA Extraction and Reverse Transcription Polymerase
Chain Reaction (RT-PCR). The RBL-2H3 cells (8 × 105 cells/well)
were seeded into six-well plates and incubated overnight in a medium
containing 200 ng/mL anti-DNP-specific IgE. The cells were washed
twice, resuspended in PIPES buffer, and stimulated with DNP-HSA
(25 ng/mL) for 1 h with and without silicon-containing compound 5c.
After incubation, the cells were washed twice with ice-cold PBS. Total
RNA was isolated using TRI reagent (Sigma-Aldrich, St. Louis, MO,
U.S.) according to the manufacturer’s instructions. The concentration
of total RNA was determined using a spectrophotometer. The total
RNA (1 μg) was used as the template for cDNA synthesis and PCR
using the Accupower RT/PCR premix kit (Bioneer, Daejeon, Korea).
The following primers were used: rat TNF-α sense 5′-CAAGGAG-
GAGAAGTTCCCAA-3′; TNF-α antisense 5′-CGGACTCCGT-
GATGTCTAAG-3′; β-actin sense 5′-ATGCCATCCTGCGTCTG-
GACCTGGC-3′; β-actin antisense 5′-AGCATTTGCGGTGCAC-
GATGGAGGG-3′. The denaturation, annealing, extension
conditions, and number of cycles were 94 °C for 60 s, 49 °C for 45
s, 72 °C for 45 s, and 35 cycles, respectively, for TNF-α. The PCR
products were electrophoresed on a 2% agarose gel and visualized by
adding ethidium bromide. The gels were examined using a
transilluminator (Vilber Lourmat, France).
ACKNOWLEDGMENTS
■
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (Grant 2010-0024877). We thank Professor
Robert West, distinguished Professor in the WCU program,
for his valuable discussions. G.D.R. also is thankful for the BK-
21 funds for the financial support of his postdoc position.
ABBREVIATIONS USED
■
RBL-2H3, rat basophilic leukemia; PCA, passive cutaneous
anaphylaxis; DNP-HSA, dinitrophenylated human serum
albumin; FcεR1, high-affinity IgE receptor; TNF-α, tumor
necrosis factor α; mRNA, messenger ribonucleic acid; DPH,
diphenylhydramine; IgE, immunoglobulin E; RT-PCR, reverse
transcription polymerase chain reaction; IACUC, Institutional
Animal Care and Use Committee; Ag, antigen
Protein Extraction and Western Blotting Analysis. To extract
protein for Western blot analysis, RBL2H3 cells (8 × 105 cells/ml)
were seeded in a six-well plate for 24 h at 37 °C with 5% CO2. The
medium was removed and replaced with fresh medium containing
various concentrations (0−200 μM) of silicon-containing compound
5c and stimulated with DNP-HSA (25 ng/mL) for 1 h. The cells were
then washed twice with ice-cold PBS (pH 7.2) and collected on ice.
The washed cell pellets were centrifuged at 12000g for 10 min at 4 °C.
The supernatant was removed from the tubes, and 150 μL of PRO-
PREP (iNtRON Biotechnology, Inc., Seongnam, Korea) was added to
each tube. The cells were placed on ice for 1 h and sonicated to
decrease viscosity. Insoluble cell debris was removed by centrifugation
at 12000g for 10 min at 4 °C. The supernatants were collected, and the
protein concentration was measured using the Bradford assay (Bio-
Rad, CA, U.S.) according to the manufacturer’s instruction. Total
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