Identification of 2,3-disubstituted pyridines as potent, orally active PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.07.007

A series of 2,3-disubstituted pyridines were synthesized and evaluated for their PDE4 inhibitory activity. We successfully modified undesirable cyano group of initial lead compound 2 to 4-pyridyl group with improvement of in vitro efficacy and optimized t

Full text of DOI:10.1016/j.bmc.2013.07.007

Bioorganic & Medicinal Chemistry 21 (2013) 5851–5854
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of 2,3-disubstituted pyridines as potent, orally active
PDE4 inhibitors
Yoshihiro Kato, Motoji Kawasaki, Tomohiro Nigo, Shunya Nakamura, Akira Fusano, Yasuhiro Teranishi,
⇑
Mari N. Ito, Takaaki Sumiyoshi
Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd, 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,3-disubstituted pyridines were synthesized and evaluated for their PDE4 inhibitory activity.
We successfully modified undesirable cyano group of initial lead compound 2 to 4-pyridyl group with
improvement of in vitro efficacy and optimized the position of nitrogen atoms in pyridine moiety and alk-
ylene linker. The most potent compound showed significant efficacy in animal models of asthma and
inflammation.
Received 13 June 2013
Revised 2 July 2013
Accepted 3 July 2013
Available online 14 July 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Phosphodiesterase 4
PDE4 inhibitors
2,3-Disubstituted pyridine
Structure–activity relationship
Anti-inflammatory agent
1. Introduction
In our drug discovery program for anti-asthmatic drugs with
bronchodilatory effect, we identified the lead compound 2 by
Cyclic nucleotide phosphodiesterases (PDEs) are a group of 11
families of metalphosphohydrolases that are responsible for
hydrolysis of cyclic adenosine 30,50-monophosphate (cAMP) and
cyclic guanosine 30,50-monophosphate (cGMP). Among the PDEs,
PDE4 specifically hydrolyzes cAMP, and its inhibition is known to
effectively increase intracellular cAMP level and to regulate various
cellular functions.1 PDE4 is reported to be expressed in key effector
cells involved in asthma, particularly airway smooth muscle cells,
and in inflammation, including neutrophils, T-lymphocytes, mac-
rophages, and eosinophils. It has been reported that PDE4 inhibi-
tors significantly increase intracellular cAMP in airway smooth
muscle cells, thereby providing a bronchodilatory effect.2–4 In
addition to the first generation PDE4 inhibitor rolipram, a number
of second generation PDE4 inhibitors (Fig. 1) have been reported
with roflumilast (Daxas™, Daliresp™) recently approved for severe
chronic obstructive pulmonary disease (COPD).5–7 Despite signifi-
cant progress in this area, PDE4 inhibitors are often associated with
side effects such as nausea, emesis, and vasculopathy, which limit
their therapeutic use.8 This highlights the need for novel pharma-
cophores that would allow the design of safer PDE4 inhibitors. We
describe here our discovery and synthesis of 2,3-disubstituted pyr-
idines as a new class of potent PDE4 inhibitors.
screening derivatives of compound 1 (Fig. 2). Detailed screening
of compound 2 revealed a moderate PDE4 inhibitory activity
(IC50: 104 nM). This result also suggested that the cyanopropoxy
moiety of this compound is essential for inhibition of PDE4 because
compound 1 did not inhibit PDE4. However, the 3-cyanopropoxy
structure is one of the undesirable structures in medicinal chemis-
try, because alkyl nitriles have potency to afford the toxic HCN. In
order to avoid this safety concern, we decided to investigate ways
to modify the 3-cyanoalkyl moiety of 2, while maintaining PDE4
inhibitory activity.
2. Chemistry
Preparation of the 2,3-disubstituted pyridines is shown in
Scheme 1. Substitution of the 2-bromo-3-hydroxypyridine by
F
NH
F
O Cl
HN
MeO
O
O
N
O
O
Cl
metal-binding unit
Roflumilast
Figure 1. Chemical structures of selected PDE4 inhibitors.
Rolipram
⇑
Corresponding author. Tel.: +81 6 6337 5906.
E-mail address: takaaki-sumiyoshi@ds-pharma.co.jp (T. Sumiyoshi).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.007

5852
Y. Kato et al. / Bioorg. Med. Chem. 21 (2013) 5851–5854
N
N
O
S
O
O
in vivo screening
Optimization
N
N
S
N
Br
Br
Br
1
2
3
PDE4 IC50: 104 nM
Not orally active
PDE4 IC50: 34 nM
Orally active
Bronchodilatory agent
(no PDE4 inhibitory activity)
Figure 2. Modification of substituted pyridine derivatives.
R= (CH2)3CN: 2
OH
S
OR
S
6
(CH2)2-4-Pyr:
(CH2)3-4-Pyr: 3
(CH2)4-4-Pyr: 7
OH
a.
b.
N
N
8
(CH2)2-3-Pyr:
N
Br
(CH2)3-3-Pyr: 9
(CH2)4-3-Pyr: 10
Br
Br
11
(CH2)3-2-Pyr:
4
5
Scheme 1. Reagents and conditions: (a) 3-bromothiophenol, THF, reflux, 37%; (b) alkylbromide, K2CO3, DMF, 80 °C; or alcohol, DIAD, PPh3, THF, rt.
Table 1
3-bromothiophenol in THF under reflux condition afforded the
2-(3-bromothiophenyl)-3-hydroxypyridine 5. Compound 5 was
then treated with alcohol under Mitsunobu reaction condition to
afford the corresponding 2,3-disubstituted pyridines 2, 3 and 6–11.
SAR study of pyridyl alkylene linker
Entry
Compound
R
n
PDE4 inhibition (%, 100 nM)
1
2
3
4
5
6
7
6
3
7
8
9
10
11
4-Pyridyl
4-Pyridyl
4-Pyridyl
3-Pyridyl
3-Pyridyl
3-Pyridyl
2-Pyridyl
2
3
4
2
3
4
3
56
80
3
60
69
5
3. Results and discussion
Our strategy to modify compound 2 is shown in Figure 2. X-ray
analysis of co-crystals of PDE4 and roflumilast showed that the ac-
tive site of PDE4 consists of three pockets, that is, the purine-selec-
tive glutamine and hydrophobic clamp pocket, the metal binding
pocket, and the solvent-filled side pocket.9 We hypothesized that
the cyano group of compound 2 would interact with the metal
binding pocket of PDE4, because the cyano group is a well-known
metal ligand. Based on this hypothesis, we decided to change the
cyano group in 2 to a pyridine, which can also bind the metal pock-
et of PDE4 and provide a drug-like structure. First, we calculated
and compared the distances of 2-, 3-, and 4-pyridines and that of
the cyano methylene group (Fig. 3). The results indicated that the
nitrogen atoms of the 3- and 4-pyridyl units are located in posi-
tions similar to the position of the nitrogen atom of the cyano
group.
Results of structure–activity relationships (SARs) for the pre-
pared pyridine derivatives are shown in Table 1. Compound 2 pos-
sessed moderate PDE4 inhibitory activity (IC50 = 104 nM).
Replacement of the cyano methylene in 2 with 4-pyridyl did not af-
fect PDE4 inhibitory activity (60% at 100 nM, entry 1). This finding
indicates that the 4-pyridyl can replace the cyano methylene as a
metal binding unit for of PDE4 inhibition. In order to obtain struc-
tural information on the interaction between the nitrogen atom of
PDE4 inhibitors and the metal binding site of PDE4, we examined
the effect of an increase in the number of carbon atoms of the alk-
ylene linker. Increase of the linker by one carbon atom improved
10
Table 2
Pharmacological data of compound 3 and rolipram
Compound PDE4 IC50 Bronchodilatory effect Cell infiltration inhibition
(nM)
(%, 10 mg/kg, p.o.)
(%, 10 mg/kg, p.o.)
Rolipram
3
54
34
N.T.
77
39
50
N.T.: not tested.
PDE4 inhibitory activity (entry 2), while adding two carbon atoms
significantly decrease PDE4 inhibitory activity (entry 3). These re-
sults indicate that the most adequate number of carbon atoms in
the linker is three. Finally, we examined the position of the nitro-
gen atom in the terminal pyridine. As expected, the 3-pyridyl com-
pounds 8–10 showed almost the same PDE4 inhibitory activity as
4-pyridyl compounds (entries 4–6), and the 2-pyridyl compound
11 showed no PDE4 inhibitory activity (entry 7).
Based on the findings of SARs, we evaluated the in vitro and
in vivo efficacy of the optimized compound 3. Compound 3 inhib-
ited inflammatory cells infiltration by up to 50% in PDE4 isozymes
isolated from guinea pig eosinophils. Oral administration of 3
(10 mg/kg) produced potent bronchodilatory effect (77%) in guinea
pigs sensitized with ovalbumin (OVA). In addition, the anti-inflam-
matory activity of 3 (50%) was stronger than that of rolipram (39%)
(Table 2).
N
N
N
N
4. Conclusion
cyano methylene
2-pyridyl
1.4 Å
3-pyridyl 4-pyridyl
2.4 Å 2.8 Å
2.6 Å
In summary, we have identified novel 2,3-disubstituted pyri-
dines as potent PDE4 inhibitors. The undesirable cyano methylene
Figure 3. Calculated distances indicated by dot-line.

Y. Kato et al. / Bioorg. Med. Chem. 21 (2013) 5851–5854
5853
group of compound 2 was successfully replaced by 4-pyridine,
leading to improved PDE4 inhibitory activity and in vivo efficacy.
Optimization of the length of the alkylene linker and the position
of the nitrogen atom of the terminal pyridine produced the 2-(3-
bromophenylthio)-3-[3-pyridin-4-yl propoxy]pyridine 3 as a po-
tent, orally active PDE4 inhibitor.
(2.0 mL) was added diisopropylazodicarboxylate (222 mg
1.1 mmol) at 0 °C and stirred at ambient temperature. After 5 h,
the mixture was evaporated, diluted with EtOAc (15 mL), and ex-
tracted with 10% HCl solution (2 ꢀ 15 mL). The aqueous phase
was then basified to pH 12 with K2CO3 and extracted with EtOAc
(2 ꢀ 15 mL). The organic phase was washed with brine
(2 ꢀ 5 mL), dried (Na2SO4), and evaporated in vacuo. The residue
was subjected to flash column chromatography (EtOAc–hexane
gradient) followed by crystallization (Et2O) to afford title com-
pound 3 as a white crystalline solid (390 mg, 97%): 1H NMR
(400 MHz, CDCl3) d 8.51 (dd, J = 1.7, 4.4 Hz, 2H), 8.01 (dd, J = 1.7,
4.6 Hz, 1H), 7.69 (dd, J = 1.7, 1.7 Hz, 1H), 7.46–7.49 (m, 2H), 7.25
(dd, J = 8.0, 8.0 Hz, 1H), 7.16 (dd, J = 1.4, 4.4 Hz, 2H), 7.05 (dd,
J = 4.4, 8.0 Hz, 1H), 7.01 (dd, J = 1.7, 8.3 Hz, 1H), 4.02 (t, J = 6.1 Hz,
2H), 2.85 (t, J = 7.3 Hz, 2H), 2.15 (tt, J = 6.1, 7.3 Hz, 2H). 13C NMR
5. Experimental section
5.1. Chemistry
5.1.1. General
Unless otherwise noted, reagents were obtained from commer-
cial suppliers and used without further purification. Melting points
were determined on a cover glass with an electrothermal melting
point apparatus and are uncorrected. Nuclear Magnetic Resonance
(NMR) spectra were recorded at ambient temperature, operating at
400 MHz for 1H NMR and 100 MHz for 13C NMR. Chemical shifts
are given in d (ppm) relative to TMS as internal standard; multi-
plicities were recorded as s (singlet), br s (broad singlet), d (dou-
(100 MHz, CDCl3)
d 151.53, 150.01, 149.85, 147.62, 141.60,
136.51, 133.13, 132.64, 131.18, 130.16, 123.94, 122.49, 121.23,
117.28, 67.15, 31.28, 29.43. Anal. Calcd for C19H17BrN2OS: C,
56.86; H, 4.27; N, 6.98. Found: C, 57.15; H, 4.35; N, 7.03. LCMS:
m/z = 400 (M, Br79)+, 402 (M, Br81)+.
blet), dd (double doublet), ddd (double double doublet),
t
5.1.5. 2-(3-Bromophenylthio)-3-(2-(pyridin-4-yl)ethoxy)pyridine
(6)
(triplet), dt (double triplet), ddt (double double triplet), q (quartet)
or m (multiplet). MS spectra were recorded under electron inpact
(EI) condition. Silica gel column chromatography was performed
on Silica gel (70–230 mesh) or prepacked amino silica gel
(40 lm, 60 Å). Reactions requiring anhydrous conditions were per-
formed under argon atmosphere.
Following the above procedure for 3, title compound 6 was pre-
pared in an identical manner from compound 5 (282 mg 1.0 mmol)
and 2-(pyridin-4-yl)ethan-1-ol (135 mg, 1.1 mmol) and was ob-
tained as a white solid (368 mg, 95%). 1H NMR (400 MHz, CDCl3)
d 8.56 (dd, J = 1.5, 4.4 Hz, 2H), 8.00 (dd, J = 3.2, 3.2 Hz, 1H), 7.66
(dd, J = 1.7, 2.0 Hz, 1H), 7.46 (ddd, J = 1.7, 2.0, 16.6 Hz, 2H), 7.28
(dd, J = 1.5, 4.6 Hz, 2H), 7.25 (dd, J = 7.8, 7.8 Hz, 1H), 7.02 (d,
J = 3.2 Hz, 2H), 4.26 (t, J = 6.4 Hz, 2H), 3.14 (t, J = 6.4 Hz, 2H). 13C
NMR (100 MHz, CDCl3) d 151.21, 149.85, 147.79, 146.85, 141.79,
136.72, 132.86, 132.82, 131.29, 130.16, 124.47, 122.46, 121.04,
117.20, 68.22, 34.92. Anal. Calcd for C18H15BrN2OS: C, 55.82; H,
3.90; N, 7.23. Found: C, 55.70; H, 3.91; N, 7.21. LCMS: m/z = 386
(M, Br79)+, 388 (M, Br81)+.
5.1.2. 2-(3-Bromophenylthio)pyridine-3-ol (5)
To a solution of 2-bromopyridin-3-ol (4) (44 g, 250 mmol) in
THF (100 mL) and DMF (100 mL) was added 3-bromothiophenol
(30 g, 160 mmol) and stirred under reflux condition. After 5 h,
the reaction mixture was then cooled and diluted with EtOAc
(1000 mL) and washed successively with 5% NaOH solution
(2 ꢀ 50 mL), and brine (2 ꢀ 100 mL). The organic phase was dried
(MgSO4) and concentrated in vacuo to afford the title compound
5 as a white solid (35 g, 78%): 1H NMR (400 MHz, CDCl3) d 8.22
(dd, J = 1.7,4.6 Hz, 1H), 7.42 (dd, J = 2.0 Hz, 1H), 7.35 (ddd, J = 1.2,
1.7, 8.0 Hz, 1H), 7.32 (dd, J = 1.7, 8.0 Hz, 1H), 7.25 (dd, J = 4.6,
8.0 Hz, 1H), 7.21 (ddd, J = 1.2, 1.7, 8.0 Hz, 1H), 7.13 (dd, 1H,
J = 8.0, 8.0 Hz, 1H), 6.65 (br s, 1H). LCMS: m/z = 281 (M, Br79)+,
283 (M, Br81)+.
5.1.6. 2-(3-Bromophenylthio)-3-(4-(pyridin-4-yl)butoxy)pyridine
(7)
Following the above procedure for 3, title compound 7 was pre-
pared in an identical manner from compound 5 (282 mg 1.0 mmol)
and 4-(pyridin-4-yl)butan-1-ol (166 mg, 1.1 mmol) and was ob-
tained as a white solid (402 mg, 97%). 1H NMR (400 MHz, CDCl3)
d 8.50 (dd, J = 1.7, 4.4 Hz, 2H), 8.00 (dd, J = 2.7, 3.7 Hz, 1H), 7.67
(dd, J = 1.7, 1.7 Hz, 1H), 7.43–7.47 (m, 2H), 7.23 (dd, J = 8.0,
8.0 Hz, 1H), 7.15 (dd, J = 1.5, 4.4 Hz, 2H), 7.04 (d, J = 1.24 Hz, 1H),
7.03 (s, 1H), 4.05 (t, J = 5.6 Hz, 2H), 2.71 (t, J = 7.2 Hz, 2H), 1.84
(m, 4H). 13C NMR (100 MHz, CDCl3) d 151.69, 150.87, 149.70,
147.60, 141.46, 136.52, 133.14, 132.66, 131.15, 130.12, 123.86,
122.44, 121.19, 117.23, 68.36, 34.73, 28.40, 26.55. Anal. Calcd for
5.1.3. 4-(2-(3-Bromophenylthio)pyridin-3-yloxy)butanenitrile
(2)
A solution of 4-bromobutyronitrile (222 mg, 1.5 mmol), 2-(3-
bromophenylthio)pyridine-3-ol (5) (282 mg 1.0 mmol) and K2CO3
(276 mg, 2.0 mmol) in DMF (2.0 mL) was stirred at 80 °C. After
5 h, the mixture was diluted with EtOAc (15 mL), and washed with
water (2 ꢀ 15 mL) and brine (5 mL), dried (Na2SO4), and evapo-
rated in vacuo. The residue was subjected to flash column chroma-
tography (EtOAc–hexane gradient) to afford the title compound 2
as a colorless oil (343 mg, 99%): 1H NMR (400 MHz, CDCl3) d 8.04
(dd,J = 2.4, 3.9 Hz, 1H), 7.67 (dd, J = 1.7, 1.7 Hz, 1H), 7.49 (ddd,
J = 0.96, 1.9, 7.8 Hz, 1H), 7.45 (ddd, J = 0.96, 1.7, 7.8 Hz, 1H), 7.28–
7.24 (m, 1H), 7.08 (s, 1H), 7.07 (d, J = 2.0 Hz, 1H), 4.17 (t,
J = 5.4 Hz, 2H), 2.63 (t, J = 7.1 Hz, 2H), 2.21–2.15 (m, 2H). Anal.
Calcd for C15H13BrN2OS: C, 51.59; H, 3.75; N, 8.02. Found: C,
51.44; H, 3.73; N, 8.00. LCMS:m/z = 349 (M, Br79)+, 351 (M, Br81)+.
C
20H19BrN2OS: C, 57.83; H, 4.61; N, 6.74. Found: C, 57.80; H,
4.63; N, 6.70. LCMS: m/z = 414 (M, Br79)+, 416 (M, Br81)+.
5.1.7. 2-(3-Bromophenylthio)-3-(2-(pyridin-3-yl)ethoxy)pyridine
(8)
Following the above procedure for 3, title compound 8 was pre-
pared in an identical manner from compound 5 (282 mg 1.0 mmol)
and 2-(pyridin-3-yl)ethan-1-ol (135 mg, 1.1 mmol) and was ob-
tained as a white solid (373 mg, 96%). 1H NMR (400 MHz, CDCl3)
d 8.60 (d, J = 2.4 Hz, 1H), 8.52 (dd, J = 1.6, 4.8 Hz, 1H), 7.98 (dd,
J = 1.6, 3.2 Hz, 1H), 7.73 (ddd, J = 1.6, 2.4, 8.0 Hz, 1H), 7.67 (dd,
J = 1.6, 3.2 Hz, 1H), 7.43–7.50 (m, 2H), 7.22–7.30 (m, 2H), 7.02 (s,
1H), 7.01 (s, 1H), 4.24 (t, J = 6.4 Hz, 2H), 3.15 (t, J = 6.4 Hz, 2H).
13C NMR (100 MHz, CDCl3) d 151.24, 150.31, 148.23, 147.81,
141.73, 136.91, 136.80, 133.40, 132.95, 132.83, 131.30, 130.17,
123.40, 122.46, 121.02, 117.13, 68.86, 32.85. Anal. Calcd for C19H17-
5.1.4. 2-(3-Bromophenylthio)-3-(3-(pyridin-4-yl)propoxy)pyridine
(3)
To
a
solution of 3-(pyridin-4-yl)propan-1-ol (151 mg,
1.1 mmol), 2-(3-bromophenylthio)pyridine-3-ol (5) (282 mg
1.0 mmol) and triphenylphosphine (289 mg, 1.1 mmol) in THF

5854
Y. Kato et al. / Bioorg. Med. Chem. 21 (2013) 5851–5854
BrN2OS: C, 55.82; H, 3.90; N, 7.23. Found: C, 55.89; H, 4.15; N, 7.64.
PDE4.10,11 The activity of PDE4 was determined by modification
of the two-step radioisotope method of Thompson et al.12 The
hydrolyzed cAMP was measured using [3H] cAMP. The IC50 values
(concentration which produced 50% inhibition of substrate hydro-
lysis) for the compounds examined were calculated from concen-
tration–response curves. Three experiments were carried out for
each agent.
LCMS: m/z = 387 (M, Br79)+, 389 (M, Br81)+.
5.1.8. 2-(3-Bromophenylthio)-3-(3-(pyridin-3-yl)propoxy)pyridine
(9)
Following the above procedure for 3, title compound 9 was pre-
pared in an identical manner from compound 5 (282 mg 1.0 mmol)
and 3-(pyridin-3-yl)propan-1-ol (151 mg, 1.1 mmol) and was ob-
tained as a white solid (392 mg, 98%). 1H NMR (400 MHz, CDCl3)
d 8.50 (d, J = 2.2 Hz, 1H), 8.47 (dd, J = 1.5, 4.6 Hz, 1H), 8.02 (dd,
J = 1.7, 4.2 Hz, 1H), 7.69 (dd, J = 1.7, 1.7 Hz, 1H), 7.55 (ddd, J = 2.0,
2.0, 7.6 Hz, 1H), 7.48 (d, J = 1.7 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H),
7.22–7.27 (m, 2H), 7.02–7.06 (m, 2H), 4.03 (t, J = 6.0 Hz, 2H), 2.86
(t, J = 7.6 Hz, 2H), 2.11-2.18 (m, 2H). 13C NMR (100 MHz, CDCl3) d
151.54, 150.00, 147.62 (2 peaks superlinposed), 141.53, 136.51,
136.29, 135.95, 133.14, 132.64, 131.15, 130.13, 123.35, 122.45,
121.21, 117.26, 67.12, 30.17, 29.05. Anal. Calcd for C19H17BrN2OS:
C, 56.86; H, 4.27; N, 6.98. Found: C, 56.87; H, 4.29; N, 7.01.
LCMS:m/z = 400 (M, Br79)+, 402 (M, Br81)+.
5.2.2. Measurement of bronchodilatory activity
Male guinea pigs (3 weeks old) were sensitized with Ovalbumin
(OVA), 6 weeks later, they were anesthetized and the trachea was
cannulated with a cannula for ventilation. The animal was venti-
lated through the cannula inserted into the trachea with room
air by a rodent ventilator (rate: 60 strokes/min). Another end of
cannula was connected to a bronchospasm transducer (UGO BA-
SILE, Italy) to measure the air-overflow according to Konzett–Röss-
ler Method.13 A vein of a hind paw was cannulated with a catheter
for administration of antigen. OVA (0.01% OVA, 0.5 mL/kg; 50 mg/
kg) was injected through the catheter to induce bronchospasm.
The response was recorded for 20 min after the challenge of OVA.
The peak response was determined within 10 min after the chal-
lenge and expressed as a percentage of the theoretical maximum
bronchospasm (i.e., ventilation volume just before challenge). Test
compounds were administered orally 1 h before the challenge. The
inhibitory % of each treatment group was calculated from in com-
parison to the vehicle control group.
5.1.9. 2-(3-Bromophenylthio)-3-(4-(pyridin-3-yl)butoxy)pyridine
(10)
Following the above procedure for 3, title compound 10 was
prepared in an identical manner from compound 5 (282 mg
1.0 mmol) and 4-(pyridin-3-yl)butan-1-ol (166 mg, 1.1 mmol)
and was obtained as a white solid (403 mg, 97%). 1H NMR
(400 MHz, CDCl3) d 8.48 (d, J = 2.4 Hz, 1H), 8.46 (dd, J = 1.6,
4.8 Hz, 1H), 7.99 (dd, J = 2.8, 3.2 Hz, 1H), 7.67 (dd, J = 1.6, 3.2 Hz,
1H), 7.53–7.56 (m, 1H), 7.42–7.48 (m, 2H), 7.22–7.26 (m, 2H),
7.03 (s,1H), 7.02 (s, 1H), 4.06 (t, J = 5.6 Hz, 2H), 2.72(t, J = 6.8 Hz,
2H), 1.82–1.90 (m, 4H). 13C NMR (100 MHz, CDCl3) d 151.70,
149.94, 147.63, 147.45, 141.44, 137.16, 136.56, 135.80, 133.13,
132.70, 131.63, 130.13, 123.33, 122.45, 121.19, 117.22, 68.40,
32.56, 28.40, 27.40. Anal. Calcd for C19H17BrN2OS: C, 57.84; H,
4.61; N, 6.74. Found: C, 57.86; H, 4.60; N, 6.77. LCMS: m/z = 415
(M, Br79)+, 417 (M, Br81)+.
5.2.3. Measurement of inhibitory activity on cell infiltration
into airway area in mice
Male mice (5 weeks old) were sensitized with OVA. Seven days
after the last injection of OVA, they were anesthetized and chal-
lenged with OVA intranasally to induce eosinophil infiltration.
Three days after the challenge, bronchoalveolar lavage was done
under NEMBUTAL anaesthetization and cells infiltrated into lung
were collected and counted. BALF was diluted with Turk’s solution
by 10 times, and living cells were counted. Test compounds were
administered orally 30 min before the challenge and the following
2 days (once a day). The inhibitory % of each treatment group was
calculated from in comparison to the vehicle control group.
5.1.10. 2-(3-Bromophenylthio)-3-(3-(pyridin-2-yl)propoxy)pyri-
dine (11)
Following the above procedure for 3, title compound 9 was pre-
pared in an identical manner from compound 5 (282 mg 1.0 mmol)
and 3-(pyridin-2-yl)propan-1-ol (151 mg, 1.1 mmol) and was ob-
tained as a white solid (315 mg, 79%). 1H NMR (400 MHz, CDCl3)
d 8.55 (ddd, J = 1.0, 1.9, 5.1 Hz, 1H), 7.99 (dd, J = 2.4, 3.9 Hz, 1H),
7.69 (dd, J = 1.8, 1.8 Hz, 1H), 7.60 (ddd, J = 2.0, 7.6, 7.6 Hz, 1H),
7.46 (dd, J = 1.7, 7.8 Hz, 2H), 7.25 (dd, J = 8.3, 8.3 Hz, 1H), 7.20 (d,
J = 7.8 Hz, 1H), 7.13 (dd, J = 4.9, 7.6 Hz, 1H), 7.04 (s, 1H), 7.03 (d,
J = 1.7 Hz, 1H) 4.08 (t, J = 6.4 Hz, 2H), 3.01 (t, J = 7.1 Hz, 2H), 2.29
(tt, J = 6.36, 7.08 Hz, 2H). 13C NMR (100 MHz, CDCl3) d 160.87,
151.75, 149.36, 147.67, 141.39, 136.60, 136.42, 133.30, 132.74,
131.15, 130.13, 123.19, 122.47, 121.23, 121.20, 117.32, 67.90,
34.31, 28.66. Anal. Calcd for C19H17BrN2OS: C, 56.86; H, 4.27; N,
6.98. Found: C, 56.63; H, 4.32; N, 7.05. LCMS: m/z = 400 (M,
Br79)+, 402 (M, Br81)+.
Acknowledgment
We thank Keiko Bando for elemental analysis and Shinya Usui
for useful discussion.
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