Discovery of new orally active phosphodiesterase (PDE4) inhibitors

Article abstract of DOI:10.1248/cpb.52.1098

A series of 4-anilinopyrazolopyridine derivatives were synthesized and biologically evaluated as inhibitors of phosphodiesterase (PDE4). Chemical modification of 3, a structurally new chemical lead that was found in our in-house library, was focused on 1- and 3-substituents. Full details of the discovery of a new orally active chemical lead 5 are presented. Structure-activity relationship data, pharmacological evaluation, and the subtype selectivity study are also presented.

Full text of DOI:10.1248/cpb.52.1098

1098
Chem. Pharm. Bull. 52(9) 1098—1104 (2004)
Vol. 52, No. 9
Discovery of New Orally Active Phosphodiesterase (PDE4) Inhibitors
Hiroshi OCHIAI,a Akiharu ISHIDA,a Tazumi OHTANI,a Kensuke KUSUMI,a Katuya KISHIKAWA,b
a
Susumu YAMAMOTO,a Hiroshi TAKEDA,a Takaaki OBATA,a Hisao NAKAI,*,a and Masaaki TODA
a Minase Research Institute, Ono Pharmaceutical Co., Ltd.; 3–1–1 Sakurai, Shimamoto, Mishima, Osaka 618–8585,
Japan: and b Development Planning, Ono Pharmaceutical Co., Ltd.; 2–1–5 Doshomachi, Chuo-ku, Osaka 541–8526,
Japan. Received April 15, 2004; accepted June 10, 2004; published online June 15, 2004
A series of 4-anilinopyrazolopyridine derivatives were synthesized and biologically evaluated as inhibitors of
phosphodiesterase (PDE4). Chemical modification of 3, a structurally new chemical lead that was found in our
in-house library, was focused on 1- and 3-substituents. Full details of the discovery of a new orally active chemi-
cal lead 5 are presented. Structure–activity relationship data, pharmacological evaluation, and the subtype selec-
tivity study are also presented.
Key words phosphodiesterase (PDE4) inhibitor; subtype selective; orally active; 4-anilinopyrazolopyridine
Phosphodiesterase 4 (PDE4) is an enzyme that is responsi- to afford chlorides 29a—z, respectively. Alkaline hydrolysis
ble for the inactivation of cyclic adenosine monophosphate of 29a—z gave carboxylic acids 30a—z, respectively. Heat-
(cAMP).1,2) The anti-inflammatory effects of PDE4 inhibitors ing of 30a—z with thionyl chloride, followed by treatment
have been well established in a variety of animal models.3) with aqueous ammonia, resulted in production of the amides
However, clinical use of these inhibitors has not been 31a—z, respectively. Replacement of the chloro moiety of
achieved yet because of dose-limiting side effects such as 31a—z with appropriate anilines led to the test compounds 5
nausea and vomiting that restrict therapeutic application.4)
and 8—26. N-Methylation of the 3-methoxyaniline moiety
In previous reports, we have described the design and syn- afforded 7. Compounds 3, 4, and 6 were prepared from 31a
thesis of bicyclo[3·3·0]octane derivatives5) and piperidine according to the procedure described for preparation of 5
derivatives,6) which are interesting new classes of PDE4 in- from 31a.
hibitors with therapeutic potential. Structure activity relation-
ship (SAR) studies of PDE4 inhibitors based on the struc- Results and Discussion
tural features of Ariflo 17) have revealed that its further modi-
A series of pyrazolopyridine derivatives were synthesized
fication yields a series of PDE4 inhibitors with an improved and evaluated for inhibition of phosphodiesterase type 4 pre-
therapeutic index relative to the classical inhibitor, rolipram 2 pared from U937 cells,12) which were derived from human
(Fig. 1). To create efficient new PDE4 inhibitors with a monocytes. The results of the assays are expressed as IC50
higher therapeutic potential, discovery and evaluation of a values, i.e., the test compound concentration that achieved
new chemical lead with a completely different structure was 50% inhibition relative to the vehicle. Test compounds were
considered to provide another approach to chemical modifi- also evaluated for inhibition of LPS-induced production of
cation. Here we report on the discovery of two new orally ac- tumor necrosis factor-a (TNF-a) in rats.13) The results are
tive pyrazolopyridines 5 and 9 (Fig. 2). SAR data are also expressed as ID50 values, i.e., the dose that showed 50% inhi-
presented.
bition relative to the vehicle.
During the course of screening of PDE4 inhibitors, com-
pound 3 showed moderate potency in both the in vitro and in
Chemistry
The synthesis of the test compounds listed in Tables 1—3 vivo assays. Preliminary SAR data are summarized in Table
is outlined in Chart 1.8—11) Compounds 27a, d—e, g—i, p, x, 1. As illustrated by the SAR study of 3—7, the meta-substi-
and z were commercially available. The other 1-methylpyra- tuted aniline analog 5 exhibited the highest activity in both
zoles, 27c and 27f, were prepared by condensation of methyl- the in vitro and in vivo evaluations. The ortho-isomer 4 also
hydrazine to the corresponding Michael acceptors 33 and 34, showed moderate in vitro activity, but the para-isomer 6 did
respectively. 3-Methylpyrazoles, 27j—o, q—w, and y were not show any in vitro activity at 0.1 mM. N-Methylation of the
prepared from 35 and the corresponding hydrazine deriva- aniline moiety of 3 afforded 7, which showed no LPDE4 in-
tives. Michael addition of 27a—z to diethyl ethoxymethylene- hibitory activity at 0.3 mM.
malonate gave 28a—z, respectively, after which ring closure
We next focused on further optimization of the 1- and 3-
was accomplished with phosphorus oxychloride under reflux methyl moieties of 5. First, optimization was started with
Fig. 2. Discovery of a New Chemical Lead 3 and Identification of Orally
Active PDE4 Inhibitors 5 and 9
Fig. 1. Structures of Ariflo 1 and Rolipram 2
* To whom correspondence should be addressed. e-mail: hi.nakai@ono.co.jp
© 2004 Pharmaceutical Society of Japan

September 2004
1099
Chart 1
Table 1. Activity Profile of Pyrazolopyridine Derivatives
Table 2. Activity Profile of Pyrazolopyridine Derivatives
Inhibition of
TNF-ab)
Inhibition of
TNF-aa)
LPDE4a)
IC50
(mM)
LPDE4a)
IC50
(mM)
R1
Compd.
R1
R2
Compd.
ID50
ID50
(mg/kg, p.o.)
(mg/kg, p.o.)
3
4
5
6
7
H
H
H
H
H
Me
0.04
0.08
0.005
ꢀ0.1
ꢀ0.3
(48%)c)
(68%)c)
2.4
8
9
10
11
12
H
Et
tBu
cPr
cPen
0.03
0.003
ꢀ0.3
0.002
0.005
NEb)
3.1
2-OMe
3-OMe
4-OMe
3-OMe
NTc)
NEb)
2.9
NTd)
NTd)
NTc)
NEb)
NTc)
a) Inhibition of PDE4 prepared from U937 cells (a cell line derived from human
monocytes). IC50 represent a mean of nꢁ2. b) ID50 for inhibition of LPS-induced
TNF-a production in rats (nꢁ7) 0.5 h after oral dosing of a test compound. c) Inhibi-
tion % at 10 mg/kg, p.o. d) Not tested.
13
14
15
0.2
0.04
0.2
chemical modification of the 3-methyl moiety of 5, as shown
in Table 2.
Replacement of the 3-methyl moiety of 5 with hydrogen,
ethyl, t-butyl, cyclopropyl, or cyclopentyl moieties gave 8—
a) See corresponding footnotes from Table 1. b) Not effective at 10 mg/kg, p.o.
c) Not tested.
12, respectively, which were nearly 6-fold less potent, nearly among compounds 13—15, did not show any in vivo activity
equipotent, far less potent, 2-fold more potent, and nearly at an oral dose of 10 mg/kg, unlike its strong in vitro activity.
equipotent as inhibitors of LPDE4, respectively. Among
Next, chemical modification of the 1-methyl moiety of 5
compounds 8—12, it was found that compounds 9 and 12 was carried out. Replacement of the 1-methyl moiety of 5
showed in vivo efficacy at ID50 values of 3.1 mg/kg, (p.o.) with an alkyl moiety, such as an ethyl, n-propyl, n-butyl,
and 2.9 mg/kg, (p.o.), respectively, while 11 showed weaker n-pentyl, cyclopentyl, or cyclohexyl moiety, gave 16—21,
in vivo activity than 12 despite its higher in vitro potency. which were nearly 6-fold less potent, equipotent, 2-fold less
Replacement of the 3-methyl moiety of 5 with aromatic sub- potent, 1.6-fold less potent, 2-fold less potent, and 20-fold
stituents such as phenyl, 2-thienyl, and para-chlorophenyl less potent inhibitors of LPDE4, respectively. Compounds
moieties resulted in 13—15, respectively, showing 40-fold, 16—20 demonstrated nearly 50% inhibition at an oral dose
8-fold, and 40-fold lower LPDE4 inhibitory activity. Com- of 10 mg/kg in the LPS-induced TNF-a production assay in
pound 14, which exhibited the strongest in vitro activity rats, while the 1-cyclohexyl analog 21, which had the weak-

1100
Vol. 52, No. 9
est in vitro activity among 16—21, did not show in vivo effi- efficacy at an oral dose of 10 mg/kg, while 25b showed 49%
cacy at 10 mg/kg. Replacement of the 1-methyl moiety of 5 inhibition of TNF-a production. In a final optimization
with an aromatic moiety, such as a phenyl, 1-(ortho-substi- process, the best substituents for position-1 and poisition-3
tuted)phenyl, 1-(meta-substituted)phenyl, or 1-(para-sub- were selected and incorporated into one molecule. Simulta-
stisuted)phenyl moiety, resulted in 22, 23a—c, 24a—c, and neous replacement of the 3-methyl moiety of 5 with a cyclo-
25a—c, respectively. Among these analogs, the 1-phenyl propyl moiety and the 1-methyl moiety with a 1-phenyl moi-
analog 22 demonstrated equipotent in vitro activity to that of ety led to 26, which showed 2-fold weaker LPDE4 inhibitory
5, but failed to show in vivo efficacy at an oral dose of activity and was much less potent in vivo (33% inhibition at
10 mg/kg. Introduction of a substituent, such as a chloro, an oral dose of 10 mg/kg). As shown in Tables 2 and 3, the
methyl, or methoxy group, into the newly introduced phenyl above chemical modification of 5 gave compounds 9, 12, 17,
moiety of 22 was found to be deleterious for both in vitro and and 19, which exhibited nearly equipotent in vitro activity
in vivo activity, as illustrated by 23a—c, 24a—c, and 25a— relative to that of 5. Among them, compounds 9 and 12 were
c. Two of these compounds, 24a and 25b, were selected for more potent and nearly equipotent in vivo, while 17 and 19
the in vivo evaluation. As a result, compound 24a showed no showed much weaker in vivo activity relative to 5. In particu-
lar, the 1-phenyl analog 22 showed no in vivo efficacy at an
Table 3. Activity Profile of Pyrazolopyridine Derivatives
oral dose of 10 mg/kg despite its equipotent in vitro activity
relative to that of 5.
Further biological evaluation of compounds 3, 5, and 9,
which were selected based on their oral activity against LPS-
induced TNF-a production, was carried out as shown in
Table 4. These compounds were evaluated for inhibition of
slow reacting substance of anaphylaxis (SRS-A)-mediated
bronchoconstriction in guinea pigs.14,15) The results are ex-
pressed as ID50 values, i.e., the dose that gave 50% inhibition
relative to the vehicle. These compounds were also evaluated
for inhibition of TNF-a production in human whole blood
(HWB)16) to estimate their clinical potential. The results are
expressed as IC50 values, i.e., the test compound concentra-
tion that gave 50% inhibition relative to the vehicle. It was
found that the inhibition of bronchoconstriction in actively
sensitized guinea pigs was not always consistent with the in-
hibition of LPS-induced TNF-a production in rats, probably
because of differences in pharmacokinetics due to cross-
species comparison. Compounds 5 and 9 demonstrated 50%
inhibition of SRS-A-induced bronchoconstriction, with ID50
values of 6.8 mg/kg, (p.o.) and 7.9 mg/kg, (p.o.), respectively,
while their ID50 values for TNF-a production were 2.4 mg/kg
and 3.1 mg/kg, respectively. Compound 3 showed 67% inhi-
bition of SRS-A-mediated bronchoconstriction at an oral
dose of 30 mg/kg, and it showed 48% inhibition of TNF-a
production in rats at an oral dose of 10 mg/kg. Regarding in-
hibition of gastric emptying in rats,17) the ID50 values of 3
and 5 were higher than that of Ariflo 1, while their IC50 val-
ues for LPS-induced TNF-a production assay in HWB were
lower than that of 1. Compounds 3, 5, and 9 were further
evaluated to access their side effect profile in a ferret emesis
model. It was shown that 3 and 5 did not cause emesis at oral
Inhibition of
TNF-aa)
LPDE4a)
IC50
(mM)
Compd.
R1
ID50
(mg/kg, p.o.)
16
17
18
19
20
21
Et
nPr
nBu
nPen
cPen
cHex
0.03
0.004
0.01
0.008
0.01
0.1
(54%)b)
(56%)b)
(47%)b)
(58%)c)
(48%)b)
NEe)
22
0.004
NEe)
23a (XꢁCl)
23b (XꢁMe)
23c (XꢁOMe)
0.04
0.08
0.07
NTd)
NTd)
NTd)
NEe)
NTd)
NTd)
NTd)
(49%)b)
NTd)
24a (XꢁCl)
24b (XꢁMe)
24c (XꢁOMe)
0.01
0.02
0.02
25a (XꢁCl)
25b (XꢁMe)
25c (XꢁOMe)
0.05
0.04
0.03
26
0.01
NEe)
a) See corresponding footnotes from Table 1. b) Inhibition % at 10 mg/kg, p.o.
c) Inhibition % at 3 mg/kg, p.o. d) Not tested. e) Not effective at 10 mg/kg, p.o.
Table 4. Activity Profile of Pyrazolopyridine Derivatives
Inhibition of
Inhibition of
TNF-a
Inhibition of
gastric
Inhibition of
TNF-a
production
in HWBd)
IC50 (mM)
Ferret emesise)
(vomiting/tested)
bronchoconstrictiona)
Compd.
productionb)
ID50
emptyingc)
ID50
ID50
(mg/kg, p.o.)
3
10
30
(mg/kg, p.o.)
(mg/kg, p.o.)
(mg/kg, p.o.)
1 (Ariflo)
4.5
(67%)g)
6.8
1.7
(48%)f)
2.4
5.7
18
13
3.0
1.2
NTh) NTh) NTh)
NTh) NTh) 0/2
3
5
9
(5%)f)
(23%)f)
(30%)f)
0/2
0/2
1/2
7.9
3.1
NTh) NTh) NTh)
a) Inhibition of SRS-A-mediated bronchoconstriction and airway microvascular leakage in actively sensitized guinea pigs (nꢁ3—6); OVA challenge 0.15 mg/kg 1 h after oral
dosing of a test compound. b) See corresponding footnotes from Table 1. c) Inhibition of gastric emptying in rats (nꢁ5). d) Inhibition of LPS-induced TNF-a production in
human whole blood. IC50 represent a mean of nꢁ3. e) Vomiting test in fasted ferrets. f) Inhibition % at 10 mg/kg, p.o. g) Inhibition % at 30 mg/kg, p.o. h) Not tested.

September 2004
1101
ing reaction was carried out under argon atmosphere. To a stirred mixture of
2-chlorophenylhydrazine hydrochloride (5.00 g, 27.9 mmol) and 35 (2.29 g,
27.9 mmol) in EtOH (160 ml) was added Et3N (3.97 ml, 27.9 mmol) under
refluxing for 2 h. The reaction mixture was poured into water and extracted
with EtOAc. The organic layer was dried over MgSO4 and concentrated in
vacuo. To a stirred solution of the residue in AcOH (16 ml) was added
AcONa (229 mg, 2.79 mmol). The reaction mixture was stirred at 90 °C for
6.5 h, poured into ice water, neutralized with 5 N NaOH and extracted with a
mixture of EtOAc/Et2O. The organic layer was washed with water and brine,
died over MgSO4 and concentrated in vacuo. The resulting residue was puri-
fied by column chromatography on silica gel (n-hexane/EtOAc, 1/1) to give
27q (4.07 g, 19.6 mmol, 70%) as an ivory powder. 1H-NMR (200 MHz,
CDCl3) 7.56—7.36 (m, 4H), 5.48 (s, 1H), 3.58 (br, 2H), 2.24 (s, 3H).
The following compounds 27r—w and 27y were prepared from the corre-
sponding hydrazine derivatives according to the same procedures as de-
scribed for the preparation of 27q from 35 and 2-chlorophenylhydrazine.
Diethyl 2-{[(1,3-Dimethyl-1H-pyrazol-5-yl)amino]methylene}malonate
(28a) A mixture of 5-amino-1,3-dimethylpyrazole 27a (20.0 g, 180 mmol)
and diethyl ethoxymethylenemalonate (38.9 g, 180 mmol) was stirred at
120 °C for 1 h. The reaction mixture was poured into n-hexane and the pre-
cipitates were collected by filtration to give 28a (45.3 g, 161 mmol, 89%) as
an ivory powder. 1H-NMR (300 MHz, CDCl3) 11.00 (bd, Jꢁ12.3 Hz, 1H),
Table 5. The Profile against PDEs of 5
Compd.
% Inhibition at 10 mM
PDE1a)
PDE2b)
PDE3b)
PDE5b)
PDE6c)
27
24
54
14
10
Source: a) bovine heart, b) human platelets, c) bovineretinal rod outersegments.
doses of up to 30 mg/kg and 10 mg/kg, respectively.
Compounds 3, 5, and 9 demonstrated unexpectedly poor
bioavailability (BA) despite their relatively good oral activity
(the BA of 3, 5, and 9 was 8.2%, 1.8%, and 3.1%, respec- 8.14 (d, Jꢁ12.3 Hz, 1H), 5.86 (s, 1H), 4.32 (q, Jꢁ7.2 Hz, 2H), 7.24 (q,
Jꢁ7.1 Hz, 2H), 3.74 (s, 3H), 2.23 (s, 3H), 1.38 (t, Jꢁ7.2 Hz, 3H), 1.32 (t,
Jꢁ7.2 Hz, 3H).
The following compounds 28b—z were prepared from the corresponding
pyrazole derivatives 27b—z, respectively according to the same procedures
tively). One of the reasons for this result was estimated to be
ascribed to their potent inhibitory activity of LPS-induced
TNF-a production in rat whole blood. For example, com-
pound 5 exhibited IC50 value of 0.18 mM (data is not given in
as described for the preparation of 28a from 27a.
Ethyl 4-Chloro-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxy-
late (29a) The following reaction was carried out under argon atmosphere.
A mixture of 28a (28.1 g, 100 mmol) and POCl3 (140 ml, 1.50 mol) was
heated at 120 °C under stirring for 18 h. The reaction mixture was poured
into ice water and the precipitates were collected by filtration to give 29a
the table) in rat whole blood.
Subtype selectivity of 5 was also investigated, as displayed
in Table 6. The inhibitory activity of 5 against PDE1, 2, 3, 5,
and 6 at 10 mM was 27%, 24%, 54%, 14% and 10%, respec-
tively.18—21) Accordingly, compound 5 seems to be a subtype-
selective PDE4 inhibitor.
1
(19.6 g, 77.5 mmol, 77%) as an ivory powder. H-NMR (300 MHz, CDCl3)
8.95 (s, 1H), 4.45 (q, Jꢁ7.2 Hz, 2H), 4.07 (s, 3H), 2.76 (s, 3H), 1.44 (t,
Jꢁ7.2 Hz, 3H).
The following compounds 29b—z were prepared from the corresponding
enamine derivatives 28b—z, respectively according to the same procedures
as described for the preparation of 29a from 28a.
Conclusion
Design and synthesis of new orally active subtype-selec-
tive PDE4 inhibitors were achieved by structural optimiza-
tion of chemical lead 3, which was found among our in-
house library. From the compounds tested, compound 5
demonstrated best results with regard to oral activity and side
effects, but it showed unexpectedly poor oral bioavailability.
Further chemical modification of 5 to improve its bioavail-
ability may be promising to identify an orally bioavailable
PDE4 inhibitor with a higher therapeutic potential.
4-Chloro-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic Acid
(30a) To a stirred mixture of 29a (19.6 g, 77.5 mmol) and 85% KOH
(50.0 g, 760 mmol) in DME (152 ml) was added H2O (120 ml). After being
stirred at room temperature for 18 h, the reaction mixture was neutralized
with 4 N HCl. The precipitates were collected by filtration and washed with
H2O to give 30a (17.3 g, 76.9 mmol, 99%) as a white powder. 1H-NMR
(300 MHz, DMSO-d6) 8.90 (s, 1H), 4.00 (s, 3H), 3.33 (br s, 1H), 2.67 (s,
3H).
The following compounds 30b—z were prepared from the corresponding
ester derivatives 29b—z, respectively according to the same procedures as
described for the preparation of 30a from 29a.
Experimental
4-Chloro-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
(31a) The following reaction was carried out under argon atmosphere. A
mixture of 30a (2.00 g, 8.89 mmol) and SOCl2 (3.24 ml, 44.5 mmol) was
heated at 80 °C under stirring for 1 h, the reaction mixture was concentrated
in vacuo. The resulting residue was used for the next step without further
purification.
To a stirred mixture of 28% NH4OH (12 ml) and THF (30 ml) was added
a solution of the acyl chloride in THF (10 ml) at 0 °C. After being stirred at
0 °C for 10 min, the reaction mixture was concentrated in vacuo. The residue
was poured into H2O and extracted with CHCl3. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The residue was triturated with
Et2O to give 31a (1.98 g, 8.84 mmol, 99%) as a white powder. 1H-NMR
(300 MHz, DMSO-d6) 8.52 (s, 1H), 8.02 (br s, 1H), 7.78 (br s, 1H), 3.98 (s,
3H), 2.64 (s, 3H).
The following compounds 31b—z were prepared from the corresponding
carboxylic acid derivatives 30b—z, respectively according to the same pro-
cedures as described for the preparation of 31a from 30a.
4-[(3-Methoxyphenyl)amino]-1,3-dimethyl-1H-pyrazolo[3,4-b]pyri-
dine-5-carboxamide (5) To a mixture of 31a (899 mg, 4.01 mmol) in
dioxane (28 ml) was added aniline 32 (1.48 g, 12.0 mmol). After being
General Procedure Analytical samples were homogeneous as con-
firmed by thin-layer chromatography (TLC), and yielded spectroscopic data
consistent with the assigned structures. All 1H-NMR spectra were obtained
with a Varian Gemini-200 or MERCURY-300 spectrometer. The chemical
shift values are reported in ppm (d) and coupling constants (J) in Hertz
(Hz). Fast atom bombardment (FAB) and electron ionisation (EI) mass spec-
tra were obtained with a JEOL JMS-DX303HF or JMS-700 spectrometer. IR
spectra were measured using a Perkin-Elmer FTIR 1760X or JASCO FTIR-
430 spectrometer. Column chromatography was carried out using silica gel
[Merck silica gel 60 (0.063—0.200 mm), Wako Gel C200, Fuji Silysia
FL60D, or Fuji Silysia BW-235]. TLC was also performed on silica gel
(Merck TLC plate, silica gel 60 F254).
3-Ethyl-1-methyl-1H-pyrazol-5-amine (27c) The following reaction
was carried out under argon atmosphere. To a stirred solution of methylhy-
drazine (3.88 ml, 73.0 mmol) in EtOH (16 ml) was added 33 (8.10 g,
73.0 mmol) under refluxing for 2 h. The reaction mixture was concentrated
in vacuo. The resulting residue was purified by column chromatography on
silica gel (EtOAc) to give 27c (6.84 g, 54.7 mmol, 75%) as a pale orange
1
powder. H-NMR (200 MHz, CDCl3) 5.38 (s, 1H), 3.61 (s, 3H), 3.45 (br s,
2H), 2.52 (q, Jꢁ7.4 Hz, 2H), 1.20 (t, Jꢁ7.4 Hz, 3H).
The following compounds 27f and 27j—o were prepared according to the stirred at 120 °C for 2 h, the reaction mixture was poured into Et2O and the
same procedures as described for the preparation of 27c.
precipitates were collected by filtration. The resulting powder was triturated
1-(2-Chlorophenyl)-3-methyl-1H-pyrazol-5-amine (27q) The follow- with H2O to give 5 (686 mg, 2.21 mmol, 55%) as white crystals. mp 259—

1102
Vol. 52, No. 9
260 °C; 1H-NMR (300 MHz, DMSO-d6) 10.97 (s, 1H), 8.74 (s, 1H), 8.21
(br s, 1H), 7.56 (br s, 1H), 7.26—7.17 (m, 1H), 6.75—6.60 (m, 3H), 3.89 (s,
3H), 3.71 (s, 3H), 1.69 (s, 3H); IR (KBr) 3363, 3196, 1676, 1619, 1585,
1521, 1500, 1385; HR-MS (EI) Calcd for C16H17N5O2 311.1382, Found
311.1369.
The following compounds 3—4, 6 and 8—26 were prepared from the cor-
responding 4-chloropyrazolopyridine derivatives 31a—z, respectively ac-
cording to the same procedures as described for the preparation of 5 from 31a.
4-Anilino-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide (3)
Yield 63%; Off-white crystals; mp 276—277 °C; 1H-NMR (300 MHz,
DMSO-d6) 11.0 (s, 1H), 8.74 (s, 1H), 8.21 (br, 1H), 7.55 (br, 1H), 7.36—
7.28 (m, 2H), 7.17—7.07 (m, 3H), 3.88 (s, 3H), 1.59 (s, 3H); IR (KBr)
3406, 3185, 2925, 1677, 1619, 1586, 1521, 1500, 1422, 1384, 1146; HR-MS
(FAB) Calcd for C15H16N5O 282.1355, Found 282.1381.
4-[(2-Methoxyphenyl)amino]-1,3-dimethyl-1H-pyrazolo[3,4-b]pyri-
dine-5-carboxamide (4) Yield 46%; White crystals; mp 260—262 °C; 1H-
NMR (300 MHz, DMSO-d6) 10.70 (s, 1H), 8.71 (s, 1H), 8.18 (br s, 1H),
7.49 (br s, 1H), 7.16—7.08 (m, 2H), 6.95 (d, Jꢁ7.5 Hz, 1H), 6.88—6.79 (m,
1H), 3.87 (s, 3H), 3.86 (s, 3H), 1.59 (s, 3H); IR (KBr) 3435, 3350, 3205,
1655, 1615, 1588, 1566, 1521, 1486, 1441, 1403, 1382, 1354, 1258, 1114,
1022; HR-MS (EI) Calcd for C16H17N5O2 311.1382, Found 311.1365.
4-[(4-Methoxyphenyl)amino]-1,3-dimethyl-1H-pyrazolo[3,4-b]pyri-
dine-5-carboxamide (6) Yield 91%; White crystals; mp 269—278 °C; 1H-
NMR (300 MHz, DMSO-d6) 11.11 (s, 1H), 8.71 (s, 1H), 8.16 (br s, 1H),
7.49 (br s, 1H), 7.08 (d, Jꢁ6.9 Hz, 2H), 6.92 (d, Jꢁ6.9 Hz, 2H), 3.85 (s, 3H),
3.75 (s, 3H), 1.54 (s, 3H); IR (KBr) 3401, 3189, 1679, 1618, 1585, 1519,
1493, 1440, 1385, 1254; HR-MS (EI) Calcd for C16H17N5O2 311.1382,
Found 311.1423.
373.1552.
4-[(3-Methoxyphenyl)amino]-1-methyl-3-(2-thienyl)-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (14) Yield 79%; Yellow crystals; mp 183—
189 °C; 1H-NMR (300 MHz, DMSO-d6) 11.12 (1H), 8.84 (s, 1H), 8.29 (br s,
1H), 7.66 (br s, 1H), 7.25 (dd, Jꢁ5.1, 0.9 Hz, 1H), 6.92 (dd, Jꢁ3.6, 0.9 Hz,
1H), 6.81 (t, Jꢁ7.5 Hz, 1H), 6.66 (dd, Jꢁ5.1, 3.6 Hz, 1H), 6.37—6.24 (m,
3H), 4.04 (s, 3H), 3.55 (s, 3H); IR (KBr) 3344, 3200, 1659, 1587, 1520,
1494, 1401, 1270, 1155; HR-MS (EI) Calcd for C19H17N5O2S 379.1103,
Found 379.1111.
3-(4-Chlorophenyl)-4-[(3-methoxyphenyl)amino]-1-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (15) Yield 74%; Yellow crystals; mp
186—189 °C; H-NMR (300 MHz, DMSO-d6) 11.22 (s, 1H), 8.85 (s, 1H),
8.30 (br s, 1H), 7.65 (br s, 1H), 7.26 (d, Jꢁ8.4 Hz, 2H), 7.10 (d, Jꢁ8.4 Hz,
2H), 6.80—6.70 (m, 1H), 6.28—6.20 (m, 3H), 4.04 (s, 3H), 3.54 (s, 3H); IR
(KBr) 3349, 3150, 1671, 1586, 1560, 1508, 1494, 1399, 1355, 1316, 1266,
1154; HR-MS (EI) Calcd for C21H18ClN5O2 407.1149, Found 407.1146.
1-Ethyl-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (16) Yield 8% in 3 steps; Gray crystals; mp
1
1
196—198 °C; H-NMR (300 MHz, DMSO-d6) 10.93 (s, 1H), 8.71 (s, 1H),
8.22—8.15 (br s, 1H), 7.60—7.50 (br s, 1H), 7.20 (dd, Jꢁ8.7, 7.8 Hz, 1H),
6.70—6.67 (m, 2H), 6.63 (d, Jꢁ8.7 Hz, 1H), 4.31 (q, Jꢁ7.2 Hz, 2H), 3.70
(s, 3H), 1.69 (s, 3H), 1.35 (t, Jꢁ7.2 Hz, 1H); IR (KBr) 3424, 3195, 2926,
1655, 1585, 1515, 1491, 1440, 1408, 1383, 1356, 1299, 1247, 1156, 1039,
948; HR-MS (EI) Calcd for C17H19N5O2 325.1539, Found 325.1547.
4-[(3-Methoxyphenyl)amino]-3-methyl-1-propyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (17) Yield 28% in 2 steps; Ivory crystals; mp
169—171 °C; H-NMR (300 MHz, DMSO-d6) 10.94 (s, 1H), 8.71 (s, 1H),
7.18 (br s, 1H), 7.55 (br s, 1H), 7.23—7.17 (m, 1H), 6.71—6.62 (m, 3H),
4.23 (t, Jꢁ6.6 Hz, 2H), 3.69 (s, 3H), 1.80 (tq, Jꢁ6.6, 7.2 Hz, 2H), 1.69 (s,
3H), 0.81 (t, Jꢁ7.2 Hz, 3H); IR (KBr) 3343, 3183, 2961, 1661, 1586, 1561,
1512, 1437, 1407, 1382, 1354, 1285, 1237, 1156, 1040, 972; HR-MS (EI)
Calcd for C18H21N5O2 339.1695, Found 339.1701.
1-Butyl-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (18) Yield 23% in 2 steps; Pale yellow crys-
tals; mp 157—159 °C; H-NMR (300 MHz, DMSO-d6) 10.94 (s, 1H), 8.71
1
4-[(3-Methoxyphenyl)amino]-1-methyl-1H-pyrazolo[3,4-b]pyridine-5-
1
carboxamide (8) Yield 67%; White crystals; mp 235—237 °C; H-NMR
(300 Hz, DMSO-d6) 11.49 (s, 1H), 8.72 (s, 1H), 8.14 (br s, 1H), 7.46 (br s,
1H), 7.38 (t, Jꢁ7.5 Hz, 1H), 7.00—6.85 (m, 3H), 6.67 (s, 1H), 3.89 (s, 3H),
3.75 (s, 3H); IR (KBr) 3384, 3026, 2925, 1653, 1601, 1522, 1490, 1407,
1343, 1283, 1269, 1196, 1160, 1049, 977; HR-MS (EI) Calcd for
C15H15N5O2 297.1226, Found 297.1229.
1
(s, 1H), 8.19 (br s, 1H), 7.55 (br s, 1H), 7.23—7.17 (m, 1H), 6.73—6.60 (m,
3H), 4.28 (t, Jꢁ7.0 Hz, 2H), 3.69 (s, 3H), 1.77 (quint., Jꢁ7.0 Hz, 2H), 1.68
(s, 3H), 1.20 (tq, Jꢁ7.0, 7.5 Hz, 2H), 0.87 (t, Jꢁ7.5 Hz, 3H); IR (KBr) 3620,
3350, 3185, 2958, 1933, 1871, 2767, 2549, 1737, 1669, 1586, 1513, 1489,
1464, 1438, 1407, 1382, 1355, 1287, 1254, 1232, 1194, 1151, 1118, 1079,
1038; HR-MS (EI) Calcd for C19H23N5O2 353.1852, Found 353.1861.
4-[(3-Methoxyphenyl)amino]-3-methyl-1-pentyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (19) Yield 75%; Pale gray crystals; mp 148—
150 °C; 1H-NMR (300 MHz, DMSO-d6) 10.92 (s, 1H), 8.70 (s, 1H), 8.18
(br, 1H), 7.54 (br, 1H), 7.23—7.17 (m, 1H), 6.71—6.67 (m, 2H), 6.65—6.61
(m, 1H), 4.26 (t, Jꢁ7.2 Hz, 2H), 3.69 (s, 3H), 1.84—1.73 (m, 2H), 1.69 (s,
3H), 1.36—1.13 (m, 4H), 0.82 (t, Jꢁ7.1 Hz, 3H); IR (KBr) 3385, 3195,
2939, 1669, 1615, 1590, 1561, 1514, 1489, 1440, 1409, 1383, 1355, 1280,
1151, 1038; HR-MS (EI) Calcd for C20H25N5O2 367.2008, Found 367.2014.
1-Cyclopentyl-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (20) Yield 73%; Ivory crystals; mp
153—157 °C; 1H-NMR (300 Hz, DMSO-d6) 10.91 (s, 1H), 8.70 (s, 1H),
8.18 (br s, 1H), 7.54 (br s, 1H), 7.19 (t, Jꢁ8.4 Hz, 1H), 6.75—6.60 (m, 3H),
5.23 (quintet, Jꢁ7.4 Hz, 1H), 3.70 (s, 3H), 2.10—1.75 (m, 6H), 1.70 (s, 3H),
1.75—1.60 (m, 2H); IR (KBr) 3341, 3184, 2957, 2870, 1657, 1586, 1559,
1509, 1441, 1382, 1354, 1283, 1257, 1196, 1154, 1045, 916; HR-MS (EI)
Calcd for C20H23N5O2 365.1852, Found 365.1862.
3-Ethyl-4-[(3-methoxyphenyl)amino]-1-methyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (9) Yield 56%; Ivory crystals; mp 209—
211 °C; 1H-NMR (300 Hz, CDCl3) 10.87 (s, 1H), 8.74 (s, 1H), 8.21 (br s,
1H), 7.56 (br s, 1H), 7.17 (t, Jꢁ8.1 Hz, 1H), 6.70—6.60 (m, 2H), 6.58 (m,
1H), 3.90 (s, 3H), 3.68 (s, 3H), 1.98 (q, Jꢁ7.2 Hz, 2H), 0.93 (t, Jꢁ7.2 Hz,
3H); IR (KBr) 3354, 3180, 2939, 1670, 1586, 1522, 1491, 1380, 1266, 1209,
1155, 1039, 975; HR-MS (EI) Calcd for C17H19N5O2 325.1539, Found
325.1549.
3-tert-Butyl-4-[(3-methoxyphenyl)amino]-1-methyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (10) Yield 100%; Ivory crystals; mp 224—
228 °C; 1H-NMR (300 MHz, DMSO-d6) 8.62 (s, 1H), 8.14 (s, 1H), 7.73
(br s, 1H), 7.39 (br s, 1H), 7.03 (t, Jꢁ8.4 Hz, 1H), 6.43—6.35 (m, 1H),
6.27—6.20 (m, 2H), 3.99 (s, 3H), 3.64 (s, 3H), 1.33 (s, 9H); IR (KBr) 3435,
3333, 3298, 3196, 1667, 1577, 1557, 1502, 1299, 1287, 1154; HR-MS (EI)
Calcd for C19H23N5O2 353.1852, Found 353.1839.
3-Cyclopropyl-4-[(3-methoxyphenyl)amino]-1-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (11) Yield 97%; Ivory crystals; mp
1
234—237 °C; H-NMR (300 MHz, DMSO-d6) 11.00 (s, 1H), 8.74 (s, 1H),
8.22 (br s, 1H), 7.57 (br s, 1H), 7.19 (t, Jꢁ7.8 Hz, 1H), 6.69—6.60 (m, 3H),
3.87 (s, 3H), 3.68 (s, 3H), 1.26—1.14 (m, 1H), 0.70—0.60 (m, 2H), 0.44—
0.34 (m, 2H); IR (KBr) 3399, 3191, 1664, 1617, 1595, 1565, 1522, 1490,
1397, 1264, 1156; HR-MS (EI) Calcd for C18H19N5O2 337.1539, Found
337.1526.
1-Cyclohexyl-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyrazolo[3,4-
1
3-Cyclopentyl-4-[(3-methoxyphenyl)amino]-1-methyl-1H-pyra- b]pyridine-5-carboxamide (21) Yield quant; White amorphous solid; H-
zolo[3,4-b]pyridine-5-carboxamide (12) Yield 75%; White crystals; mp
183—185 °C; H-NMR (300 MHz, DMSO-d6) 10.82 (s, 1H), 8.74 (s, 1H),
NMR (300 MHz, CDCl3) 10.54 (s, 1H), 8.51 (s, 1H), 7.22—7.15 (m, 1H),
6.73—6.66 (m, 3H), 5.90—5.70 (br s, 2H), 4.78—4.68 (m, 1H), 3.76 (s,
1
8.21 (br s, 1H), 7.56 (br s, 1H), 7.17 (d, Jꢁ8.1 Hz, 1H), 6.70—6.61 (m, 2H), 3H), 2.05—1.85 (m, 6H), 1.80 (s, 3H), 1.75—1.20 (m, 4H); IR (KBr) 3346,
6.56 (d, Jꢁ8.1 Hz, 1H), 3.91 (s, 3H), 3.69 (s, 3H), 2.25—2.10 (m, 1H), 3193, 2933, 2856, 1656, 1587, 1509, 1454, 1383, 1357, 1308, 1261, 1235,
1.65—1.43 (m, 6H), 1.35—1.15 (m, 2H); IR (KBr) 3350, 3175, 1668, 1586, 1155, 1046, 971; HR-MS (EI) Calcd for C21H25N5O2 379.2008, Found
1561, 1519, 1490, 1455, 1398, 1318, 1266, 1156; HR-MS (EI) Calcd for 379.2004.
C20H23N5O2 365.1852, Found 365.1845.
4-[(3-Methoxyphenyl)amino]-3-methyl-1-phenyl-1H-pyrazolo[3,4-
4-[(3-Methoxyphenyl)amino]-1-methyl-3-phenyl-1H-pyrazolo[3,4- b]pyridine-5-carboxamide (22) Yield 62%; White crystals; mp 189—
b]pyridine-5-carboxamide (13) Yield 99%; White crystals; mp 209—
193 °C; 1H-NMR (300 MHz, CDCl3) 10.56 (s, 1H), 8.60 (s, 1H), 8.12—8.09
213 °C; 1H-NMR (300 MHz, CDCl3) 10.47 (s, 1H), 8.64 (s, 1H), 7.35—7.31 (m, 2H), 7.53—7.48 (m, 2H), 7.26—7.18 (m, 2H), 6.78—6.69 (m, 3H),
(m, 2H), 7.11—7.06 (m, 3H), 6.75 (t, Jꢁ8.1 Hz, 1H), 6.37—6.32 (m, 1H), 5.90—5.70 (br s, 2H), 3.77 (s, 3H), 1.77 (s, 3H); IR (KBr) 3396, 3195, 1659,
6.26—6.19 (m, 2H), 5.90—5.75 (br s, 2H), 4.14 (s, 3H), 3.59 (s, 3H); IR
1593, 1561, 1508, 1490, 1465, 1439, 1404, 1384, 1361, 1308, 1229, 1197,
(KBr) 3351, 3194, 1677, 1587, 1519, 1494, 1470, 1400, 1357, 1309, 1268, 1151, 1127, 1037, 945; HR-MS (EI) Calcd for C21H19N5O2 373.1539, Found
1241, 1156, 1036, 974; HR-MS (EI) Calcd for C21H19N5O2 373.1539, Found 373.1535.

September 2004
1103
159—163 °C; 1H-NMR (300 MHz, CDCl3) 10.50 (s, 1H), 8.61 (s, 1H), 8.12
(t, Jꢁ7.5 Hz, 2H), 7.49 (t, Jꢁ8.1 Hz, 2H), 7.31—7.17 (m, 2H), 6.78—6.62
(m, 3H), 6.00—5.60 (br s, 2H), 1.37—1.25 (m, 1H), 0.90—0.81 (m, 2H),
0.53—0.48 (m, 2H); IR (KBr) 3376, 1655, 1593, 1559, 1506, 1397, 1306,
1151, 1051, 954; HR-MS (EI) Calcd for C23H21N5O2 399.1695, Found
399.1717.
1-(2-Chlorophenyl)-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (23a) Yield 78%; Beige crystals; mp
230—231 °C; H-NMR (300 MHz, DMSO-d6) 10.93 (br, 1H), 8.67 (s, 1H),
8.23 (br, 1H), 7.70 (dd, Jꢁ7.7, 1.8 Hz, 1H), 7.66—7.49 (m, 4H), 7.25 (dd,
Jꢁ7.7, 7.7 Hz, 1H), 6.78—6.67 (m, 3H), 3.72 (s, 3H), 1.78 (s, 3H); IR
(KBr) 3331, 3196, 1651, 1592, 1489, 1384, 1359, 1300, 1264, 1225, 1195,
1151, 1105, 1036, 956; HR-MS (EI) Calcd for C21H18ClN5O2 407.1149,
Found 407.1172.
1
4-[(3-Methoxyphenyl)(methyl)amino]-1,3-dimethyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide (7) The following reaction was carried out
under argon atmosphere. To a stirred mixture of 5 (100 mg, 0.322 mmol),
toluene (10 ml) and MeCN (5 ml) were added Ag2O (112 mg, 0.485 mmol)
and MeI (568 mg, 4.00 mmol). After being stirred at 0 °C for 15 h, the pre-
cipitates were removed by filtration through a pad of celite, and washed with
toluene. The filtrate was concentrated in vacuo. The resulting residue was
purified by column chromatography on silica gel (CHCl3/MeOH, 50/1) to
4-[(3-Methoxyphenyl)amino]-3-methyl-1-(2-methylphenyl)-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (23b) Yield 71%; Ivory crystals; mp
1
133—136 °C; H-NMR (300 MHz, DMSO-d6) 10.95 (s, 1H), 8.67 (s, 1H),
8.21 (br s, 1H), 7.59 (br s, 1H), 7.45—7.30 (m, 4H), 7.24 (t, Jꢁ8.1 Hz, 1H),
6.80—6.60 (m, 3H), 3.72 (s, 3H), 2.05 (s, 3H), 1.78 (s, 3H); IR (KBr) 3188,
1654, 1587, 1560, 1509, 1302, 1296, 1151; HR-MS (EI) Calcd for
C22H21N5O2 387.1695, Found 387.1698.
1
give 7 (98.4 mg, 0.303 mmol, 94%) as a yellow amorphous solid. H-NMR
1-(2-Methoxyphenyl)-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (23c) Yield 79% in 3 steps; White
crystals; mp 205—206 °C; 1H-NMR (300 MHz, DMSO-d6) 10.87 (s, 1H),
8.63 (s, 1H), 8.25—8.15 (br s, 1H), 7.62—7.53 (br s, 1H), 7.52—7.46 (m,
1H), 7.38—7.34 (m, 1H), 7.27—7.21 (m, 2H), 7.10—7.05 (m, 1H), 6.74—
6.66 (m, 3H), 3.73 (s, 3H), 3.70 (s, 3H), 1.77 (s, 3H); IR (KBr) 3431, 3176,
1668, 1584, 1561, 1516, 1467, 1438, 1406, 1383, 1357, 1298, 1278, 1238,
1152, 1043, 1021, 951; HR-MS (EI) Calcd for C22H21N5O3 403.1644, Found
403.1663.
1-(3-Chlorophenyl)-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (24a) Yield 99%; White crystals; mp
93—95 °C; 1H-NMR (300 MHz, DMSO-d6) 10.95 (br, 1H), 8.84 (s, 1H),
8.39 (t, Jꢁ2.0 Hz, 1H), 8.28 (br, 1H), 8.22—8.18 (m, 1H), 7.70 (br, 1H),
7.55 (dd, Jꢁ8.1, 8.1 Hz, 1H), 7.38—7.33 (m, 1H), 7.22 (t, Jꢁ8.0 Hz, 1H),
6.79—6.68 (m, 3H), 3.72 (s, 3H), 1.79 (s, 3H); IR (KBr) 3413, 3195, 1710,
1685, 1589, 1561, 1508, 1484, 1435, 1407, 1385, 1291, 1152, 1038; HR-MS
(EI) Calcd for C21H18C11N5O2 407.1149, Found 407.1134.
4-[(3-Methoxyphenyl)amino]-3-methyl-1-(3-methylphenyl)-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (24b) Yield 92%; Brown crystals;
mp 133—140 °C; 1H-NMR (300 MHz, DMSO-d6) 10.92 (s, 1H), 8.81 (s,
1H), 8.26 (br s, 1H), 8.00—7.95 (m, 2H), 7.67 (br s, 1H), 7.40 (t, Jꢁ7.8 Hz,
1H), 7.21 (t, Jꢁ7.8 Hz, 1H), 7.12 (d, Jꢁ7.8 Hz, 1H), 6.80—6.65 (m, 3H),
3.71 (s, 3H), 2.39 (s, 3H), 1.80 (s, 3H); IR (KBr) 3429, 2923, 1731, 1652,
1588, 1559, 1506, 1489, 1402, 1383,1358, 1298, 1223, 1197, 1150, 1044;
HR-MS (EI) Calcd for C22H21N5O2 387.1695, Found 387.1697.
1-(3-Methoxyphenyl)-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (24c) Yield 28%; Brown crystals; mp
88—89 °C; 1H-NMR (300 MHz, DMSO-d6) 10.92 (s, 1H), 8.82 (s, 1H),
8.35—8.20 (br s, 1H), 7.82—7.79 (m, 2H), 7.73—7.60 (br s, 1H), 7.42 (t,
Jꢁ8.1 Hz, 1H), 7.22 (t, Jꢁ8.1 Hz, 1H), 6.89—6.85 (m, 1H), 6.75—6.68 (m,
3H), 3.82 (s, 3H), 3.71 (s, 3H), 1.80 (s, 3H); IR (KBr) 3424, 2925, 1656,
1594, 1561, 1507, 1491, 1404, 1385, 1359, 1285, 1225, 1151, 1043; HR-MS
(EI) Calcd for C22H21N5O3 403.1644, Found 403.1638.
(300 MHz, DMSO-d6) 8.64 (s, 1H), 7.62 (br s, 1H), 7.43 (br s, 1H), 7.03 (t,
Jꢁ8.1 Hz, 1H), 6.37—6.33 (m, 1H), 6.17—6.10 (m, 2H), 3.96 (s, 3H), 3.64
(s, 3H), 3.27 (s, 3H), 2.02 (s, 3H); IR (KBr) 3463, 3385, 3142, 1678, 1580,
1491, 1378, 1271, 1214, 1169, 1118, 1052, 959; HR-MS (EI) Calcd for
C17H19N5O2 325.1539, Found 325.1534.
Assay of Human PDE4 Activity The method of Reeves et al.22) was
modified to isolate phosphodiesterase type 4 isozyme (PDE4). The enzyme
was prepared from U937 cells derived from human monocytes, and was
stored at ꢂ20 °C after preparation. Measurement of PDE4 activity was per-
formed using this stored enzyme after it was diluted with distilled water con-
taining bovine serum albumin. The substrate solution was prepared by
3
adding H-cAMP (300000 dpm (5000 Bq)/assay) and 100 mmol/l cAMP so-
lution to 100 mmol/l Tris–HCl (pH 8.0) containing 5 mmol/l ethylene glycol-
bis (b-aminoethyl ether) and N,N,Nꢃ,Nꢃ-tetraacetic acid. The substrate solu-
tion was mixed with the enzyme solution containing a test compound dis-
solved in dimethylsulfoxide (DMSO), and incubation was done for 30 min at
30 °C. Assays were performed in duplicate at three to four different concen-
trations of each test compound, and the IC50 values were determined.
Inhibition of LPS-Induced Plasma TNF-a Production in Rats Male
Crj:CD(SD)IGS rats aged 6 weeks (nꢁ7) were fasted overnight, and the test
compounds (0.01—0.1 mg/10 ml/kg) were administered orally at 1 h before
intravenous injection of 1 mg/kg of LPS (Escherichia coli Serotype 055 B5).
The plasma TNF-a level was measured with a commercially available
ELISA kit (R&D Systems) at 90 min after LPS challenge. The percent inhi-
bition (the dosage required to inhibit plasma TNF-a production by 50%)
was determined by the following formula:
% inhibitionꢁ100ꢂ(C-S)/(L-S)ꢄ100
C: Plasma TNF-a concentration in LPS-treated animals pretreated with a
test compound. L: Plasma TNF-a concentration in LPS-treated animals pre-
treated with saline. S: Plasma TNF-a concentration in saline-treated animals
pretreated with saline.
SRS-A Mediated Bronchoconstriction in Guinea Pigs Male Hartley
guinea pigs aged 7 weeks (nꢁ5) were actively sensitized by intraperitoneal
administration of 1 mg of ovalbumin (OVA) containing 5ꢄ109 killed Borde-
tella pertussis organisms on day 0. On day 14, the bronchoconstrictor re-
sponse was measured using a modified version of the method of Konzett and
Rüssler. Bronchoconstriction was induced by an intravenous injection of
OVA (0.15—0.5 mg/kg). Sensitized animals were treated with both a cy-
clooxygenase inhibitor (indomethacin at 5 mg/kg i.v., 3 min before OVA)
and an antihistamine (pyrilamine at 1 mg/kg i.v., 1 min before OVA) to en-
sure that endogenous SRS-A was solely responsible for bronchoconstriction.
Test compounds were administered orally at 1 h before antigen challenge.
Bronchoconstrictor response was measured for 15 min and the result was
represented as the area under the curve (AUC 0—15 min).
Gastric Emptying in Rats Male Sprague-Dawley rats were fasted
overnight and were orally administered test compounds or 0.5 w/v% methyl-
cellulose (10 ml/kg). In addition, 0.05 mg/ml of phenol red solution was
orally administered in a volume of 1.5 ml at twenty minutes after dosing
with the test compounds. Forty minutes after administration of the test com-
pounds, both the cardia and pylorus of the stomach were ligated under anes-
thesia with sodium pentobarbital (75 mg/kg, i.p.), and then the stomach was
isolated without leakage of phenol red. The stomach was cut open and the
phenol red solution was drained into a beaker containing 100 ml of 0.1 N
NaOH. Part of the solution was filtrated (pore size: 0.45 mm) and the ab-
sorbance at 546 nm was measured to determine the amount of dye remaining
in the stomach. Then the gastric emptying rate was calculated by the follow-
ing formula:
1-(4-Chlorophenyl)-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (25a) Yield 42%; Beige crystals; mp
1
180—181 °C; H-NMR (300 MHz, DMSO-d6) 10.94 (br, 1H), 8.81 (s, 1H),
8.29 (br, 1H), 8.26 (d, Jꢁ9.0 Hz, 2H), 7.69 (br, 1H), 7.58 (d, Jꢁ9.0 Hz, 2H),
7.22 (dd, Jꢁ8.1, 8.1 Hz, 1H), 6.77—6.67 (m, 3H), 3.71 (s, 3H), 1.79 (s, 3H);
IR (KBr) 3478, 3178, 1665, 1595, 1561, 1507, 1491, 1464, 1404, 1386,
1303, 1259, 1221, 1153, 1091, 1037, 952; HR-MS (EI) Calcd for
C21H18ClN5O2 407.1149, Found 407.1156.
4-[(3-Methoxyphenyl)amino]-3-methyl-1-(4-methylphenyl)-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (25b) Yield 81%; White crystals; mp
1
131—133 °C; H-NMR (300 MHz, DMSO-d6) 10.91 (s, 1H), 8.79 (s, 1H),
8.26 (br s, 1H), 8.03 (d, Jꢁ8.1 Hz, 2H), 7.66 (br s, 1H), 7.31 (d, Jꢁ8.1 Hz,
2H), 7.21 (t, Jꢁ8.4 Hz, 1H), 6.75—6.67 (m, 3H), 3.71 (s, 3H), 2.35 (s, 3H),
1.79 (s, 3H); IR (KBr) 3364, 2925, 1655, 1590, 1559, 1514, 1491, 1440,
1404, 1383, 1358, 1305, 1150, 1044, 943; HR-MS (EI) Calcd for
C22H21N5O2 387.1695, Found 387.1714.
1-(4-Methoxyphenyl)-4-[(3-methoxyphenyl)amino]-3-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (25c) Yield 73% in 3 steps; Gray
crystals; mp 205—207 °C; 1H-NMR (300 MHz, DMSO-d6) 10.93 (s, 1H),
8.78 (s, 1H), 8.30—8.20 (br s, 1H), 8.00 (d, Jꢁ9.0 Hz, 2H), 7.67—7.58 (br s,
1H), 7.22 (t, Jꢁ8.1 Hz, 1H), 7.08 (d, Jꢁ9.0 Hz, 2H), 6.75—6.67 (m, 3H),
3.80 (s, 3H), 3.71 (s, 3H), 1.79 (s, 3H); IR (KBr) 3464, 3351, 2837, 1660,
1594, 1561, 1516, 1491, 1408, 1385, 1297, 1242, 1171, 1153, 1038, 952;
HR-MS (EI) Calcd for C22H21N5O3 403.1644, Found 403.1638.
3-Cyclopropyl-4-[(3-methoxyphenyl)amino]-1-phenyl-1H-pyra-
zolo[3,4-b]pyridine-5-carboxamide (26) Yield 82%; White crystals; mp

1104
gastric emptying rateꢁ100ꢄ(0.75ꢂconcentration of dye in the
Vol. 52, No. 9
R. R., Manning C. D., Grous M., Hillegas L. M., Bartus J. O., Ryan M.
D., Eggleston D. S., Haltiwanger R. C., Torphy T. J., J. Med. Chem.,
41, 821—835 (1998).
stomach)/0.75
A value of 0.75 mg/ml was equal to a concentration of 0.05 mg/ml, which
was achieved by adding 1.5 ml of phenol red to 100 ml of 0.1 N NaOH. The
50% inhibition rate for gastric emptying by the test compounds was calcu-
lated by defining gastric emptying after vehicle administration as 100%.
Inhibitory Activity on LPS-Induced TNF-a Production in Human
Whole Blood Under the supervision of a physician, blood was collected
into a heparinized tube (final concentration: 10 U/ml heparin sodium) from a
forearm vein in three healthy male donors. A solution of the test compound
(10 ml) dissolved in DMSO was added to 180 ml of whole blood, and the
mixture was pre-incubated for 30 min at 37 °C. Then 10 ml of 2 mg/ml
of LPS was added and incubated for 6 h at 37 °C, after which the plasma
TNF-a concentration was measured with a human TNF-a ELISA kit
(DIACLONE). Assays were performed in duplicate at three to four different
concentrations of each test compound, and the IC50 values were determined.
Ferret Emetic Study Male ferrets (weighting about 1.2 kg) were fasted
overnight and test compounds were administered orally. Their behavior was
observed throughout a 1 h period after gavage. Results were expressed as the
number of animals that vomited relative to the animals tested.
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