Design, synthesis and biological evaluation of a novel series of potent, orally active adenosine A1 receptor antagonists with high blood-brain barrier permeability

Article abstract of DOI:10.1248/cpb.49.988

A novel series of 3-(2-substituted-3-oxo-2,3-dihydropyridazin-6-yl) -2-phenylpyrazolo[1,5-a]pyridines (5-38) were synthesized and evaluated for their in vitro adenosine A₁ and A_2A receptor binding activities, and in vitro metabolism by rat liver in order to search for orally active compounds. Most of the test compounds were potent adenosine A₁ receptor antagonists with high A₁ selectivity and the A₁ affinity and A₁ selectivity of carbonyl derivatives (5-11) was particularly high. In particular, compound 7 was an extremely potent and selective adenosine A₁ antagonist with high A₁ selectivity (Ki=0.026 nM, A_2A/A₁=5400). In terms of metabolic stability, 2-oxopropyl (5), 2-hydroxypropyl (12), N-methylacetamide (16), 2-(piperidin-1-yl)ethyl (28) and 1-methylpiperidin-4-yl (32, FR194921) were the most stable compounds in this series of analogues. Further in vivo evaluation indicated that compounds 5, 13, 17, 28 and 32 were detected in both plasma and brain after oral administration in rats. In particular, 32 displayed good plasma and brain concentrations (dose: 32mg/kg (n=3); after 30 min, plasma conc.=3390±651 nM, brain conc.=3670±496 nM; after 60 min, plasma conc.=1580±348 nM, brain conc.=2143±434 nM), and a good brain/plasma ratio (1.11±0.060 (30 min), 1.39±0.172 (60 min)). As a result, we could show that 32 is a good candidate for an orally active adenosine A₁ receptor antagonist with high blood-brain barrier permeability and good bioavailability (Ki=6.6 nM, A_2A/A₁=820, BA=60.6±4.9% (32 mg/kg)).

Full text of DOI:10.1248/cpb.49.988

988
Chem. Pharm. Bull. 49(8) 988—998 (2001)
Vol. 49, No. 8
Design, Synthesis and Biological Evaluation of a Novel Series of Potent,
Orally Active Adenosine A1 Receptor Antagonists with High Blood–Brain
Barrier Permeability
Satoru KURODA,* Fujiko TAKAMURA, Yoshiyuki TENDA, Hiromichi ITANI, Yasuyo TOMISHIMA,
Atsushi AKAHANE, and Kazuo SAKANE
Medicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1–6, Kashima 2-chome, Yodogawa-ku,
Osaka-shi, Osaka 532–8514, Japan. Received March 7, 2001; accepted May 10, 2001
A novel series of 3-(2-substituted-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridines (5—38)
were synthesized and evaluated for their in vitro adenosine A1 and A2A receptor binding activities, and in vitro
metabolism by rat liver in order to search for orally active compounds. Most of the test compounds were potent
adenosine A1 receptor antagonists with high A1 selectivity and the A1 affinity and A1 selectivity of carbonyl deriv-
atives (5—11) was particularly high. In particular, compound 7 was an extremely potent and selective adenosine
A1 antagonist with high A1 selectivity (Ki؍0.026 nM, A2A/A1؍5400). In terms of metabolic stability, 2-oxopropyl
(5), 2-hydroxypropyl (12), N-methylacetamide (16), 2-(piperidin-1-yl)ethyl (28) and 1-methylpiperidin-4-yl (32,
FR194921) were the most stable compounds in this series of analogues. Further in vivo evaluation indicated
that compounds 5, 13, 17, 28 and 32 were detected in both plasma and brain after oral administration in rats.
In particular, 32 displayed good plasma and brain concentrations (dose: 32 mg/kg (n؍3); after 30 min,
plasma conc.؍3390؎651 nM, brain conc.؍3670؎496 nM; after 60 min, plasma conc.؍1580؎348 nM, brain
conc.؍2143؎434 nM), and a good brain/plasma ratio (1.11؎0.060 (30 min), 1.39؎0.172 (60 min)). As a result, we
could show that 32 is a good candidate for an orally active adenosine A1 receptor antagonist with high blood–
brain barrier permeability and good bioavailability (Ki؍6.6 nM, A2A/A1؍820, BA؍60.6؎4.9% (32 mg/kg)).
Key words adenosine A1 antagonist; bioavailability; blood–brain barrier permeability; FR194921
FK838 was not detected in brain after p.o. administration,12)
In the central nervous system (CNS), adenosine regulates
a variety of important physiological functions as a modulator
despite the fact that FK838 has good BA.10) It was reasoned
that the poor brain permeability of FK838 was due to the
presence of the carboxylic acid group, a feature also present
in FR166124. Moreover, a number of FR166124 derivatives
were found to be orally active compounds, but were not de-
tected in brain after p.o. administration in rats.12) It was thus
necessary to search for novel derivatives with the goal of dis-
covery of compounds having good brain permeability.
through G-protein coupled extracellular adenosine receptors,
which have a seven-transmembrane structure.1) Adenosine
receptors were classified into four subtypes A1, A2A, A2B and
A3, and these four subtypes of human receptors have been
cloned.2) The A1 and A2A receptors are high affinity receptors
for adenosine, while the A2B and A3 receptors are low affinity
adenosine receptors.2) With regards to the distribution of
adenosine receptors in brain, A1 receptors are widely present
in brain and are especially present in high density in cortex
and hippocampus. On the other hand, A2A receptors are pre-
sent in high density in striatum.3) On the basis of pharmaco-
logical studies in the CNS, it has been suggested that adeno-
sine A1 receptors play a role in learning and memory.4) Fur-
ther investigations on adenosine A1 receptor antagonists in
several animal models have pointed towards novel therapeu-
tic agents5) for cognitive enhancement,6) depression7) and for
the treatment of dementia such as senile dementia and
Alzheimer’s disease.8) In recent years, several xanthine type
adenosine A1 receptor antagonists have been reported as
agents acting in the central nervous system.5) Apaxifylline
(1),9) the active enantiomer of KFM-19, has been developed
for the treatment of Alzheimer’s disease, senile dementia and
cognitive defects, and shows high oral bioavailability (BA) in
rats (Chart 1).
Concerning the penetration of small molecule drugs into
the CNS, lipophilicity is known to be an important determi-
nant, since lipid-mediated blood–brain barrier transport of
lipophilic compounds is an important pathway.13) Moreover,
metabolic stability is also an important factor, since
lipophilic drugs are readily metabolized to more polar
species, which causes a reduction of lipophilicity.13a) Further-
more, metabolic stability of drugs is essential, since first path
effect by the liver is of fundamental importance for good oral
BA. From the results of structure–activity relationships
(SAR) study on FR166124 derivatives, various alcohol, ester,
nitrile and amides were found to be potent adenosine A1 re-
ceptor antagonists with high A1 selectivity and these deriva-
tives were more lipophilic compounds than FR166124.11)
Therefore, in order to discover orally active agents with
blood–brain barrier permeability, we designed a novel series
of 3-(2-substituted-3-oxo-2,3-dihydropyridazin-6-yl)-2-
phenylpyrazolo[1,5-a]pyridines, including carbonyl (5—11),
hydroxy (12, 13), amide (15—27), amine (28—30) and
piperidine derivatives (32—38) as N2 substituents, in the ex-
pectation that such derivatives would have sufficient
lipophilicity for blood–brain barrier permeability and would
be resistant to metabolism. In this paper, we wish to report
the synthesis, adenosine A1 and A2A binding activities, stabil-
ity in vitro rat liver metabolism, and plasma and brain con-
We recently reported the discovery of FK838 (2)10) and
FR166124 (3),11) potent 3-(2-substituted-3-oxo-2,3-dihydro-
pyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridines as adeno-
sine A1 receptor antagonists with strong diuretic activities
after both intravenous (i.v.) and oral ( p.o.) administration
(Chart 1). We had initially hoped that FK838 may be poten-
tially useful for CNS-related diseases, however, from the re-
sults of pharmacokinetic studies in rats, it was elucidated that
To whom correspondence should be addressed. e-mail: satoru_kuroda@po.fujisawa.co.jp
© 2001 Pharmaceutical Society of Japan

August 2001
989
Chart 1. Adenosine A1 Antagonists
Reagents: (a) chloroacetone, t-BuOK, 18-crown-6-ether, DMF; (b) 40 or BrCH2COR1, NaH, DMF; (c) CH2(O)CR2R3, NaOH, Bn(Et)3NϩClϪ, CH2Cl2–H2O;
(d) 1. BrCH2CO2Et, NaH, DMF, 2. 1 N NaOH, EtOH; (e) amine (R5NH), EDC·HCl, HOBt, DMF; (f) amine hydrochloride (R5NH·HCl), EDC, HOBt, DMF;
(g) 42 or Cl(CH2)2NR6·HCl, NaOH, Bn(Et)3NϩClϪ, CHCl3–H2O; (h) 1. 1-Boc-4-hydroxypiperidine, PPh3, DEAD, THF, 2. 6 N HCl; (i) 44 or R7OH, PPh3,
DEAD, THF; (j) isopropyl iodide, NaH, DMF; (k) Ac2O, Pyridine.
Chart 2
centrations in rats after p.o. administration of this series and
the discovery of 32, which was found to be a potent, orally
active adenosine A1 receptor antagonist with high blood–
brain barrier permeability.
Chemistry Synthetic routes for the novel 3-(2-substi-
tuted-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyra-
zolo[1,5-a]pyridines 5—38 prepared in this work are summa-
Chart 3
rized in Chart 2. Starting 6-(2-phenylpyrazolo[1,5-a]pyridin-
3-yl)-3(2H)-pyridazinone (4) was prepared according to the
previously described method.10a,14) Carbonyl derivatives 5—
C).
Compound 14 was obtained in 87% yield as a synthetic
precursor of amide derivatives by alkylation of 4 with ethyl
bromoacetate followed by alkaline hydrolysis of the resulting
ester (method D). Preparation of amide derivatives 15—27
was accomplished by activation of intermediate 14 as the 1-
hydroxybenzotriazole (HOBt) ester followed by treatment
with the appropriate amine (method E, 57—96% yields) or
amine hydrochloride (method F, 47—96% yields).
Amine derivatives 28 and 29 were prepared by reaction of
4 with 1-(2-chloroethyl)piperidine hydrochloride and 2,6-cis-
1-(2-chloroethyl)-2,6-dimethylpiperidine (42) using NaOH
as a base and benzyltriethylammonium chloride as a phase-
transfer catalyst17) in a mixture of CHCl3 and H2O, followed
by treatment with HCl in ethanol (EtOH), in 72 and 93%
yields, respectively (method G). Compound 30 was also pre-
pared in 70% yield by the reaction of 4 with 2-(hexamethyl-
11 were prepared from 4 by method A or B. The details of
which are as follows: 5 was obtained in 79% yield by alkyla-
tion of 4 with chloroacetone using potassium tert-butoxide
(tert-BuOK) as a base (method A). Compounds 6—11 were
obtained by alkylation of 4 with the appropriate bromoketone
using NaH as a base in 16—86% yields (method B). The
starting bromoketones were obtained by standard methods.
1-Bromo-3-methyl-2-butanone was prepared according to the
reported method15) and the preparation of bromoketones
40a—c was achieved in a similar manner from the appropri-
ate ketone 39a—c in 78—95%yields (Chart 3). Commer-
cially available 1-bromo-2-butanone and 1-bromopinacolone
were used for preparation of 7 and 8, respectively. Hydroxy
derivatives 12 and 13 were prepared by the reaction of 4 with
propylene oxide and isobutylene oxide according to the re-
ported method16) in 71 and 59% yields, respectively (method

990
Vol. 49, No. 8
eneimino)ethyl chloride according to method G, without HCl with acetic anhydride (Ac2O) in 90% yield (method L).
treatment (method H). Preparation of 42 was achieved in Biology The compounds prepared were evaluated for
62% yield by acylation of 2,6-cis-2,6-dimethylpiperidine their in vitro adenosine A1 and A2A receptor binding activi-
with ethyl oxalyl chloride, and reduction with lithium alu- ties, using receptors cloned from human hippocampus and
minum hydride (LiAlH4) in refluxing tetrahydrofuran (THF) expressed in Chinese hamster ovary (CHO) cells. Adenosine
followed by treatment with thionyl chloride (Chart 4).
A1 and A2A receptor binding was measured using 4.5 nM
Piperidine derivatives 32—34, 36 and 37 were prepared [3H]-(8-cyclopentyl-1,3-dipropylxanthin ([3H]-DPCPX) and
from 4 with the appropriate 1-substituted-4-hydroxy piperi- 20 nM [3H]-2-[4-[(2-carboxethyl)phenyl]ethylamino]-5Ј-N-
dine using Mitsunobu reaction conditions18) in 28—71% ethylcarbonyladenosine ([3H]-CGS21680), respectively. The
yields (method J). Commercially available 4-hydroxy-1- in vitro A1 and A2A receptor binding activities are expressed
methylpiperidine and 1-benzyl-4-hydroxypiperidine were as Ki values which were calculated from the IC50 values. Se-
used for preparation 32 and 37, respectively, and 1-substi- lectivity (A2A/A1) is expressed as the ratio of Ki values ob-
tuted-4-hydroxy piperidines 44a—c were prepared in 83— tained from the receptor binding assays. Stability to in vitro
98% yields from 4-hydroxypiperidine (43) by acylation with metabolism in rat liver microsomes and cytosol was mea-
the appropriate acyl chloride, followed by reduction of the re- sured by HPLC analysis, and is presented as percentage of
sulting amide (Chart 5). Compound 31 was obtained in 62% remaining concentration versus the initial concentration of
yield as a synthetic precursor of 35 and 38 via Method I: test compounds after incubation for 10 min at 37 °C. Stability
Mitsunobu reaction of 4 with 1-tert-butoxycarbonyl-4-hy- of compounds 5—11 was examined in both microsomes and
droxypiperidine followed by acidic deprotection of the tert- cytosol, whilst the other compounds were only evaluated in
butoxycarbonyl group. Preparation of 35 was achieved by the presence of microsomes.
alkylation of 31 with 2-iodopropane using NaH as a base
Selected compounds having potent A1 affinity, high A1 se-
(method K). Compound 38 was prepared by acetylation of 31 lectivity and good metabolic stability were then examined for
plasma and brain concentrations via p.o. administration.
Plasma and brain concentrations, 30 min after p.o. adminis-
tration of test compounds to fasted Sprague-Dawley (SD)
male rats were measured by HPLC analysis. The brain/
plasma (B/P) ratio is expressed as the ratio of brain and
plasma concentrations. Pharmacokinetic parameters of the
orally active adenosine A1 antagonist having the highest
Chart 4
brain concentration in rats were also studied.
Results and Discussion
Binding Assays In order to investigate potency as
adenosine A1 antagonists and to search for orally active com-
pounds, we introduced various substituents such as carbonyl
(5—11), hydroxy (12, 13), amide (15—27), amine (28—30)
and piperidine moiety (32—38) at the N2 of the pyridazi-
Chart 5
Table 1. Binding Assay and in Vitro Metabolism by Rat Liver of Carbonyl Derivatives
Adenosine receptor bindingb)
Ki (nM)
% of initial concentrationd)
Selectivityc)
A2A/A1
Compound
R1
Method
Yield (%)
clog Pa)
A1
4.8
0.19
0.73
0.026
0.12
A2A
Microsomes
Cytosol
1
5
6
7
8
1.33
1.52
2.04
2.35
2.75
530
170
650
140
340
110
900
890
5400
2800
N.T.e)
76.6
33.2
8.2
N.T.e)
1.8
45.5
97.5
98.7
Me
Et
i-Pr
t-Bu
A
B
B
B
79
83
80
86
32.8
9
B
B
31
16
3.55
3.61
0.082
0.088
87
1100
1400
6.4
3.5
90.0
98.0
10
120
11
B
78
4.17
0.078
54
690
2.6
101.1
a) The clog P values were calculated using MacLog P from Biobyte Corporation: clog P for Macintosh version 2.0.3. b) Inhibition of specific binding; A1: [3H]-DPCPX
(4.5 nM), A2A: [3H]-CGS 21680 (20 nM). c) Ratio of Ki values obtained by receptor binding assay. d) Test compounds were incubated for 10 min at 37 °C. Substrate concentra-
tion; microsomes: 1 mM, cytosol: 10 mM; Identification limit: 0.05 mg/ml or 0.05 mg/tissue. e) N.T.: not tested.

August 2001
991
Table 2. Binding Assay and in Vitro Metabolism by Rat Liver of Hydroxy Derivatives
Adenosine receptor bindingb)
% of initial
Ki (nM)
Selectivityc) concentrationd)
A2A/A1
Compound
R2
R3
Method
Yield (%)
clog Pa)
A1
A2A
Microsomes
12e)
13
Me
Me
H
Me
C
C
71
59
1.73
2.13
5.1
0.36
250
160
49
440
83.2
74.6
a—d) See corresponding footnotes of Table 1. e) Racemate.
Table 3. Binding Assay and in Vitro Metabolism by Rat Liver of Amide Derivatives
Adenosine receptor bindingb)
% of initial
Ki (nM)
Selectivityc)
A2A/A1
concentrationd)
Compound
NR5
Method
Yield (%)
clog Pa)
A1
A2A
Microsomes
15
16
17
18
19
20
21
22
NH2
NHMe
NMe2
F
F
F
F
E
E
E
E
47
96
85
95
83
86
57
88
0.54
0.78
1.05
1.30
1.61
2.01
1.58
2.11
5.5
2.5
18
460
1900
3800
1100
490
432
1000
920
84
760
210
610
1700
570
240
420
N.T.e)
84.0
73.0
40.7
29.8
3.4
NHEt
1.8
NHPri
NHBut
N(Me)Et
NEt2
0.29
0.76
4.1
33.9
1.5
2.2
23
24
25
26
27
E
F
E
E
E
76
90
86
76
91
1.69
0.66
2.24
1.22
1.94
6.8
2900
10000 (48)f )
620
430
Ͼ290
480
14.3
N.T.e)
14.8
1.0
34
1.3
34
9.1
10000 (38)f )
830
Ͼ290
91
1.0
a—e) See corresponding footnotes of Table 1. f ) (%) inhibition at 10000 nM.
none ring and in vitro adenosine A1 and A2A receptor binding
activities were measured (Tables 1—5). As shown in Table 1,
all carbonyl derivatives (5—11) were highly potent adeno-
sine A1 antagonists with high A1 selectivity. Amongst them,
compound 7 was the most potent adenosine A1 antagonist
(Kiϭ0.026 nM, A2A/A1ϭ5400). Introduction of sterically hin-
dered alkyl groups on the side chain increased affinity for the
A1 receptor, but introduction of a cyclohexyl group increased
affinity for the A2A receptor (9, 11). Hydroxy derivatives such
as tri-substituted 13 showed high A1 affinity and good A1 se-
lectivity (Kiϭ0.36 nM, A2A/A1ϭ440), but di-substituted ana-
logue 12 had reduced A1 affinity and A1 selectivity
(Kiϭ5.1 nM, A2A/A1ϭ49) (Table 2). Moreover, hydroxy de-
rivatives showed lower A1 selectivity than carbonyl deriva-
tives and reduction of the carbonyl group to a hydroxy group
decreased A1 affinity, but retained A2A affinity (5 versus 12).
Amide derivatives 15—27 also showed high A1 affinity and
good A1 selectivity (Table 3), 19 (Kiϭ0.29 nM, A2A/A1ϭ
1700) and 20 (Kiϭ0.76 nM, A2A/A1ϭ570) displayed very
high A1 affinity, and 19 showed very high A1 selectivity. In
the series of amide derivatives, mono-substituted amides (16,
18—20) showed higher A1 affinity and A1 selectivity than di-
substituted (17, 21, 22) and cyclic amide derivatives (23—
27). Table 4 shows the results for amine derivatives 28—30.
Compounds 28 (Kiϭ2.4 nM, A2A/A1Ͼ420) and 30 (Kiϭ
2.6 nM, A2A/A1ϭ730) showed high A1 affinity and A1 selec-
tivity. Introduction of additional methyl groups at the carbon
adjacent to the nitrogen in the piperidine ring reduced A1
affinity (28 versus 29). As shown in Table 5, piperidine deriv-
atives displayed high A1 affinity and A1 selectivity, except for
36 and 37. Compound 38 showed the highest A1 affinity
(Kiϭ0.55 nM) amongst them. Introduction of long alkyl (36)
and aromatic (37) moieties to the amino group of the piperi-
dine ring reduced A1 affinity but retained A2A affinity.

992
Vol. 49, No. 8
Table 4. Binding Assay and in Vitro Metabolism by Rat Liver of Amine Derivatives
Adenosine receptor bindingb)
% of initial
Selectivityc) concentrationd)
A2A/A1
Ki (nM)
Compound
NR6
n
Method
Yield (%)
clog Pa)
A1
2.4
A2A
Microsomes
28
1
G
G
H
72
93
70
3.58
4.62
4.14
1000 (23)f )
Ͼ420
Ͼ360
730
25.9
2.0
29e)
30
1
0
28
10000 (18)f )
1900
2.6
7.0
a—d) See corresponding footnotes of Table 1. e) Single diastereoisomer. f ) (%) inhibition at 1000 or 10000 nM.
Table 5. Binding Assay and in Vitro Metabolism by Rat Liver of Piperidine Derivatives
Adenosine receptor bindingb)
Ki (nM)
% of initial
Selectivityc)
A2A/A1
concentrationd)
Compound
R7
Method
Yield (%)
clog Pa)
A1
A2A
Microsomes
32
33
34
35
36
37
38
Me
Et
n-Pr
J
J
J
K
J
79
28
45
63
39
71
90
2.95
3.48
4.01
3.79
5.07
4.94
1.94
6.6
4.1
1.2
3.1
37
35
0.55
5400
2300
550
3500
1700
2000
150
820
560
460
1100
46
85.1
75.5
58.6
74.7
N.T.e)
N.T.e)
27.6
i-Pr
(CH2)4CH3
CH2Ph
Ac
J
L
57
270
a—e) See corresponding footnotes of Table 1.
Furthermore, a relationship was found for the analogues
prepared here between A1 affinity and lipophilicity, as ex-
pressed by calculated partition coefficient (clog P) values.
Figure 1 and Eqs. 1—3 show the relationship between adeno-
sine A1 affinity and the clog P value.
Carbonyl derivatives
Ϫlog Kiϭ0.19(clog P)ϩ0.41
nϭ7, rϭ0.41, sϭ0.44
(1)
(2)
(3)
Hydroxy and amide derivatives
Ϫlog Kiϭ0.62(clog P)Ϫ1.48
nϭ15, rϭ0.54, sϭ0.56
Amine and piperidine derivatives
Fig. 1. Relationship between Adenosine A1 Receptor Affinity and clog P
Ϫlog KiϭϪ0.52(clog P)ϩ1.24
nϭ10, rϭ0.76, sϭ0.43
of the carbonyl derivatives and the hydroxy and amide deriv-
In the Eqs. 1—3, n is the number of compounds, r is the cor- atives having potent A1 affinity, increasing clog P tends to in-
relation coefficient and s is the standard error. In both series crease A1 affinity. In contrast, A1 affinity of amine and piperi-

August 2001
993
Table 6. Plasma and Brain Concentrations in Rats of Novel 3-(2-Substituted-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo-[1,5-a]pyridine Adeno-
sine A1 Antagonists
After 30 min of p.o. administration
Plasma and Brain concentrations
After 60 min of p.o. administration
Plasma and Brain concentrations
Dose
(mg/kg)
Compound
B/P
B/P
Plasma conc.a)
(nM)
Brain conc.a)
(nM)
ratio
Plasma conc.a)
(nM)
Brain conc.a)
(nM)
ratio
1
1
5
7
8
13
17
25
28
32
32
1.0
10
10
10
10
10
10
10
10
3.2
32
4750Ϯ371
34500Ϯ3670
412Ϯ75
N.D.b)
1330Ϯ187
415Ϯ75d)
N.D.b)
N.T.c)
N.T.c)
N.T.c)
N.T.c)
0.038Ϯ0.002
0.478e)
244Ϯ128d)
N.D.b)
73Ϯ73f )
N.D.b)
0.497g)
N.D.b)
N.D.b)
286Ϯ19
3590Ϯ190
128Ϯ70d)
165Ϯ14
N.D.b)
N.D.b)
N.D.b)
172Ϯ5
0.605Ϯ0.039
0.124Ϯ0.008
205Ϯ105d)
1790Ϯ161
189Ϯ36
N.D.b)
114Ϯ55d)
51Ϯ51f )
N.D.b)
0.562h)
0.072i)
445Ϯ43
N.D.b)
397Ϯ80
361Ϯ5
3670Ϯ496
2.39Ϯ0.347
0.930Ϯ0.046
1.11Ϯ0.060
223Ϯ89d)
N.T.c)
389Ϯ23
3390Ϯ651
N.T.c)
1580Ϯ348
2143Ϯ434
1.39Ϯ0.172
a) MeanϮstandard error, nϭ3, Identification limit: 0.05 mg/ml or 0.05 mg/tissue. b) N.D.: not detected. c) N.T.: not tested. d) 1/3 N.D. e) nϭ2, plasma and brain
conc.: No. 1; 558 and 229 nM, No. 2; 383 and 209 nM. f ) 2/3 N.D. g) nϭ1, plasma and brain conc.: 433 and 215 nM. h) nϭ2, plasma and brain conc.: No. 1; 267 and 170 nM,
No. 2; 347 and 170 nM. i) nϭ1, plasma and brain conc.: 2100 and 152 nM.
dine derivatives was decreased due to higher clog P values
Plasma and Brain Concentrations, Pharmacokinetics
with good correlation. In this series of 3-(2-substituted-3- In the next step, we investigated concentrations in plasma
oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5- and brain in rats dosed by p.o. administration with selected
a]pyridines, it is thus assumed that a suitable clog P value compounds. In consideration of the adenosine A1 affinity, A1
for high A1 affinity ranges from 1.5 to 3.5. Moreover, it is selectivity and metabolic stability, we selected 5, 7 and 8 as
thus concluded that 3-(3-oxo-2,3-dihydropyridazin-6-yl)- carbonyl derivatives, 13 as a hydroxy derivative, 17 and 25 as
2-phenylpyrazolo[1,5-a]pyridine is an excellent pharma- amide derivatives, 28 as an amine derivative and 32 as a
cophore for the expression of high A1 affinity.
piperidine derivative for this study, and the results are sum-
Metabolism Concerning the oral BA and metabolic sta- marized in Table 6. In the case of carbonyl derivatives, 5
bility to maintain lipophilicity, first path effect by the liver is (clog Pϭ1.52) was detected in both plasma and brain after 30
an important factor. Therefore, in vitro metabolism by rat and 60 min of p.o. (10 mg/kg) administration (plasma conc.ϭ
liver microsomes as a model of major metabolism was exam- 412Ϯ75 nM (30 min); 244Ϯ128 nM (60 min), brain conc.ϭ
ined, since microsomes contain cytochrome P450s, which 415Ϯ75 nM (30 min); 73Ϯ73 nM (60 min)), but 7 (clog Pϭ
display an important role in drug metabolism involving a va- 2.35) and 8 (clog Pϭ2.75) were not detected, even in plasma.
riety of reactions such as oxidation and reduction.19) Deter- Thinking about the metabolic stability of carbonyl deriva-
mination of the metabolites and metabolizing enzymes are tives, it was thus suggested that stability in microsomes was a
important aspects of downstream drug development, how- more important factor than that in cytosol for the expression
ever, it is enough at the early stages to measure metabolic of oral activity. The same result was observed for amide de-
stability in terms of percentage of remaining concentration rivatives 17 (clog Pϭ1.05) and 25 (clog Pϭ2.24). Compound
versus initial concentration of test compounds. In the case of 17, more stable in microsomes than 25, had the highest
carbonyl derivatives (5—11), in vitro metabolism by rat liver plasma concentration of all tested compounds and good brain
cytosol was also measured, because it was known that car- concentrations after 30 and 60 min of p.o. (10 mg/kg) admin-
bonyl groups can be reduced to alcohol groups by metabo- istration (plasma conc.ϭ3590Ϯ190 nM (30 min); 1790Ϯ
lism in cytosol. The results are summarized in Tables 1—5. 161 nM (60 min), brain conc.ϭ445Ϯ43 nM (30 min); 51Ϯ
As shown in Table 1, 5 was the most stable in microsomes, 51 nM (60 min)). For the amine derivative 28 (clog Pϭ3.58)
but disappeared quickly in cytosol. It was observed that more and piperidine derivative 32 (clog Pϭ2.95), 28 showed the
sterically hindered carbonyl derivatives than 5 had reduced best B/P ratio after 30 min of p.o. (10 mg/kg) administration
stability in microsomes, but increased stability in cytosol. (plasma conc.ϭ165Ϯ14 nM, brain conc.ϭ397Ϯ80 nM, B/Pϭ
Both hydroxy derivatives 12 and 13 were more stable in mi- 2.39Ϯ0.347) and 32 showed the highest brain concentration
crosomes than the carbonyl derivatives (Table 2). As shown and a good B/P ratio after 30 and 60 min of p.o. (32 mg/kg)
in Tables 3—5, it is apparent that in the series of amide administration (plasma conc.ϭ3390Ϯ651 nM (30 min);
(15—27), amine (28—30) and piperidine (32—38) deriva- 1580Ϯ348 nM (60 min), brain conc.ϭ3670Ϯ496 nM (30 min);
tives, the more sterically hindered the side chain moiety, the 2143Ϯ434 nM (60 min), B/Pϭ1.11Ϯ0.060 (30 min); 1.39Ϯ
more stability in microsomes was reduced, to a similar level 0.172 (60 min)) amongst the tested compounds. Furthermore,
as the carbonyl derivatives. Furthermore, it is thus demon- 32 showed a good brain concentration and good B/P ratio
strated that hydroxy and piperidine derivatives have good sta- even at a dose of 3.2 mg/kg ( p.o.) after 30 min of administra-
bility in rat liver microsomes and may be expected to be new tion (plasma conc.ϭ389Ϯ23 nM, brain conc.ϭ361Ϯ5 nM,
compounds with potentially good oral BA and brain perme- B/Pϭ0.930Ϯ0.046). From the results described above, it was
ability.
suggested that the presence of the amino moiety on the N2

994
Vol. 49, No. 8
Table 7. Oral Bioavailability of FR194921 (32) of Ratsa)
All J values are given in Hz. Mass (MS) spectra were measured on a Hitachi
Model M-1000H mass spectrometer using APCI for ionization. Elemental
analyses were carried out on a Perkin Elmer 2400 CHN Elemental Analyzer.
Column chromatography was performed with the indicated solvents using
Merck Silica gel 60 (70—230 mesh). Monitoring of reactions was carried
out using Merck 60 F254 Silica gel, glass-supported thin layer chromatogra-
phy plates, followed by visualization with UV light (254, 365 nm) and stain-
ing with iodine vapor. Reagents and solvents were used as obtained from
commercial suppliers without further purification unless otherwise noted.
Method A. 2-(2-Oxopropyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
3(2H)-pyridazinone (5) To a mixture of tert-BuOK (5.0 g, 44.5 mmol)
and 18-crown-6-ether (740 mg, 2.8 mmol) in N,N-dimethylformamide
(DMF, 80 ml) was added 4 (8.0 g, 27.7 mmol) at 5 °C under a nitrogen at-
mosphere. After 30 min, to the reaction mixture was added dropwise
chloroacetone (3.53 ml, 44.5 mmol), and the reaction was then allowed to
warm to ambient temperature and stirred for 1 h. The reaction mixture was
partitioned between CH2Cl2 and H2O, and the organic layer was separated,
washed with 1 N NaOH and brine, dried over magnesium sulfate (MgSO4),
and purified by column chromatography on silica-gel (CH2Cl2–methanol
(MeOH) 8 : 1 and 4 : 1) and recrystallization from EtOH to give 5 (7.5 g,
AUCb) (mg·h/ml)
6.91Ϯ0.521
Cmaxc) (mg/ml)
2.13Ϯ0.091
Tmaxd) (h)
BA (%)
0.63Ϯ0.13
60.6Ϯ4.9
a) Dose: 32 mg/kg ( p.o., Fasted): MeanϮS.E., nϭ4. b) AUC: area under concen-
tration curve. c) Cmax: maximal blood concentration. d) Tmax: time to maximal
blood concentration.
substituent is a more important factor than the lipophilicity
of test compounds for the expression of high blood–brain
barrier permeability after p.o. administration.
Finally, concerning the results from the studies of plasma
and brain concentrations in rats after oral administration, we
selected 32 as the best compound for further pharmacokinet-
ics studies in rats. As shown in Table 7, 32 showed good BA
(60.6Ϯ4.9%) at a dose of 32 mg/kg ( p.o.). As a result, 32
was found to be the first 3-(2-substituted-3-oxo-2,3-dihy-
1
79%) as a white solid. mp 190—191 °C. H-NMR (CDCl3) d: 2.33 (3H, s),
5.07 (2H, s), 6.80 (1H, d, Jϭ9.7 Hz), 6.93 (1H, td, Jϭ6.9, 1.4 Hz), 7.05 (1H,
dropyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridine adeno- d, Jϭ9.7 Hz), 7.24—7.30 (1H, m), 7.44—7.49 (3H, m), 7.61—7.66 (2H, m),
7.90 (1H, td, Jϭ8.9, 1.1 Hz), 8.53 (1H, td, Jϭ6.9, 1.0 Hz). IR (KBr) cmϪ1
:
sine A1 antagonist with good oral bioavailability and good
blood–brain permeability. Thus, 32 is a good candidate for
further pharmacological evaluations.
1713, 1664, 1587, 1524, 1493. MS m/z 345: (MϩH)ϩ. Anal. Calcd for
C20H16N4O2: C, 69.76; H, 4.68; N, 16.27. Found: C, 69.36; H, 4.63; N,
16.15.
Method B. 2-(2-Oxobutyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
3(2H)-pyridazinone (6) To a suspension of NaH (60% dispersion in min-
eral oil, 200 mg, 5.0 mmol) in DMF (150 ml) was added 4 (1.0 g, 3.5 mmol)
at 5 °C under a nitrogen atmosphere. After 30 min, to the reaction mixture
was added dropwise 1-bromo-2-butanone (0.46 ml, 4.5 mmol), followed by
warming to ambient temperature and stirred for 1 h. The reaction mixture
was partitioned between 25% n-hexane in ethyl acetate (EtOAc) and H2O,
and the organic layer was separated, washed with 1 N NaOH and brine, and
dried over MgSO4. Evaporation of the solvent gave a residual solid which
was recrystallized from EtOH to give 6 (1.04 g, 83%) as a white solid. mp
171—172 °C. 1H-NMR (CDCl3) d: 1.18 (3H, t, Jϭ7.3 Hz), 2.63 (2H, q,
Jϭ7.3 Hz), 5.06 (2H, s), 6.80 (1H, d, Jϭ9.7 Hz), 6.86—6.94 (1H, m), 7.05
(1H, d, Jϭ9.7 Hz), 7.23—7.33 (1H, m), 7.44—7.48 (3H, m), 7.60—7.66
Conclusion
In summary, we have prepared a novel series of 3-(2-sub-
stituted-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyra-
zolo[1,5-a]pyridines and evaluated them for in vitro adeno-
sine A1 and A2A receptor binding activities, and most were
found to be potent adenosine A1 receptor antagonists with
high A1 selectivity. The SAR study in this series of com-
pounds revealed the following main features: 1) a carbonyl
moiety is a good substituent for high A1 affinity and replace-
ment of the carbonyl group by a hydrophilic moiety such as
hydroxy or amide groups decreased both affinity for the
adenosine A1 receptor and A1 selectivity. 2) In the carbonyl
derivatives, A2A affinity was increased by introduction of a
cyclohexyl group. 3) Mono-substituted amide derivatives
(2H, m), 7.90 (1H, d, Jϭ8.9 Hz), 8.52 (1H, d, Jϭ7.0 Hz). IR (KBr) cmϪ1
:
1728, 1660, 1589, 1529, 1495, 1468. MS m/z 359: (MϩH)ϩ. Anal. Calcd for
C21H18N4O2: C, 70.38; H, 5.06; N, 15.63. Found: C, 70.10; H, 5.01; N,
15.37.
Compounds 7—11 were prepared following a procedure similar to
showed higher A1 affinity and A1 selectivity than di-substi- Method B.
2-(3-Methyl-2-oxobutyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-
pyridazinone (7): White solid (80%). mp 171—173 °C (EtOH). H-NMR
tuted and cyclic amide derivatives. 4) In the case of piperi-
dine derivatives, introduction of a lipophilic moiety such as a
long chain alkyl or aromatic moiety, reduced A1 affinity but
retained A2A affinity. 5) Lipophilicity is an important factor
for expression of high A1 affinity, with a suitable clog P value
being in the range 1.5 to 3.5.
The studies of in vitro rat liver metabolism and concentra-
tions in plasma and brain after p.o. administration to rats in-
dicated the following features: 1) introduction of a sterically
hindered moiety on the N2 substituent reduced the stability
in microsomes. 2) An amino group was the best N2 sub-
stituent for the expression of blood–brain barrier permeabil-
ity after p.o. administration. It is thus concluded that 32 can
possibly lead to a new neuronal therapeutic agent, and is pro-
gressed to further pharmacological studies in rats, such as
passive avoidance test4a) and Morris water maze test,6) the re-
sults of which will be reported in due course.
1
(DMSO-d6) d: 1.13 (6H, d, Jϭ6.8 Hz), 2.78—3.00 (1H, m), 5.19 (2H, s),
6.91 (1H, d, Jϭ9.7 Hz), 7.00—7.15 (2H, m), 7.38—7.65 (6H, m), 7.87 (1H,
d, Jϭ8.9 Hz), 8.82 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1726, 1666, 1591,
1527. MS m/z 373: (MϩH)ϩ. Anal. Calcd for C22H20N4O2: C, 70.95; H,
5.41; N, 15.04. Found: C, 70.59; H, 5.41; N, 14.93.
2-(3,3-Dimethyl-2-oxobutyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
1
3(2H)-pyridazinone (8): White solid (86%). mp 219—220 °C (EtOH). H-
NMR (CDCl3) d: 1.32 (9H, s), 5.22 (2H, s), 6.78 (1H, d, Jϭ9.7 Hz), 6.89
(1H, td, Jϭ6.9, 1.4 Hz), 7.04 (1H, d, Jϭ9.7 Hz), 7.26 (1H, td, Jϭ6.8,
1.1 Hz), 7.41—7.47 (3H, m), 7.59—7.66 (2H, m), 7.87 (1H, d, Jϭ8.9 Hz),
8.51 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1718, 1668, 1652, 1593, 1529,
1491, 1470. MS m/z 387: (MϩH)ϩ. Anal. Calcd for C23H22N4O2: C, 71.48;
H, 5.74; N, 14.50. Found: C, 71.44; H, 5.81; N, 14.48.
2-(2-Cyclohexenyl-2-oxoethyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
1
3(2H)-pyridazinone (9): White solid (31%). mp 138—139 °C (EtOH). H-
NMR (CDCl3) d: 1.10—2.10 (10H, m), 2.50—2.70 (1H, m), 5.12 (2H, s),
6.78 (1H, d, Jϭ9.7 Hz), 6.84—6.93 (1H, m), 7.03 (1H, d, Jϭ9.7 Hz), 7.21—
7.30 (1H, m), 7.43—7.48 (3H, m), 7.61—7.66 (2H, m), 7.87 (1H, d,
Jϭ8.9 Hz), 8.51 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1721, 1664, 1633, 1589,
1527, 1498. MS m/z 413: (MϩH)ϩ. Anal. Calcd for C25H24N4O2·0.25H2O:
C, 72.01; H, 5.92; N, 13.44. Found: C, 72.13; H, 5.84; N, 13.48.
Experimental
Chemistry All melting points (mp) were determined with a Büchi 535
apparatus in open capillaries and are uncorrected. Infrared (IR) spectra were
recorded on a Horiba Spectradesk FT-210 or FT-710 spectrometer. 1H-NMR
spectra were measured with a Bruker AC200P (200 MHz). Chemical Shifts
are given in parts per million (ppm) using tetramethylsilane as the internal
standard for spectra obtained in CDCl3 or dimethylsulfoxide-d6 (DMSO-d6).
2-(3-Cyclopentyl-2-oxobutyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
1
3(2H)-pyridazinone (10): White solid (16%). mp 150—151 °C (EtOH). H-
NMR (CDCl3) d: 1.11 (10H, m), 2.50—2.70 (1H, m) 5.12 (2H, s), 6.78 (1H,
d, Jϭ9.7 Hz), 6.84—6.93 (1H, m), 7.03 (1H, d, Jϭ9.7 Hz), 7.21—7.30 (1H,
m), 7.43—7.48 (3H, m), 7.61—7.66 (2H, m), 7.87 (1H, d, Jϭ8.9 Hz), 8.51

August 2001
995
(1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1730, 1660, 1635, 1593, 1529, 1497. MS 6.20—6.40 (1H, m), 6.80 (1H, d, Jϭ9.7 Hz), 6.92—6.97 (1H, m), 7.06 (1H,
m/z 413: (MϩH)ϩ. Anal. Calcd for C25H24N4O2: C, 72.79; H, 5.86; N, 13.58. d, Jϭ9.7 Hz), 7.27—7.45 (1H, m), 7.45—7.50 (3H, m), 7.59—7.65 (2H, m),
Found: C, 72.45; H, 5.84; N, 13.46.
8.10 (1H, d, Jϭ8.9 Hz), 8.53 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1680, 1657,
2-(3-Cyclohexyl-2-oxobutyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)- 1585, 1551, 1527. MS m/z 388: (MϩH)ϩ. Anal. Calcd for
1
3(2H)-pyridazinone (11): White solid (78%). mp 141—142 °C (EtOH). H- C22H21N5O2·0.25H2O: C, 67.42; H, 5.53; N, 17.87. Found: C, 67.51; H,
NMR (CDCl3) d: 0.90—1.45 (5H, m), 1.65—2.10 (6H, m), 2.46 (2H, d,
Jϭ6.9 Hz), 5.04 (2H, s), 6.79 (1H, d, Jϭ9.7 Hz), 6.85—6.93 (1H, m), 7.04
5.41; N, 17.86.
Compounds 20—23 and 25—27 were prepared following a procedure
(1H, d, Jϭ9.7 Hz), 7.21—7.30 (1H, m), 7.43—7.49 (3H, m), 7.60—7.66 similar to Method E.
(2H, m), 7.87 (1H, d, Jϭ8.9 Hz), 8.51 (1H, d, Jϭ7.0 Hz). IR (KBr) cmϪ1
:
N-tert-Butyl-2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
1728, 1659, 1633, 1593, 1529, 1497. MS m/z 427: (MϩH)ϩ. Anal. Calcd for pyridazin-1-yl]acetamide (20): White solid (86%). mp 225—227 °C (EtOH).
C26H26N4O2·0.4H2O: C, 72.00; H, 6.23; N, 12.92. Found: C, 72.19; H, 6.20;
N, 12.85.
1H-NMR (CDCl3) d: 1.39 (9H, s), 4.84 (2H, s), 6.16 (1H, br s), 6.79 (1H, d,
Jϭ9.7 Hz), 6.87—6.96 (1H, m), 7.05 (1H, d, Jϭ9.7 Hz), 7.26—7.36 (1H,
Method C. 2-(2-Hydroxypropyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3- m), 7.44—7.48 (3H, m), 7.60—7.65 (2H, m), 8.09 (1H, d, Jϭ8.9 Hz), 8.52
yl)-3(2H)-pyridazinone (12) A mixture of 4 (1.0 g, 3.5 mmol), propylene (1H, d, Jϭ6.9 Hz); IR (KBr) cmϪ1: 1693, 1662, 1595, 1525. MS m/z 402:
oxide (1.5 ml, 21.4 mmol), NaH (60% dispersion in mineral oil, 170 mg, (MϩH)ϩ. Anal. Calcd for C23H23N5O2·0.25H2O: C, 68.05; H, 5.83; N,
4.2 mmol) and benzyltriethylammonium chloride (80 mg, 0.35 mmol) in a 17.25. Found: C, 67.92; H, 5.71; N, 17.21.
mixture of H2O (20 ml) and CH2Cl2 (20 ml) was stirred for 24 h. The organic
N-Ethyl-N-methyl-2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
layer was separated, washed with H2O and brine, and dried over MgSO4. 1(6H)-pyridazin-1-yl]acetamide (21): Pale yellow solid (57%). mp 190—
Evaporation of the solvent gave a residual solid which was recrystallized 192 °C (EtOH). 1H-NMR (CDCl3) d: 1.17, 1.81 (3H (1 : 1), 2ϫt, Jϭ7.2 Hz),
from EtOH to give 12 (850 mg, 71%) as a white solid. mp 203—204 °C. 1H- 3.02, 3.11 (3H (1 : 1), 2ϫs), 3.40—3.57 (2H, m), 5.06, 5.09 (2H (1 : 1), 2ϫs,
NMR (CDCl3) d: 1.35 (3H, d, Jϭ6.0 Hz), 3.71 (1H, s), 4.35—4.37 (3H, m),
6.82 (1H, d, Jϭ9.7 Hz), 6.88—6.97 (1H, m), 7.06 (1H, d, Jϭ9.7 Hz), 7.27—
Jϭ7.2 Hz), 6.80 (1H, d, Jϭ9.7 Hz), 6.85—6.92 (1H, m), 7.03 (1H, d,
Jϭ9.7 Hz), 7.22—7.31 (1H, m), 7.43—7.47 (3H, m), 7.62—7.66 (2H, m),
7.36 (1H, m), 7.44—7.48 (3H, m), 7.58—7.64 (2H, m), 7.97 (1H, d, 8.01 (1H, d, Jϭ8.9 Hz), 8.50 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1664, 1593,
Jϭ8.9 Hz), 8.54 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1651, 1579, 1524, 1495,
1527, 1493. MS m/z 388: (MϩH)ϩ. Anal. Calcd for C22H21N5O2·0.25H2O:
1466. MS m/z 347: (MϩH)ϩ. Anal. Calcd for C20H18N4O2: C, 69.35; H, C, 67.42; H, 5.53; N, 17.87. Found: C, 67.41; H, 5.36; N, 17.89.
5.24; N, 16.17. Found: C, 69.24; H, 5.15; N, 15.85.
N,N-Diethyl-2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazin-1-yl]acetamide (22): Pale yellow solid (88%). mp 137—138 °C
Compound 13 was prepared following a procedure similar to Method C.
2-(2-Hydroxy-2-methylpropyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)- (EtOH). 1H-NMR (CDCl3) d: 1.20 (3H, t, Jϭ7.1 Hz), 1.32 (3H, t,
1
3(2H)-pyridazinone (13): White solid (59%). mp 199—220 °C (EtOH). H- Jϭ7.1 Hz), 3.39—3.54 (4H, m), 5.08 (2H, s), 6.80 (1H, d, Jϭ9.7 Hz),
NMR (DMSO-d6) d: 1.18 (6H, s), 4.19 (2H, s), 4.79 (1H, s), 6.88 (1H, d, 6.85—6.93 (1H, m), 7.04 (1H, d, Jϭ9.7 Hz), 7.22—7.31 (1H, m), 7.43—
Jϭ9.6 Hz), 7.03—7.11 (2H, m), 7.35—7.70 (6H, m), 8.12 (1H, d,
Jϭ8.9 Hz), 8.81 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1649, 1579, 1522, 1493,
1462. MS m/z 361: (MϩH)ϩ. Anal. Calcd for C21H20N4O2·0.2H2O: C,
69.29; H, 5.64; N, 15.39. Found: C, 69.26; H, 5.58; N, 15.39.
7.49 (3H, m), 7.62—7.68 (2H, m), 8.03 (1H, d, Jϭ8.9 Hz), 8.51 (1H, d,
Jϭ6.9 Hz). IR (KBr) cmϪ1: 1660, 1589, 1527, 1491. MS m/z 402: (MϩH)ϩ;
Anal. Calcd for C23H23N5O2·0.5H2O: C, 67.30; H, 5.89; N, 17.06. Found: C,
67.53; H, 5.69; N, 17.14.
Method D. [6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazin-1-yl]acetic Acid (14) To a suspension of NaH (60% dispersion
2-[2-Oxo-2-(pyrrolidin-1-yl)ethyl]-6-(2-phenylpyrazolo[1,5-a]pyridin-3-
yl)-3(2H)-pyridazinone (23): Pale yellow solid (76%). mp 170—171 °C
in mineral oil, 2.3 g, 57.5 mmol) in DMF (150 ml) was added 4 (15.0 g, (EtOH). 1H-NMR (CDCl3) d: 1.84—2.12 (4H, m), 3.54—3.63 (4H, m), 5.01
52 mmol) at 5 °C under a nitrogen atmosphere. After 30 min, to the reaction (2H, s), 6.80 (1H, d, Jϭ9.7 Hz), 6.90 (1H, t, Jϭ6.9 Hz), 7.03 (1H, d,
mixture was added dropwise ethyl bromoacetate (6.3 ml, 57.5 mmol), which
Jϭ9.7 Hz), 7.19—7.29 (1H, m), 7.43—7.49 (3H, m), 7.62—7.68 (2H, m),
was allowed to warm to ambient temperature and stirred overnight. The re- 8.05 (1H, d, Jϭ8.9 Hz), 8.52 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1664, 1591,
action mixture was partitioned between 25% n-hexane in EtOAc and H2O, 1527, 1467. MS m/z 400: (MϩH)ϩ. Anal. Calcd for C23H21N5O2·0.25H2O:
and the organic layer was separated, washed with H2O and brine, and dried
over MgSO4. Evaporation of the solvent gave a residual solid, which was
C, 68.59; H, 5.36; N, 17.38. Found: C, 68.33; H, 5.19; N, 17.24.
2-[2-Oxo-2-(piperidin-1-yl)ethyl]-6-(2-phenylpyrazolo[1,5-a]pyridin-3-
triturated with a mixture of EtOAc and n-hexane to give ethyl [6-oxo-3-(2- yl)-3(2H)-pyridazinone (25): Pale yellow solid (86%). mp 205—206 °C
1
phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazin-1-yl]acetate (94%) as (EtOH). H-NMR (DMSO-d6) d: 1.40—1.70 (6H, m), 3.40—3.55 (4H, m),
a pale yellow solid. mp 155—157 °C. 1H-NMR (CDCl3) d: 1.33 (3H, t, 5.07 (2H, s), 6.89 (1H, d, Jϭ9.7 Hz), 7.03—7.12 (2H, m), 7.37—7.53 (4H,
Jϭ7.1 Hz), 4.31 (2H, q, Jϭ7.1 Hz), 5.01 (2H, s), 6.80 (1H, d, Jϭ9.7 Hz), m), 7.60—7.66 (2H, m), 7.92 (1H, d, Jϭ8.9 Hz), 8.82 (1H, d, Jϭ6.9 Hz). IR
6.91 (1H, td, Jϭ6.9, 1.4 Hz), 7.04 (1H, d, Jϭ9.7 Hz), 7.24—7.35 (1H, m), (KBr) cmϪ1: 1660, 1593, 1527, 1497. MS m/z 414: (MϩH)ϩ. Anal. Calcd
7.44—7.50 (3H, m), 7.60—7.66 (2H, m), 7.96 (1H, td, Jϭ8.9, 1.2 Hz), 8.52
(1H, td, Jϭ6.9, 1.0 Hz). IR (KBr) cmϪ1: 1745, 1674, 1593, 1525. MS m/z
375: (MϩH)ϩ. Anal. Calcd for C21H18N4O3: C, 67.37; H, 4.85; N, 14.96.
for C24H23N5O2: C, 69.72; H, 5.61; N, 19.64. Found: C, 69.46; H, 5.47; N,
16.19.
2-[2-Oxo-2-(morpholin-4-yl)ethyl]-6-(2-phenylpyrazolo[1,5-a]pyridin-3-
Found: C, 67.10; H, 4.97; N, 14.59. To a solution of the above ethyl ester in yl)-3(2H)-pyridazinone (26): Pale yellow solid (76%). mp 205—206 °C
EtOH (400 ml) was added 1 N NaOH (105 ml) and the mixture refluxed for (EtOH). 1H-NMR (CDCl3) d: 3.58—3.80 (8H, m), 5.09 (2H, s), 6.80 (1H, d,
2 h with stirring. Evaporation of the solvent gave a residue, which was dis- Jϭ9.7 Hz), 6.86—6.95 (1H, m), 7.06 (1H, d, Jϭ9.7 Hz), 7.24—7.34 (1H,
solved in H2O and washed with EtOAc. The aqueous layer was separated, m), 7.44—7.49 (3H, m), 7.62—7.67 (2H, m), 8.00 (1H, d, Jϭ8.9 Hz), 8.52
pH was adjusted to pH 1.0 with concentrated HCl. Insoluble material was (1H, d, Jϭ7.0 Hz). IR (KBr) cmϪ1: 1664, 1587, 1529, 1500. MS m/z 416:
collected by filtration, washed with H2O and EtOH, and dried to give 14 (2 (MϩH)ϩ. Anal. Calcd for C23H21N5O3: C, 66.49; H, 5.09; N, 16.86. Found:
steps, 15.6 g, 87%) as a pale yellow solid. mp 247 °C (dec.). 1H-NMR C, 66.54; H, 5.23; N, 16.73.
(DMSO-d6) d: 4.91 (2H, s), 6.93 (1H, d, Jϭ9.7 Hz), 7.04—7.14 (2H, m),
2-[2-Oxo-2-(thiomorpholin-4-yl)ethyl]-6-(2-phenylpyrazolo[1,5-a]-
7.38—7.66 (6H, m), 7.97 (1H, d, Jϭ8.9 Hz), 8.82 (1H, d, Jϭ6.9 Hz), 13.17 pyridin-3-yl)-3(2H)-pyridazinone (27): Pale yellow solid (91%). mp 207—
(1H, br s). IR (KBr) cmϪ1: 1740, 1649, 1574, 1527, 1500, 1466. MS m/z 208 °C (EtOH). H-NMR (CDCl3) d: 2.60—2.80 (4H, m), 3.80—4.00 (4H,
1
347. (MϩH)ϩ. Anal. Calcd for C19H14N4O3·0.25H2O: C, 65.04; H, 4.16; N,
m), 5.07 (2H, s), 6.79 (1H, d, Jϭ9.7 Hz), 6.85—6.94 (1H, m), 7.05 (1H, d,
Jϭ9.7 Hz), 7.23—7.32 (1H, m), 7.43—7.49 (3H, m), 7.61—7.67 (2H, m),
15.97. Found: C, 65.21; H, 4.06; N, 15.74.
Method E. N-Isopropyl-2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3- 7.99 (1H, d, Jϭ8.9 Hz), 8.52 (1H, d, Jϭ7.0 Hz). IR (KBr) cmϪ1: 1664, 1647,
yl)-1(6H)-pyridazin-1-yl]acetamide (19) To a solution of 14 (1.0 g, 1585, 1527, 1497. MS m/z 432: (MϩH)ϩ. Anal. Calcd for C23H21N5O2S: C,
2.89 mmol) in DMF (10 ml) was added successively HOBt (510 mg,
3.77 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
64.02; H, 4.91; N, 16.23. Found: C, 63.97; H, 4.81; N, 16.09.
Method F. N,N-Dimethyl-2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-
(EDC·HCl, 723 mg, 3.77 mmol) and isopropylamine (0.32 ml, 3.74 mmol) 3-yl)-1(6H)-pyridazin-1-yl]acetamide (17) To a solution of 14 (1.0 g,
at ambient temperature under a nitrogen atmosphere and stirred overnight. 2.89 mmol) in DMF (10 ml) was added successively HOBt (510 mg,
Evaporation of the solvent gave a residue, which was partitioned between 3.77 mmol), EDC (0.69 ml, 3.78 mmol) and dimethylamine hydrochloride
CHCl3 and H2O. The organic layer was separated, washed with saturated (307 mg, 3.77 mmol) at ambient temperature under a nitrogen atmosphere
NaHCO3 in H2O and brine, dried over MgSO4, evaporated, and crystallized and stirred overnight. Evaporation of the solvent gave a residue, which was
from EtOH to give 19 as a white solid (935 mg, 83%). mp 231—232 °C. 1H- partitioned between CHCl3 and H2O. The organic layer was separated,
NMR (CDCl3) d: 1.19 (6H, d, Jϭ6.5 Hz), 4.03—4.21 (1H, m), 4.89 (2H, s),
washed with saturated NaHCO3 in H2O and brine, dried over MgSO4, evapo-

996
Vol. 49, No. 8
rated, and crystallized from EtOH to give 17 (920 mg, 85%) as a pale yellow H2O and brine, and dried over MgSO4. Evaporation of the solvent gave a
1
solid. mp 189—190 °C. H-NMR (DMSO-d6) d: 2.90 (3H, s), 3.08 (3H, s),
5.07 (2H, s), 6.89 (1H, d, Jϭ9.7 Hz), 7.04—7.12 (2H, m), 7.37—7.66 (6H,
residual solid, which was recrystallized from EtOH to give 30 as a pale yel-
low solid (2.0 g, 70%). mp 132—133 °C. 1H-NMR (DMSO-d6) d: 1.50 (8H,
m), 7.93 (1H, d, Jϭ8.9 Hz), 8.82 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1664, br s), 2.63—2.70 (4H, m), 2.88 (2H, t, Jϭ6.7 Hz), 4.22 (2H, t, Jϭ6.7 Hz),
1587, 1525, 1497. MS m/z 374: (MϩH)ϩ. Anal. Calcd for C21H19N5O2·
6.86 (1H, d, Jϭ9.6 Hz), 7.00—7.11 (2H, m), 7.39—7.63 (6H, m), 7.95 (1H,
0.5H2O: C, 65.96; H, 5.27; N, 18.31. Found: C, 66.07; H, 5.27; N, 18.15.
d, Jϭ8.9 Hz), 8.81 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1659, 1589, 1529,
Compounds 15, 16, 18 and 24 were prepared following a procedure simi- 1495, 1468. MS m/z 414: (MϩH)ϩ. Anal. Calcd for C25H27N5O: C, 72.61;
lar to Method F.
H, 6.58; N, 16.947. Found: C, 72.47; H, 6.50; N, 16.91.
2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazin-1-
Method I. 6-(2-Phenylpyrazolo[1,5-a]pyridin-3-yl)-2-(piperidin-4-yl)-
1
yl]acetamide (15): Pale yellow solid (47%). mp 238—239 °C (EtOH). H- 3(2H)-pyridazinone (31) To a solution of 4 (17.9 g, 62.1 mmol), 1-tert-
NMR (DMSO-d6) d: 4.76 (2H, s), 6.89 (1H, d, Jϭ9.7 Hz), 7.07 (1H, d, butoxycarbonyl-4-hydroxypiperidine (15.0 g, 74.5 mmol) and triphenylphos-
Jϭ9.7 Hz), 7.04—7.10 (1H, m), 7.35—7.75 (8H, m), 7.97 (1H, d,
phine (PPh3, 24.4 g, 93.0 mmol) in THF (600 ml) was added dropwise di-
ethyl azodicarboxylate (DEAD, 14.7 ml, 93.3 mmol) at Ϫ5 to 0 °C under a
Jϭ8.9 Hz), 8.82 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1689, 1660, 1633, 1583,
1531, 1518, 1491. MS m/z 368: (MϩNa)ϩ. Anal. Calcd for C19H15N5O2· nitrogen atmosphere. After addition, the reaction mixture was allowed to
0.4H2O: C, 64.73; H, 4.52; N, 19.86. Found: C, 64.83; H, 4.34; N, 19.69.
N-Methyl-2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyri-
warm to ambient temperature and stirred overnight. Evaporation of the sol-
vent gave a residue, to which was added 6 N HCl and refluxed for 8 h. The
dazin-1-yl]acetamide (16): Pale yellow solid (96%). mp 231—232 °C resulting solution was washed with EtOAc, pH of the aqueous layer was ad-
(EtOH). 1H-NMR (CDCl3) d: 2.78 (3H, d, Jϭ4.9 Hz), 4.93 (2H, s), 6.54 justed to pH 11.5 with 30% NaOH while keeping the temperature at 5 to
(1H, br s), 6.80 (1H, d, Jϭ9.7 Hz), 6.93 (1H, t, Jϭ6.9 Hz), 7.07 (1H, d,
15 °C, followed by extraction three times with CHCl3, dried over MgSO4,
Jϭ9.7 Hz), 7.30—7.38 (1H, m), 7.45—7.49 (3H, m), 7.59—7.63 (2H, m), and purified by column chromatography on silica-gel (CHCl3–MeOH 40 : 1,
8.10 (1H, d, Jϭ8.9 Hz), 8.53 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1670, 1585, 20 : 1 and 10 : 1). Evaporation of the solvent gave a residual solid, which was
1529. MS m/z 360: (MϩH)ϩ. Anal. Calcd for C20H17N5O2·0.25H2O: C, crystallized from 50% aqueous EtOH (aq. EtOH) to give 31 (14.35 g, 62%)
66.01; H, 4.85; N, 19.25. Found: C, 66.02; H, 4.73; N, 19.26.
N-Ethyl-2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyri-
as a pale yellow solid. mp 115—117 °C. 1H-NMR (DMSO-d6) d: 1.70—
1.85 (4H, m), 2.50—2.70 (2H, m), 3.00—3.10 (2H, m), 4.80—5.00 (1H, m),
dazin-1-yl]acetamide (18): Pale yellow solid (95%). mp 252—254 °C 6.87 (1H, d, Jϭ9.6 Hz), 7.05—7.13 (2H, m), 7.43—7.63 (6H, m), 7.91 (1H,
1
(EtOH). H-NMR (CDCl3) d: 1.17 (3H, t, Jϭ7.3 Hz), 3.28—3.43 (2H, m), d, Jϭ8.9 Hz), 8.82 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1657, 1585, 1529,
4.92 (2H, s), 6.50 (1H, br s), 6.80 (1H, d, Jϭ9.7 Hz), 6.89—6.97 (1H, m),
7.07 (1H, d, Jϭ9.7 Hz), 7.29—7.38 (1H, m), 7.44—7.49 (3H, m), 7.59—
7.65 (2H, m), 8.10 (1H, d, Jϭ8.9 Hz), 8.55 (1H, d, Jϭ6.9 Hz). IR (KBr)
1495, 1464, 1421. MS m/z 372: (MϩH)ϩ. Anal. Calcd for C22H21N5O·
2.25H2O: C, 64.21; H, 6.25; N, 17.02. Found: C, 64.26; H, 6.28; N, 16.94.
Method J. 2-(1-Methylpiperidin-4-yl)-6-(2-phenylpyrazolo[1,5-a]-
cmϪ1: 1684, 1659, 1585, 1556, 1527. MS m/z 374: (MϩH)ϩ. Anal. Calcd pyridin-3-yl)-3(2H)-pyridazinone (32) To a solution of 4 (6.0 g, 20.8
for C21H19N5O2·0.5H2O: C, 65.96; H, 5.27; N, 18.31. Found: C, 65.78; H, mmol), 1-methyl-4-hydroxypiperidine (3.1 g, 26.9 mmol) and PPh3 (8.7 g,
5.02; N, 18.23.
33.2 mmol) in THF (120 ml) was added dropwise DEAD (5.3 ml,
2-[2-((3R)-3-Hydroxypyrrolidin-1-yl)-2-oxoethyl]-6-(2-phenylpyra- 33.6 mmol) at Ϫ5 to 0 °C under a nitrogen atmosphere and stirred for 4 h.
zolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone (24): Pale yellow solid (90%).
mp 177—178 °C (EtOH). H-NMR (CDCl3) d: 1.95—2.20 (2H, m), 2.79
Evaporation of the solvent gave a residue, which was partitioned between
EtOAc and 2 N HCl. The aqueous layer was separated and the pH was ad-
1
(1H, br s), 3.50—3.90 (4H, m), 4.45—4.55 (1H, m), 4.94—5.07 (2H, m), justed to pH 10.7 with 30% NaOH while keeping the temperature at 5 to
6.79 (1H, d, Jϭ9.7 Hz), 6.88 (1H, t, Jϭ6.9 Hz), 7.04 (1H, d, Jϭ9.7 Hz), 15 °C. Insoluble material was collected by filtration, washed with H2O, dried,
7.20—7.31 (1H, m), 7.41—7.48 (3H, m), 7.61—7.67 (2H, m), 8.02 (1H, d, dissolved in CHCl3 and purified by column chromatography on silica-gel
Jϭ8.9 Hz), 8.50 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1659, 1583, 1527, 1498.
(EtOAc, CHCl3, CHCl3–MeOH 40 : 1 and 10 : 1). Evaporation of the solvent
MS m/z 416: (MϩH)ϩ. Anal. Calcd for C23H21N5O3·0.25H2O: C, 65.78; H, gave a residual solid, which was crystallized from 50% aq. EtOH to give 32
1
5.16; N, 16.68. Found: C, 65.87; H, 5.05; N, 16.66.
(6.3 g, 79%) as a pale yellow solid. mp 139—140 °C. H-NMR (DMSO-d6)
Method G. 6-(2-Phenylpyrazolo[1,5-a]pyridin-3-yl)-2-(2-piperidin-1- d: 1.50—2.10 (6H, m), 2.20 (3H, s), 2.80—3.00 (2H, m), 4.70—4.90 (1H,
yl)ethyl-3(2H)-pyridazinone Hydrochloride (28) A mixture of 4 (2.0 g, m), 6.87 (1H, d, Jϭ9.7 Hz), 7.05—7.13 (2H, m), 7.40—7.65 (6H, m), 7.90
6.9 mmol), 1-(2-chloroethyl)piperidine hydrochloride (2.7 g, 14.7 mmol), (1H, d, Jϭ8.9 Hz), 8.83 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1660, 1589,
NaH (60% dispersion in mineral oil, 1.12 g, 28 mmol) and benzyltriethylam- 1531, 1497, 1466. MS m/z 386: (MϩH)ϩ. Anal. Calcd for C23H23N5O·H2O:
monium chloride (158 mg, 0.69 mmol) in a mixture of H2O (50 ml) and C, 68.47; H, 6.25; N, 17.36. Found: C, 68.71; H, 6.08; N, 17.37.
CHCl3 (50 ml) was stirred for 24 h. The organic layer was separated, washed
Compounds 33, 34, 36 and 37 were prepared following a procedure simi-
with H2O and brine, dried over MgSO4, and purified by column chromatog- lar to Method J.
raphy on silica-gel (EtOAc and CHCl3–MeOH 30 : 1). Evaporation of the
solvent gave a residual solid, which was treated with HCl in EtOH to give 28
2-(1-Ethylpiperidin-4-yl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-
pyridazinone (33): Pale yellow solid (28%). mp 134—135 °C (50% aq.
1
as a pale yellow solid (2.18 g, 72%). Recrystallization afford an analytical EtOH). H-NMR (DMSO-d6) d: 1.01 (3H, t, Jϭ7.1 Hz), 1.80—2.15 (6H,
sample. mp 267—268 °C (EtOH). 1H-NMR (DMSO-d6) d: 1.15—1.60 (1H,
m), 2.36 (2H, q, Jϭ7.1 Hz), 2.90—3.10 (2H, m), 4.70—4.90 (1H, m), 6.87
m), 1.60—1.90 (5H, m), 2.80—3.10 (2H, m), 3.30—3.65 (4H, m), 4.58 (2H, (1H, d, Jϭ9.7 Hz), 7.05—7.15 (2H, m), 7.45—7.65 (6H, m), 7.89 (1H, d,
t, Jϭ6.5 Hz), 6.93 (1H, d, Jϭ9.7 Hz), 7.05—7.16 (2H, m), 7.38—7.60 (4H, Jϭ8.9 Hz), 8.83 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1660, 1589, 1529, 1493,
m), 7.60—7.70 (2H, m), 8.07 (1H, d, Jϭ9.0 Hz), 8.83 (1H, d, Jϭ6.9 Hz). IR 1466. MS m/z 400: (MϩH)ϩ. Anal. Calcd for C24H25N5O·0.4H2O: C, 70.88;
(KBr) cmϪ1: 1664, 1630, 1593, 1525, 1493. MS m/z 400: (MϩH)ϩ. Anal. H, 6.39; N, 17.22. Found: C, 71.02; H, 6.35; N, 17.07.
Calcd for C24H25N5O·HCl: C, 66.12; H, 6.01; N, 16.06. Found: C, 66.03; H,
6.06; N, 16.04.
Compound 29 was prepared following a procedure similar to Method G.
6-(2-Phenylpyrazolo[1,5-a]pyridin-3-yl)2-(1-propylpiperidin-4-yl)-3(2H)-
pyridazinone (34): Pale yellow solid (45%). mp 104—106 °C (50% aq.
EtOH). H-NMR (DMSO-d6) d: 0.86 (3H, t, Jϭ7.3 Hz), 1.35—1.55 (2H,
1
2-[2-(2,6-cis-2,6-Dimethylpiperidin-1-yl)ethyl]-6-(2-phenylpyra- m), 1.70—2.35 (8H, m), 2.90—3.05 (2H, m), 4.70—4.90 (1H, m), 6.89 (1H,
zolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone Hydrochloride (29) Pale d, Jϭ9.7 Hz), 7.05—7.30 (2H, m), 7.40—7.60 (6H, m), 7.88 (1H, d,
1
yellow solid (93%). mp Ͼ270 °C (EtOH). H-NMR (DMSO-d6) d: 1.30—
2.00 (11H, m), 3.30—3.55 (4H, m), 4.40—4.50 (2H, m), 6.97 (1H, d,
Jϭ9.7 Hz), 7.09 (1H, d, Jϭ6.9 Hz), 7.23 (1H, d, Jϭ9.7 Hz), 7.40—7.65 (6H,
m), 7.99 (1H, d, Jϭ8.9 Hz), 8.84 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1689,
Jϭ8.9 Hz), 8.83 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1657, 1589, 1531, 1468.
MS m/z 414: (MϩH)ϩ. Anal. Calcd for C25H27N5O·1.1H2O: C, 69.29; H,
6.79; N, 16.16. Found: C, 69.17; H, 6.98; N, 16.27.
2-(1-Pentylpiperidin-4-yl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
1660, 1633, 1583, 1531, 1518, 1491. MS m/z 428: (MϩH)ϩ. Anal. Calcd for 3(2H)-pyridazinone (36): Pale yellow solid (39%). mp 105—106 °C (50%
C26H29N5O·HCl: C, 64.79; H, 6.69; N, 14.53. Found: C, 64.74; H, 6.72; N, aq. EtOH). H-NMR (DMSO-d6) d: 0.83—0.91 (3H, m), 1.20—1.45 (6H,
1
14.49.
m), 1.75—2.15 (6H, m), 2.20—2.35 (2H, m), 2.90—3.05 (2H, m), 4.70—
Method H. 2-(2-Azepan-1-yl)ethyl-6-(2-phenylpyrazolo[1,5-a]pyridin- 4.90 (1H, m), 6.88 (1H, d, Jϭ9.6 Hz), 7.05—7.15 (2H, m), 7.43—7.51 (4H,
3-yl)-3(2H)-pyridazinone (30) A mixture of 4 (2.0 g, 6.9 mmol), 2-(hexa- m), 7.57—7.62 (2H, m), 7.88 (1H, d, Jϭ8.9 Hz), 8.83 (1H, d, Jϭ6.9 Hz). IR
methyleneimino)ethyl chloride hydrochloride (1.93 g, 9.7 mmol), NaH (60% (KBr) cmϪ1: 1660, 1589, 1531, 1497, 1466. MS m/z 442: (MϩH)ϩ. Anal.
dispersion in mineral oil, 0.84 g, 21 mmol) and benzyltriethylammonium Calcd for C27H31N5O·0.25H2O: C, 72.70; H, 7.12; N, 15.70. Found: C,
chloride (158 mg, 0.69 mmol) in a mixture of H2O (50 ml) and CHCl3 72.73; H, 7.10; N, 15.72.
(50 ml) was stirred for 4 h. The organic layer was separated, washed with
2-(1-Benzylpiperidin-4-yl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-

August 2001
997
3(2H)-pyridazinone (37): Pale yellow solid (71%). mp 184—186 °C (50%
aq. EtOH). 1H-NMR (DMSO-d6) d: 1.70—2.20 (6H, m), 2.93 (2H, br d,
fluxing mixture of LiAlH4 4.4 g, 0.116 mol) in THF (130 ml) was added
dropwise a solution of the above ethyl 2,6-cis-2-(2,6-dimethylpiperidin-1-
Jϭ12.4 Hz), 3.51 (2H, s), 4.70—4.90 (1H, m), 6.88 (1H, d, Jϭ9.6 Hz), yl)-2-oxoacetate (13.5 g, 63.3 mmol) in THF (35 ml) under a nitrogen atmos-
7.00—7.17 (2H, m), 7.20—7.65 (11H, m), 7.88 (1H, d, Jϭ8.8 Hz), 8.84 phere. After stirring for 2 h, the reaction mixture was allowed to cool to 0 °C
(1H, d, Jϭ7.0 Hz). IR (KBr) cmϪ1: 1662, 1589, 1525, 1491, 1460. MS m/z
and added carefully H2O (4.4 ml), followed by 4 N NaOH (4.4 ml) and H2O
462: (MϩH)ϩ. Anal. Calcd for C29H27N5O·0.3H2O: C, 74.59; H, 5.96; N, (13.3 ml) successively. Insoluble material was removed by filtration and the
15.00. Found: C, 74.37; H, 6.08; N, 15.36. filtrate was concentrated under reduced pressure to give 2,6-cis-2,6-di-
Method K. 2-(1-Isopropylpiperidin-4-yl)-6-(2-phenylpyrazolo[1,5- methyl-1-(2-hydroxyethyl)piperidine (10.0 g, quant.) as a pale yellow oil.
a]pyridin-3-yl)-3(2H)-pyridazinone (35) To a suspension of NaH (60%
dispersion in mineral oil, 65 mg, 1.63 mmol) in DMF (20 ml) was added 31
1H-NMR (CDCl3) d: 1.10 (6H, d, Jϭ6.3 Hz), 1.15—1.72 (6H, m), 2.40—
2.60 (2H, m), 2.72 (2H, t, Jϭ6.4 Hz), 2.92 (1H, br s), 3.54 (2H, t, Jϭ6.4 Hz).
(500 mg, 1.35 mmol) at ambient temperature under a nitrogen atmosphere MS m/z 158: (MϩH)ϩ. A stirred solution of 2,6-cis-2,6-dimethyl-1-(2-hy-
and stirred for 30 min. To the reaction mixture was added 2-iodopropane droxyethyl)piperidine (10.0 g, 63.5 mmol) and thionyl chloride (9.3 ml,
(0.55 ml, 5.53 mmol) and stirred for an additional 18 h. To the resulting mix- 127 mmol) in toluene (100 ml) was stirred for 2.5 h at 80 °C under a nitrogen
ture was added excess triethylamine (Et3N) and H2O. Evaporation of the sol- atmosphere. Evaporation of the solvent gave a residual solid, which was
vent gave a residue, which was partitioned between EtOAc and 1 N HCl. The
aqueous layer was separated, adjusted to pH 10 with 4 N NaOH, and ex-
washed with diisopropylether, collected by filtration, and dried under re-
duced pressure to give 42 (12.8 g, 95%) as a pale yellow solid. H-NMR
1
tracted with EtOAc. The organic layer was separated, washed with H2O, (DMSO-d6) d: 1.29—1.38 (6H, m), 1.44—1.90 (6H, m), 3.20—3.60 (4H,
dried over MgSO4, evaporated, and crystallized from 50% aq. EtOH to give m), 3.38—4.05 (2H, m). MS m/z 176: (MϩH)ϩ.
35 (350 mg, 63%) as a pale yellow solid. mp 158—159 °C. 1H-NMR
4-Hydroxy-1-propylpiperidine (44b) To a suspension of 4-hydroxy-
(DMSO-d6) d: 0.98 (6H, d, Jϭ6.5 Hz), 1.75—2.20 (4H, m), 2.20—2.40 (2H, piperidine (43, 5.0 g, 49.4 mmol) and Et3N (7.6 ml, 54.5 mmol) in CH2Cl2
m), 2.65—2.95 (3H, m), 4.70—4.90 (1H, m), 6.87 (1H, q, Jϭ9.6 Hz), (100 ml) was added dropwise propionyl chloride (4.5 ml, 51 mmol) at Ϫ70
7.05—7.13 (2H, m), 7.40—7.60 (6H, m), 7.88 (1H, d, Jϭ8.9 Hz), 8.83 (1H, to Ϫ60 °C under a nitrogen atmosphere. After stirring for 1 h, insoluble ma-
d, Jϭ7.0 Hz). IR (KBr) cmϪ1: 1660, 1589, 1531, 1468. MS m/z 414: terial was removed by filtration and the filtrate was concentrated under re-
(MϩH)ϩ. Anal. Calcd for C25H27N5O·0.5H2O: C, 71.06; H, 6.68; N, 16.57. duced pressure, to which was added EtOAc, followed by stirring for 30 min.
Found: C, 71.25; H, 6.45; N, 16.78.
Insoluble material was removed by filtration and the filtrate was concen-
Method L. 2-(1-Acetylpiperidin-4-yl)-6-(2-phenylpyrazolo[1,5-a]- trated under reduced pressure to give 4-hydroxy-1-propionylpiperidine
1
pyridin-3-yl)-3(2H)-pyridazinone (38) To
a stirred solution of 31 (7.7 g, 99%) as a yellow oil. H-NMR (CDCl3) d: 1.15 (3H, t, Jϭ7.5 Hz),
(500 mg, 1.35 mmol) in pyridine (30 ml) was added dropwise Ac2O (1.28 ml, 1.37—1.59 (2H, m), 1.82—1.96 (2H, m), 2.36 (2H, q, Jϭ7.5 Hz), 3.10—
13.6 mmol) at ambient temperature under a nitrogen atmosphere and stirred 3.30 (2H, m), 3.65—4.20 (3H, m). MS m/z 158: (MϩH)ϩ. To a suspension
overnight. Evaporation of the solvent gave a residue, which was partitioned of LiAlH4 (1.8 g, 47.4 mmol) in THF (100 ml) was added dropwise a solu-
between EtOAc and 2 N HCl. The organic layer was separated, washed with tion of 4-hydroxy-1-propionylpiperidine (5.0 g, 31.8 mmol) in THF (30 ml)
saturated NaCl solution in H2O, dried over MgSO4, evaporated, and crystal- at ambient temperature under a nitrogen atmosphere. After stirring for 1 h,
lized from 50% aq. EtOH to give 38 (500 mg, 90%) as a pale yellow solid. the reaction mixture was refluxed for 4 h, and then cooled to 4 °C. To the re-
mp 114—117 °C. 1H-NMR (DMSO-d6) d: 1.50—2.00 (4H, m), 2.02 (3H, s),
sulting mixture was added carefully H2O (1.8 ml), 4 N NaOH (1.8 ml) and
2.60—2.80 (1H, m), 3.15—3.35 (1H, m), 3.90 (1H, br d, Jϭ12.2 Hz), 4.49 H2O (5.4 ml) successively. Insoluble material was removed by filtration and
(1H, br d, Jϭ13.2 Hz), 5.00—5.20 (1H, m), 6.92 (1H, d, Jϭ9.6 Hz), 7.03— the filtrate was concentrated under reduced pressure to give 44b (4.37 g,
7.12 (1H, m), 7.20 (1H, d, Jϭ9.6 Hz), 7.40—7.60 (6H, m), 7.84 (1H, d, 96%) as pale yellow oil. 1H-NMR (CDCl3) d: 0.89 (3H, t, Jϭ7.4 Hz),
Jϭ8.9 Hz), 8.83 (1H, d, Jϭ6.9 Hz). IR (KBr) cmϪ1: 1657, 1628, 1585, 1531,
1458. MS m/z 414: (MϩH)ϩ. Anal. Calcd for C24H23N5O2·2.5H2O: C,
62.87; H, 6.15; N, 15.27. Found: C, 63.13; H, 5.94; N, 15.41.
1.45—1.69 (4H, m), 1.84—2.19 (5H, m), 2.24—2.33 (2H, m), 2.73—2.84
(2H, m), 3.62—3.76 (1H, m). MS m/z 144: (MϩH)ϩ.
Compounds 44a and 44c were prepared following a procedure similar to
1-Bromo-2-cyclohexenyl-2-ethanone (40a) To an ice-cooled solution 44b.
of acetylcyclohexane (39a, 5.0 g, 39.6 mmol) in MeOH (30 ml) was added
1-Ethyl-4-hydroxypiperidine (44a): Pale yellow oil (2 steps, 83%). 1H-
Br2 (2.0 ml, 39.6 mmol) in a single portion and the reaction temperature was NMR (DMSO-d6) d: 0.96 (3H, t, Jϭ7.2 Hz), 1.24—1.43 (2H, m), 1.60—
kept below 15 °C until the red color of the solution turned colorless. To the 1.75 (2H, m), 1.85—2.00 (2H, m), 2.26 (2H, q, Jϭ7.2 Hz), 2.60—2.75 (2H,
reaction mixture was added H2O (30 ml) and the mixture stirred overnight at m), 3.33—3.50 (1H, m), 4.52 (1H, d, Jϭ4.2 Hz). MS m/z 130: (MϩH)ϩ.
1
ambient temperature. The resulting solution was extracted three times with
4-Hydroxy-1-pentylpiperidine (44c): Pale yellow oil (2 steps, 98%). H-
25% n-hexane in EtOAc. The extracts were combined, washed twice with NMR (CDCl3) d: 0.89 (3H, t, Jϭ6.4 Hz), 1.20—1.65 (8H, m), 1.85—1.95
10% K2CO3 solution in H2O, and dried over sodium sulfate. Evaporation of (2H, m), 2.00—2.35 (5H, m), 2.72—2.83 (2H, m), 3.50—3.75 (1H, m). MS
the solvent gave a residue, which was distilled under reduced pressure to m/z 172: (MϩH)ϩ.
give 40a (6.68 g, 82%) as a pale yellow oil. bp 120—125 °C (18—19 mm.).
1H-NMR (CDCl3) d: 1.10—1.95 (10H, m), 2.64—2.80 (1H, m), 3.96 (2H, brane Preparation All reagents were biochemical grade. DNA modifica-
s). MS m/z 205, 207: (MϩH)ϩ.
tion enzymes were obtained from Boehringer Mannheim, GIBCO-BRL
Compounds 40b and 40c were prepared following a procedure similar to Takara Shuzo, and TOYOBO. Human hippocampus cDNA library was from
Biological Methods. cDNA Cloning and Expression Cells and Mem-
40a.
Clontech. Adenosine antagonists and agonists were from Research Bio-
chemicals, Inc. Radioligands were from DuPon-NEN. The phage library
DNA was subjected to 35 cycles of amplification with each of two oligonu-
cleotide primers for A1 and A2A receptors cDNA, and each reaction cycle
1-Bromo-3-cyclopentyl-2-propanone (40b): Pale yellow oil (95%). bp
115—120 °C (18—19 mm.). 1H-NMR (CDCl3) d: 1.10—1.90 (9H, m), 2.67
(2H, d, Jϭ7.1 Hz), 3.88 (2H, s). MS m/z 205, 207: (MϩH)ϩ.
1-Bromo-3-cyclohexyl-2-propanone (40c): Pale yellow oil (78%). bp consisted of incubations at 94 °C for 0.5 min, 60 °C for 1 min, and 72 °C for
140—145 °C (18 mm.). 1H-NMR (CDCl3) d: 0.85—1.94 (11H, m), 2.52 2 min, with AmpliTaq DNA polymerase (Perkin Elmer). The amplified DNA
(2H, d, Jϭ6.8 Hz), 3.86 (2H, s). MS m/z 219, 221: (MϩH)ϩ.
fragments were subcloned into pUC18, and then isolated clones were se-
2,6-cis-1-(2-Chloroethyl)-2,6-dimethylpiperidine Hydrochloride (42) quenced by an Applied Biosystems model 370 DNA sequencer. The expres-
To a suspension of NaH (60% dispersion in mineral oil, 4.4 g, 0.11 mol) in sion plasmids were constructed with identified adenosine receptor cDNAs,
DMF (150 ml) was added dropwise 2,6-cis-2,6-dimethylpiperidine (41,
and the expression cells were established by the Calcium-Phosphate precipi-
13.5 ml, 83 mmol) at 40 °C under a nitrogen atmosphere. After stirring for tation method. Stable expression cells, derived from CHO, were cultured in
1 h, the reaction mixture was allowed to cool to 0 °C, to which was added minimum essential medium without nucleosides (GIBCO-BRL) containing
dropwise ethyl oxalyl chloride (11.2 ml, 0.1 mol) and stirred overnight at 10% fetal bovine serum and 100 nM methotrexate, in an atmosphere of 5%
ambient temperature. The resulting mixture was partitioned between EtOAc CO2 in air at 37 °C. Transient expression cells, derived from COS 7, were
and H2O, the organic layer was separated, washed with H2O and saturated cultured in Dulbecco’s modified Eagle’s medium. Cells were washed twice
NaCl solution in H2O, and dried over MgSO4. Evaporation of the solvent with phosphate-buffered saline (PBS) and detached from roller bottle with
gave a residue, which was purified by column chromatography on silica-gel PBS. Cell suspensions were centrifuged at 1500ϫg at 4 °C for 10 min and
(n-hexane–EtOAc 9 : 1 4 : 1 and 2 : 1) to give ethyl 2,6-cis-2-(2,6-dimethyl-
resuspended in 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2, 2 mM Dithiothreitol,
and 1 mM phenylmethanesulfonyl fluoride (buffer A). The cells were allow to
piperidin-1-yl)-2-oxoacetate (14.0 g, 66%) as a pale yellow oil. 1H-NMR
(CDCl3) d: 1.22—1.44 (9H, m), 1.50—2.00 (6H, m), 3.75—3.95 (1H, m), stand on ice for 30 min, and then homogenized on ice by ultrasonication.
4.20—4.45 (2H, m), 4.60—4.80 (1H, m). MS m/z 214: (MϩH)ϩ. To a re- The homogenate was centrifuged at 39000ϫg at 4 °C for 20 min and washed

998
Vol. 49, No. 8
three times with buffer A. After centrifugation, the precipitate was sus- References and Notes
pended in 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2. Protein concentrations
were determined by the Bradford method using protein assay kit (Bio Rad).
The membrane suspension was stored at Ϫ80 °C until assayed.
1) a) Williams M. (ed.), “Adenosine and Adenosine Receptors,” The Hu-
mana Press, Clifton, New Jersey, 1990, pp. 1—15; b) Dalziei H. H.,
Westfall D. P., Pharmacol. Rev., 46, 449—466 (1994).
Receptor Binding Adenosine A1 and A2A receptor binding were as-
sayed in 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2 containing 10 and 20 mg
membrane prepared with 3 and 6 U/ml adenosine deaminase, 4.5 nM
[3H]-DPCPX (95.3 Ci/mmol; DuPon-NEN.) and 20 nM [3H]-CGS21680
(39.5 Ci/mmol; DuPon-NEN.), and appropriate concentrations of test com-
pounds respectively. Non-specific binding was determined in the presence of
10 mM unlabelled DPCPX and CGS21680. Incubation was carried out at
25 °C for 2 h in a volume of 0.25 ml and terminated by rapid filtration under
vacuum through Whatman GF/B filters that had been pretreated with 0.3%
polyethyleneimine. The filter was washed with 5ϫ4 ml of ice-cold 50 mM
Tris–HCl (pH 7.4), 5 mM MgCl2 and the retained radioactivity was quanti-
tated by liquid scintillation counting in 5 ml of Aquasol (National Diagnos-
tics).
In Vitro Metabolism Microsomes and cytosol from liver of male SD
rats were prepared according to the method described by Sugiura et al.20) In-
cubation mixture (0.5 ml) contained microsomes (0.5 mg of protein/ml) or
cytosol (50 mg of liver/ml), 1 or 10 mM test compounds, 2 mM nicotinamide
adenine dinucleotide phosphate, 10 mM glucose-6-phosphate, 5 mM MgCl2,
0.5 units glucose-6-phosphate dehydrogenase and 100 mM phosphate buffer
(pH 7.4). After preincubation at 37 °C for 5 min, the reaction was started by
addition of test compounds. Incubation was carried out at 37 °C for 10 min
and then the reaction was stopped by addition of 0.1 N NaOH (0.5 ml) and
CHCl3 (4 ml). The resulting mixture was shaken for 10 min and centrifuged
at 1700 g for 5 min. The organic layer (3 ml) was separated and evaporated
in a stream of nitrogen. The residue was dissolved in methanol (100 ml) and
analyzed by HPLC.
2) a) Fredholm B. B., Abbracchio M. P., Burnstock G., Daly J. W.,
Harden K., Jacobson K. A., Leff P., Williams M., Pharmacol. Rev., 46,
143—156 (1994); b) Collis M. G., Hourani S. M. O., Trends. Pharma-
col. Sci., 14, 360—366 (1993).
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Reppert S. M., Mol. Endocr., 6, 384—393 (1992); b) Palmer T. M.,
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619—641 (1998).
6) Dudley M., Hitchcock J., Sorensen S., Chaney S., Zwolshen J., Lentz
N., Borcherding D., Peet N., Drug Dev. Res., 31, 226 (1994).
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9) a) Suzuki F., Shimada J., Shiozaki S., Ichikawa S., Ishii A., Nakamura
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349 (Suppl.), R96 (1994).
Quantitation in Plasma and Brain, Pharmacokinetics SD male rats 10) a) Akahane A., Katayama H., Mitsunaga T., Kato T., Kinoshita T., Kita
(7—9 weeks) fasted for 24 h were administered with test compound in 0.5%
methyl cellulose. After 30 or 60 min, rats were anesthetized with ether to
allow blood sampling from the abdominal aorta. Where indicated, rats were
then perfused with 0.9% saline via an aortic catheter before the cerebrum
was removed. The heparinized plasma and cerebrum were stored at Ϫ20 °C
until measurement of the concentration by HPLC. The heparinized plasma
(0.1 ml) or 20% cerebrum homogenate (0.5 ml) was mixed with methanol
(50 ml) and then to the mixture was added 0.1 N NaOH (0.5 ml) and CHCl3
Y., Kusunoki T., Terai T., Yoshida K., Shiokawa Y., J. Med. Chem., 42,
779—783 (1999); b) Horiai H., Yoshida K., Minoura H., Takeda M.,
Nakano K., Hanaoka K., Kusunoki T., Terai T., Ohtsuka M., Shimo-
mura K., Can. J. Physiol. Pharmacol., 72 (Suppl. 1), 505 (1994); c)
Kusunoki T., Kita Y., Akahane A., Shiokawa Y., Kohno Y., Horiai H.,
Sendoh H., Yoshida K., Tanaka H., ibid., 72 (Suppl. 1), 505 (1994); d)
Takeda M., Kohno Y., Esumi K., Horiai H., Ohtsuka M., Shimomura
K., Imai M., Jpn. J. Pharmacol., 64 (Suppl. 1), 176 p (1994).
(4 ml). The resulting mixture was shaken for 10 min and centrifuged at 11) a) Kuroda S., Akahane A., Itani H., Nishimura S., Durkin K., Ki-
1700 g for 5 min. The organic layer (3 ml) was separated and evaporated in a
stream of nitrogen. The residue was dissolved in 150 ml of HPLC mobile
phase and analyzed by HPLC. Quantitation was achieved using a standard
curve which was made by adding standard solutions of test compounds to
noshita T., Tenda Y., Sakane K., Bioorg. Med. Chem. Lett., 9, 1979—
1984 (1999); b) Kuroda S., Akahane A., Itani H., Nishimura S.,
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(2000).
control plasma or brain homogenate instead of methanol. The lower limit of 12) Unpublished result.
quantitation was 0.05 mg/ml for plasma and 0.05 mg/g tissue for brain.
HPLC Analysis The HPLC system consisted of a Waters LC Module 1
and a Millennium 2010J chromatography manager. All samples were re-
13) a) Wiliam M. P., Adv. Drug Del. Rev., 15, 5—36 (1995): b) Gibson G.
G. (ed.), “Progress in Drug Metabolism,” Taylor & Francis, London
Washington DC, 1992, pp. 141—178.
solved using a CAPCELL PAC C18 UG120 column (4.6ϫ150 mm, Shiseido) 14) Zanka A., Hashimoto N., Uematsu R., Okamoto T., Org. Process Res.
maintained at 30 °C. The column was eluted at 1 ml/min with various mix-
tures of 20 mM phosphate buffer (pH 6.5) and acetonitrile. The column elu-
ent was monitored at 250 nm.
Dev., 2, 320—324 (1998).
15) Gaudry M., Marquet A., Org. Synth., Coll. Vol. 6, 193—195 (1988).
16) Kuroda S., Akahane A., Itani H., Nishimura S., Durkin K., Kinoshita
T., Nakanishi I., Sakane K., Tetrahedron, 55, 10351—10364 (1999).
Acknowledgments The authors would like to acknowledge Dr. Susumu 17) Yamada T., Nobuhara Y., Shimamura H., Tsukamoto Y., Yoshihara K.,
Satoh, Molecular Biological Research Laboratory, for helpful discussions Yamaguchi A., Ohki M., J. Med. Chem., 26, 373—381 (1983).
and Dr. David Barrett, Medicinal Chemistry Research Laboratories, for criti- 18) Mitsunobu O., Synthesis, 1—28 (1981).
cal evaluation of this manuscript. 19) Ioannides C. (ed.), “Cytochromes P450: Metabolic and Toxicological
Aspects,” CRC Press, Florida, 1996, pp. 29—53.
20) Sugiura M., Iwasaki K., Noguchi H., Kato R., Life Sci., 15, 1433—
1442 (1974).