Novel adenosine A1 receptor antagonists. Synthesis and structure-activity relationships of a novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5 -a]pyridines.

Article abstract of DOI:10.1016/S0968-0896(99)00258-8

A novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazol o[1,5-a]pyridines was synthesized and evaluated for in vitro adenosine A1 and A2A receptor binding activities. Most of the cyclohexenyl derivatives (7a-e, 8a-s) were found to be potent adenosine A1 receptor antagonists. In a series of analogues of FR166124 (3a), alcohol 7c, nitrile 7e and amide derivatives (7d, 8c, 8r) were found to be more potent A1 antagonists with higher A2A/A1 selectivity than FR166124. Amongst them, 8r showed considerable water solubility (33.3 mg/mL), but lower than that of the sodium salt of FR166124 (> 200 mg/mL). Additionally, FR166124 had strong diuretic activity by both p.o. and iv administration in rats (minimum effective dose=0.1 and 0.032 mg/kg, respectively).

Full text of DOI:10.1016/S0968-0896(99)00258-8

Bioorganic & Medicinal Chemistry 8 (2000) 55±64
Novel Adenosine A1 Receptor Antagonists. Synthesis and
Structure±Activity Relationships of a Novel Series of
3-(2-Cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-
phenylpyrazolo[1,5-a]pyridines
Satoru Kuroda, a,* Atsushi Akahane, a Hiromichi Itani, a Shintaro Nishimura, b
Kieran Durkin, a Yoshiyuki Tenda c and Kazuo Sakane a
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1-6, 2-chome, Kashima, Yodogawa-ku,
Osaka 532-8514, Japan
bBasic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1-6, 2-chome, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
cMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1-6, 2-chome, Kashima, Yodogawa-ku,
Osaka 532-8514, Japan
Received 9 July 1999; accepted 24 August 1999
AbstractÐA novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridines was synthesized
and evaluated for in vitro adenosine A1 and A2A receptor binding activities. Most of the cyclohexenyl derivatives (7a±e, 8a±s) were
found to be potent adenosine A1 receptor antagonists. In a series of analogues of FR166124 (3a), alcohol 7c, nitrile 7e and amide
derivatives (7d, 8c, 8r) were found to be more potent A1 antagonists with higher A2A/A1 selectivity than FR166124. Amongst them,
8r showed considerable water solubility (33.3 mg/mL), but lower than that of the sodium salt of FR166124 (>200 mg/mL). Addi-
tionally, FR166124 had strong diuretic activity by both p.o. and iv administration in rats (minimum e€ective dose=0.1 and
0.032 mg/kg, respectively). # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
discovery of FK453 (1)13±17 and FK838 (2),18±21 two
potent 3-substituted 2-phenylpyrazolo[1,5-a]pyridine
adenosine A1 receptor antagonists with strong diuretic
activities (Fig. 1). Moreover, we have also reported the
discovery of FR166124 (3a, Fig. 1), a more potent deri-
vative with higher A2A/A1 selectivity that has very high
water solubility as the sodium salt (>200 mg/mL).22
Adenosine plays a variety of important physiological
roles through extracellular adenosine receptors which
are classi®ed into four subtypes called A1, A2A, A2B and
A3.1±4 With regards to possible therapeutic applications
of adenosine antagonists, e€ects on the cardiovascular
system, kidney and central nervous system have been
studied, and extensive pharmacological studies have
suggested that selective antagonists of the A1 receptor
may lead to novel therapeutic agents for the treatment
of certain kidney and central nervous system dis-
eases.2,5±10 The low water solubility of the known ade-
nosine A1 receptor antagonists6 has made it especially
dicult to develop therapeutic agents, however, several
xanthine derivatives are known to be water-soluble A1
receptor antagonists.11,12 We recently reported the
The design process leading to the discovery of
FR166124 involved introduction of various cyclic acid
groups to the N20 of the pyridazinone ring, in place of
the butyric acid group of FK838, as substituents to
induce rigidity and mimic the postulated conformation
of FK453.22 A cyclohexenyl acetic acid group was
found to be especially e€ective. In order to discover
even more potent and A2A/A1 selective analogues, we
then designed a series of FR166124 derivatives in order
to further elucidate the importance of the cyclohexenyl
acetic acid moiety. In this paper, we wish to report the
synthesis and structure±activity relationships (SAR) of a
novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyr-
idazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridines, including
Keywords: antagonists; receptors.
*Corresponding author. Tel.: +81-6-6390-5497; fax: +81-6-6304-
5435; e-mail: satoru_kuroda@po.fujisawa.co.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00258-8

56
S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
Figure 1. 3-Substituted-2-phenylpyrazolo[1,5-a]pyridine adenosine A1 receptor antagonists.
alcohol 7c, nitrile 7e, amide derivatives (7d,8a±8s), and a
series of ole®n isomers of FR166124.
tert-butyl esters 6b±d which were obtained in 65±76%
yields by the previously described methods (eq. 1).23
ꢀ1†
Chemistry
Synthetic routes for the novel 3-(2-cyclohexenyl-3-oxo-
2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyr-
idines 3, 7 and 8 prepared in this work are shown in
Schemes 1±3. The ole®n isomers (3b±d) of 3a were
prepared by acidic deprotection of the corresponding
One carbon homologated compound 7a was prepared
from 3a in 31% yield by Arndt±Eistert reaction using
trimethylsilyldiazomethane and benzylalcohol followed
Scheme 1. Synthesis of ole®n isomers of FR166124 (3a).
Scheme 2. Synthesis of N20-cyclohexenyl analogues. Reagents and conditions: (i) 1. SOCl2/CH2Cl2; 2. trimethylsilyldiazomethane/THF±CH3CN; 3.
.
2,4,6-trimethylpyridine, PhCH2OH; 4. 1 N NaOH aq, THF±H2O (7a, 7b). (ii) BH3 THF, THF (7c). (iii) 1-Ethyl-3-(3-dimethylaminopropyl)carbo-
diimide (EDC), HOBt, NH4Cl/THF±CH2Cl2 (7d). (iv) TFAA, pyridine, CH2Cl2 (7e).

S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
57
3
3
.
.
Scheme 3. Synthesis of amide derivatives of FR166124 (3a). Reagents and conditions: (i) R H HCl, EDC, HOBt/THF±CH2Cl2 (8a, b, f, j, l); R H,
3
3
.
EDC HCl, HOBt/THF±CH2Cl2 (8c, p, q) or DMF (8n, o); (COCl)2/DMF±CH2Cl2, then R H HCl, Et3N/CH2Cl2 (8d, h); R H, EDC HCl, HOBt,
.
.
Et3N/DMF, then 4 N HCl/EtOAc (8r); 1-triphenylmethylpiperazine, EDC HCl, HOBt/THF±CH2Cl2, then HCO2H, c-HCl (8s) (ii) TFA/CH2Cl2
(8e, i), (iii) 1 N NaOH/1,4-dioxane-H2O (8g, k, m).
by alkaline hydrolysis. Two carbon homologated com-
pound 7b was prepared from 7a in 80% yield by repeti-
tion of the same procedure. Alcohol 7c was prepared in
branes using 1 nM [3H]-cyclohexyladenosine (CHA).
Adenosine A2A receptor binding was measured in rat
striatal membranes using 5 nM [3H]-N-ethylcarbox-
amideadenosine (NECA) in the presence of 50 nM
cyclopentyladenosine (CPA) to eliminate the adenosine
A1 component. The in vitro A1 and A2A receptor bind-
ing activities are expressed as the nanomolar con-
centration of a compound required for 50% inhibition
(IC50) of speci®c binding of [3H]-CHA and [3H]-NECA,
respectively. Selectivity (A2A/A1) is expressed as the
ratio of IC50 values obtained by receptor binding assay.
Table 1 shows the results of binding assay and the
selectivity of these new N20-cyclohexenyl analogues, in
comparison with DPCPX (xanthine type adenosine A1
receptor antagonist),25 FK453 and FK838. The results
of binding and selectivity for the new amide derivatives
of FR166124 are shown in Table 2.
.
70% yield by reduction of 3a using BH3 THF complex.
The preparation of amide derivatives 7d, 8a±c and 8n±q
was accomplished by activation of 3a as the HOBt
(HOBt) ester, followed by treatment with an appro-
priate amine or amine hydrochloride in 40±92% yields.
Compound 7d was easily converted to 7e by dehydra-
tion using tri¯uoroacetic anhydride (TFAA) in 62%
yield. Compounds 8e and 8i were prepared by reaction
of the acid chloride generated from 3a with (COCl)2 and
the appropriate amino ester, followed by acidic depro-
tection of the resulting tert-butyl esters 8d and 8h using
tri¯uoroacetic acid (TFA), in 55 and 43% yields,
respectively. Compounds 8g, 8k and 8m were prepared
in 37±60% yields by reaction of the HOBt ester of 3a
with the appropriate amino ester, followed by alkaline
hydrolysis of the resulting esters 8f, 8j and 8l, respec-
tively. Compound 8r was prepared in 59% yield by
reaction of the HOBt ester of 3a with 1-methylpiper-
azine followed by treatment with hydrogen chloride in
EtOAc. Compound 8s was prepared in 76% yield by
reaction of the HOBt ester of 6a with 1-triphenyl-
methylpiperazine24 followed by deprotection of the tri-
phenylmethyl group with a mixture of concd HCl in
HCO2H.
In order to investigate the potency and selectivity for
adenosine A1 receptors of this type of compounds, we
initially synthesized and evaluated the in vitro activity
of N20-cyclohexenyl analogues 3 and 7, which are ole®n
isomers and related compounds, derived from
FR166124 (Table 1). With regards to the stereochem-
istry of the double bond, ole®n isomers 3b and 3d dis-
played 2-fold lower anity for the adenosine A1
receptor (IC50=33 and 32 nM, respectively), and the
A2A/A1 selectivity was also 2-fold lower (230 and 210-
fold, respectively) than that of FR166124. The anity
of isomer 3c was 5-fold less potent (IC50=72 nM) and
3-fold less selective (130-fold) than FR166124. Thus, it
was elucidated that the position of the double bond of
the cyclohexenyl group and the orientation of the car-
boxylic acid group were critical for A1 anity and
Results and Discussion
The prepared compounds were evaluated for in vitro
adenosine A1 and A2A receptor binding activities
according to the reported method.16 Adenosine A1
receptor binding was measured in rat cortical mem-

58
S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
Table 1. Binding assay and selectivity of N20-cyclohexenyl analogues
Table 2. Binding assay and selectivity of amide derivatives of
FR166124
Adenosine receptor
bindingb IC50 (nM)
Selectivityc
A2A/A1
Adenosine receptor Selectivityc
bindingb IC50 (nM)
Compounda
R1
A1
A2A
DPCPXd
FK453 (1)
FK838 (2)
4.7
17
120
1100
11000
5900
230
650
49
Compounda
R3
A1
A2A
A2A/A1
8a
8b
8c
8e
8g
8i
8k
8m
8n
NHMe
NMe2
6.8
1.7
2.0
16
59
9.2
30
12
3.1
1900
530
890
7600
14000
1900
1300
1400
640
280
310
450
480
240
210
43
NH(CH2)2OH
NHCH2CO2H
N(Me)CH2CO2H
NH(CH2)2 CO2H
NMe(CH2)2 CO2H
NH(CH2)3 CO2H
3a
(FR166124)
15
33
6200
7500
410
230
3be
120
210
8o
8p
9.2
5.0
1600
1200
170
240
3ce
3de
72
32
9300
6600
130
210
8q
8r
8.7
7.9
1800
4200
210
530
7a
7b
28
30
7600
3900
270
130
8s
21
6100
290
aAll test compounds were dissolved in DMSO.
bInhibition of [3H]-CHA speci®c binding to rat cortical membranes
(A1 receptor) and [3H]-NECA speci®c binding to rat striatal mem-
branes (A2A receptor) (n=3).
7c
7d
2.1
2500
740
1200
410
cRatio of IC50 values obtained by receptor binding assay.
1.8
6.4
8.6
cyclohexenylcarboxylic acids (3a±d, 7a, 7b). tert-Butyl
ester 6a, which is the synthetic precursor of
FR166124,23 showed high A1 anity (IC50=8.6 nM),
but the A2A/A1 selectivity was low (200-fold). On the
other hand, alcohol 7c, amide 7d and nitrile 7e showed
very high A1 anity and very high A2A/A1 selectivity.
In particular, 7c was 7-fold more potent (IC50=2.1 nM)
and 3-fold more selective (1200-fold) than FR166124;
7d was 8-fold more potent (IC50= 1.8 nM) than
FR166124. From the results obtained in this series, we
thus selected 7d as a new lead compound since a variety
of derivatives can be synthesized by chemical modi®ca-
tion of the amide moiety, despite the fact that 7d also
displayed 8-fold more potent adenosine A2A receptor
binding (IC50=740 nM) than FR166124.
7e
6a
3300
1700
520
200
aAll test compounds were dissolved in DMSO.
bInhibition of [3H]-CHA speci®c binding to rat cortical membranes (A1
receptor) and [3H]-NECA speci®c binding to rat striatal membranes (A2A
receptor) (n=3).
cRatio of IC50 values obtained by receptor binding assay.
dDPCPX: 8-cyclopentyl-1,3-dipropylxanthine.
eRacemic compounds.
especially for A2A/A1 selectivity. The one-carbon
homologated compound 7a was 2-fold less potent
(IC50=28 nM) and 2-fold less selective (270-fold) than
FR166124. The two-carbon homologated compound 7b
was 2-fold less potent (IC50=30 nM) and 3-fold less
selective (130-fold) than FR166124. It is thus clear that
FR166124 is the best compound in the series of N20-
In order to optimize the amide substituent, a variety of
derivatives were examined and the results are collected
in Table 2. Although introduction of two methyl groups
or a hydroxyethyl group to the amide nitrogen of 7d
retained A1 anity and A2A/A1 selectivity (8b, 8c),
introduction of only one methyl group reduced potency
and A2A/A1 selectivity (8a). Introduction of a carboxyl

S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
59
group into the side chain of the amide group reduced
Table 3. Water solubility of selected compounds
A1 anity (8e, 8g, 8i, 8k, 8m) and A2A/A1 selectivity
(8g, 8i, 8k, 8m). Moreover, when an additional methyl
group was introduced to the amide nitrogen of 8e and
8i, the A1 anity and A2A/A1 selectivity were reduced
(8e versus 8g, 8i versus 8k). Amides derived from cyclic
amines (8n±s) had lower A1 anity than 7d, however 8r
derived from N-methylpiperazine showed high A2A/A1
selectivity (530-fold) compared to 7d (410-fold). Despite
the fact that replacement of the C4000 of piperidine (8o)
by heteroatoms such as oxygen (8p), sulfur (8q), methyl-
amino (8r) and amino (8s) had little e€ect on A1 recep-
tor binding, the A2A/A1 selectivity of 8r was higher than
that of 7d.
Compound
Solubility
1 (FK453)
2 (FK838)a
3a (FR166124)a
7c
7d
7e
8c
8r
11.9 mg/mL
10 mg/mL
>200 mg/mLb
6.4 mg/mL
25.6 mg/mL
2.8 mg/mL
185.6 mg/mL
33.3 mg/mL
aWater solubility was measured as sodium salt.
bUpper limit was not determined.
Table 4. Diuretic activity of FR166124 in ratsa
Minimum e€ective doseb (mg/kg)
As a result of these investigations, alcohol 7c, nitrile 7e
and amide derivatives (7d, 8c, 8r) were found to be more
potent adenosine A1 receptor antagonists with higher
A2A/A1 selectivity than FR166124. In vitro adenosine
A1 receptor anity of the cyclohexenyl derivatives (7, 8)
decreased in the order: CH2CONMe2 (8b), CH2CONH2
(7d), CH2CONH(CH2)2OH (8c), (CH2)2OH (7c)
<CH2CON(-C4H8-) (8n)>CH2CN (7e), CH2CONHMe
(8a)>CH2CON(-C2H4-X-C2H4-), (8o±s, e.g. X=O, S,
NH), CH2CONH(CH2)nCO2H (8e, 8i, 8m, n=1±3)
>(CH2)nCO2H (7a, 7b, n=1, 2)>CH2CON(Me)
(CH2)nCO2H (8g, 8k, n=1, 2). Concerning the receptor
binding of FR166124 derivatives, introduction of a
more hydrophobic moiety than a carboxylic acid group
(e.g. 5a, 7c±e, 8b, 8c, 8n) at the C100 of the cyclohexene
ring increased both A1 and A2A anity. Introduction of
hydrophilic groups such as carboxylic acid or amino
moieties at the amide portion decreased A2A anity
(e.g. 8g, 8s). In the case of amide derivatives of
FR166124, it is inferred that hydrophobicity is an
important factor for increasing A1 anity and A2A/A1
selectivity rather than steric factors.
Compound
p.o.
iv
2 (FK838)
3a (FR166124)
0.1
0.1
0.1
0.032
aTest compounds were administered (n=3) and the urine was collected
for 3 h (iv) and 6 h (p.o.).
bStatistical evaluation was performed by Dunnett's multiple compar-
ison test (P<0.05 were considered signi®cant).
therapeutic agent, diuretic activity was determined
according to the reported method.15 Table 4 shows the
minimum e€ective dose of FR166124 in rats by p.o. and
iv administration. It was found that FR166124 and
FK838 had equal diuretic activity under the conditions
of p.o. administration, but in the case of iv administra-
tion, FR166124 was three fold more potent than
FK838. More potent adenosine A1 receptor antagonists
with high A2A/A1 selectivity derived from FK838 have
stronger diuretic activity. It was thus concluded that
FR166124 can possibly lead to a new therapeutic agent,
and was progressed to further pharmacological study,
the results of which will be reported in due course.
In terms of solubility, 8e, 8g, 8i, 8k, 8m and 8r can be
expected to have high water solubility, since they pos-
sess a carboxylic acid moiety, which can be converted to
the sodium salt, or amino groups, obtained as a hydro-
chloride salt, in the amide moiety. In spite of these
features, sodium salts of carboxylates (8e, 8g, 8i, 8k, 8m)
were so hygroscopic that suitable solids for measure-
ment of solubility could not be obtained. After con-
sideration of A1 anity, A2A/A1 selectivity and
chemical properties, the water solubilities of 7c, 7d, 7e,
8c and 8r were measured by adding the test compound
to water and agitating at ambient temperature until
insoluble material remained (Table 3). Although, 7c is a
highly potent adenosine A1 receptor antagonist with
high A2A/A1 selectivity, solubility in water was very low
(6.4 mg/mL). Compound 8r showed good water solubi-
lity (33.3 mg/mL) but lower than that of the sodium salt
of FR166124 (>200 mg/mL).
Conclusion
In summary, we have prepared a novel series of 3-(2-
cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenyl-
pyrazolo[1,5-a]pyridines and evaluated their in vitro
adenosine A1 and A2A receptor binding activities, lead-
ing to alcohol 7c, nitrile 7e and amide derivatives (7d,
8c, 8r), which were found to be more potent adenosine
A1 receptor antagonists with higher A2A/A1 selectivity
than FR166124. Amongst them, 7c, 7e and 8r were
found to be highly potent and highly A2A/A1 selective
adenosine A1 receptor antagonists. Furthermore, 8r has
high water solubility, but less than that of the sodium
salt of FR166124. The SAR study in this series of com-
pounds revealed the following main features. (1) The
position of the double bond of the cyclohexenyl group
and the chain length of the carboxylic acid group were
critical for high A1 anity and high A2A/A1 selectivity.
(2) Replacement of the carboxylic acid group of
FR166124 by hydrophobic moieties such as hydroxy,
nitrile and carbamoyl groups increased A1 anity and
A2A/A1 selectivity. (3) Certain amide derivatives of
FR166124 with an additional amino group on the
Whilst a number of these analogues of FR166124 were
found to be more potent adenosine A1 receptor antago-
nists with high A2A/A1 selectivity than FR166124, after
considering water solubility and synthetic cost,
FR166124 was selected as the best compound for
further evaluation. To investigate the potential as a

60
S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
amide moiety have high water solubility. (4) 3-(2-
Cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenyl-
pyrazolo[1,5-a]pyridines were potent adenosine A1
receptor antagonists with high A2A/A1 selectivity. (5)
Diuretic activity in rats of FR166124 (iv) was more
potent than FK838.
2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinyl]-(Z)-cyclohexylidene-1-acetic
acid
(3c).
White solid (65%); mp 256 ꢀC (dec.) (aq EtOH); FT-IR
1
(KBr) 1714, 1651, 1585, 1527, 1495 cm
;
1H NMR
(CDCl3) d 1.41±1.70 (m, 2H), 1.80±2.60 (m, 6H), 5.85±
6.19 (m, 2H), 6.93 (t, 2H, J=6.2 Hz), 7.17±7.34 (m, 2H),
7.43±7.46 (m, 3H), 7.55±7.56 (m, 2H), 7.80 (d, 1H,
J=8.8 Hz), 8.56 (d, 1H, J=6.9 Hz); MS m/z 427
(M+H)+. Anal. calcd for C25H22N4O3: C, 70.41; H,
5.20; N, 13.14. Found: C, 70.25; H, 5.26; N, 12.86.
Experimental
General procedures
6-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
acid
All melting points (mp) were determined with a Buchi
535 apparatus in open capillaries and are uncorrected.
Infrared spectra were recorded on a Horiba Spectradesk
FT-210 spectrometer (FT-IR) or Hitachi 260-10 spec-
trometer (IR) as KBr disks, neat or in solution as indi-
pyridazinyl]-(E)-cyclohexylidene-1-acetic
(3d).
White solid (76%); mp 250±253 ꢀC (aq EtOH); FT-IR
1
(KBr) 1714, 1651, 1595, 1527 cm 1; H NMR (DMSO-
d6) d 1.20±2.20 (m, 7H), 3.80±4.00 (m, 1H), 4.93 (s, 1H),
5.40±5.11 (m, 1H), 6.98 (d, 1H, J=9.7 Hz), 7.04±7.11
(m, 1H), 7.21 (d, 1H, J=9.7 Hz), 7.40-7.63 (m, 6H), 7.85
(d, 1H, J=8.9 Hz), 8.84 (d, 1H, J=6.9 Hz), 12.21 (br-s,
1H); MS m/z 427 (M+H)+. Anal. calcd for C25H22
N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C, 70.18; H,
5.02; N, 13.51.
1
cated. H 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 DMSO-d6 and CDCl3.
All J values are given in Hz. Mass (MS) spectra were
measured on a Hitachi model M-1000H mass spectro-
meter using APCI for ionization. Elemental analyses
were carried out on a Perkin±Elmer 2400 CHN ele-
mental analyzer. Column chromatography was per-
formed with the indicated solvents using Merck silica
gel 60 (70±230 mesh). Monitoring of reactions was car-
ried out using Merck 60 F254 silica gel, glass-supported
TLC plates, followed by visualization with UV light
(254 and 365 nm) and staining with iodine vapor.
Reagents and solvents were used as obtained from
commercial suppliers without further puri®cation.
3-[2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinyl]-1-cyclohexenyl]propionic acid (7a). To a
suspension of 3a (0.2 g, 0.47 mmol) in CH2Cl2 (4 mL)
was added SOCl2 (0.046 mL, 0.61 mmol) at ambient
temperature under a nitrogen atmosphere. After stirring
for 2.5 h, the reaction mixture was evaporated under
reduced pressure. The resultant residue was dissolved in
a mixture of THF (2 mL) and CH3CN (2 mL) and to
this mixture was added a solution of 10% trimethyl-
silyldiazomethane in n-hexane (2.2 g, 1.5 mmol) at 0 ꢀC
under a nitrogen atmosphere, then the mixture was
stirred for an additional 3 h. Evaporation of volatiles
gave a residue which was treated with benzyl alcohol
(1.0 mL) and 2,4,6-trimethylpyridine (1.0 mL), then the
mixture was stirred at 180±185 ꢀC for 7 min under a
nitrogen atmosphere. The reaction mixture was parti-
tioned between EtOAc and 10% aqueous citric acid
solution, and the organic layer was separated, washed in
turn with water and brine, dried over MgSO4, evapo-
rated, and the residue was puri®ed by column chroma-
tography on silica gel (CH2Cl2:MeOH 100:1 elution) to
give benzyl 3-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyr-
idin-3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]propionate
6-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinyl]-1-cyclohexenylacetic acid (3b). To a solu-
tion of tert-butyl 6-[6-oxo-3-(2-phenylpyrazolo[1,5-a]-
pyridin-3-yl)-1(6H)-pyridazinyl]-1-cyclohexenylacetate 6b
(15 g, 31 mmol) in CH2Cl2 (30 mL) was added dropwise
TFA (24 mL, 310 mmol) at 5 ꢀC under a nitrogen atmo-
sphere. After the addition, the reaction mixture was
allowed to warm to ambient temperature and stirred for
2 h. Evaporation of the solvent gave a residue which was
azeotroped twice with toluene (100 mL). The resultant
residue was partitioned between EtOAc and 1 N aqu-
eous NaOH solution. The aqueous layer was separated,
its pH was adjusted to 3.3 with 2 N aqueous HCl and
extracted with CH2Cl2, which was then washed with
water, dried over MgSO4 and evaporated. The resultant
solid was recrystallized from 70% aqueous EtOH to
give 3b (9.96 g, 75%) as a white solid. Mp 164.0±
(87.0 mg, 35%) as a solid. Mp 111±113 ꢀC (Et2O); IR
1
(Nujol) 1735, 1710, 1670, 1630, 1595, 1530 cm
;
1H
NMR (CDCl3) d 1.65±2.00 (m, 4H), 2.10±2.60 (m, 8H),
5.02 (s, 2H), 6.76 (d, 1H, J=9.7 Hz), 6.88 (dt, 1H,
J=1.4, 6.9 Hz), 7.00 (d, 1H, J=9.7 Hz), 7.20±7.30 (m,
6H), 7.43±7.47 (m, 3H), 7.60±7.66 (m, 2H), 7.89 (d, 1H,
J=8.9 Hz), 8.50 (d, 1H, J=6.9 Hz); MS m/z 531
(M+H)+. A mixture of the above benzyl ester (74 mg,
0.14 mmol), 1 N aqueous NaOH solution (0.28 mL) and
THF (4 mL) was re¯uxed with stirring for 5 h. Eva-
poration of the solvent gave a residue which was parti-
tioned between EtOAc and water. The aqueous layer
was separated, acidi®ed with 1 N aqueous HCl and
extracted with EtOAc. The extract was washed with
brine and dried over MgSO4. Evaporation of the solvent
gave a residual solid which was recrystallized from
aqueous EtOH to give 7a as a white solid (54.0 mg,
166.0ꢀC; FT-IR (KBr) 1720, 1641, 1571, 1529 cm 1; H
1
NMR (DMSO-d6) d 1.50±2.00 (m, 6H), 2.77 (ABq, 2H,
J=15.9, 15.8 Hz), 5.67 (br-s, 1H), 5.97 (s, 1H), 6.86 (d,
1H, J=9.6 Hz), 7.03±7.11 (m, 2H), 7.38±7.63 (m, 6H),
7.93 (d, 1H, J=8.9 Hz), 8.81 (d, 1H, J=6.9 Hz), 12.13
(br-s, 1H); MS m/z 427 (M+H)+. Anal. calcd for
C25H22N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C,
70.58; H, 5.27; N, 13.51.
Preparation of 3c and 3d was carried out by a method
similar to that described for 3b from the corresponding
tert-butyl esters 6c and 6d, respectively.

S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
61
88%). Mp 184±185 ꢀC; IR (Nujol) 1695, 1655, 1625,
a€orded an analytical sample. Mp 204±205 ꢀC (EtOAc);
1
1
1585, 1520 cm 1; H NMR (DMSO-d6) d 1.55±1.81 (m,
FT-IR (KBr) 1672, 1659, 1589, 1527 cm 1; H NMR
4H), 2.00±2.35 (m, 8H), 6.91 (d, 1H, J=9.7 Hz), 7.07
(dt, 1H, J=1.3, 6.9 Hz), 7.14 (d, 1H, J=9.7 Hz), 7.20±
7.70 (m, 6H), 7.79 (d, 1H, J=8.9 Hz), 8.82 (d, 1H,
J=6.9 Hz), 12.06 (s, 1H); MS m/z 441 (M+H)+. Anal.
calcd for C26H24N4O3: C, 70.89; H, 5.49; N, 12.72.
Found: C, 70.57; H, 5.60; N, 12.42.
(DMSO-d6) d 1.60±1.85 (m, 4H), 2.05±2.40 (m, 4H),
2.71 (ABq, 2H, J=14.7, 20.7 Hz), 6.96 (d, 1H,
J=9.7 Hz), 7.04±7.18 (m, 2H), 7.38±7.70 (m, 6H), 7.92
(d, 1H, J=8.9 Hz), 8.82 (d, 1H, J=6.9 Hz); MS m/z 426
+
.
(M+H) . Anal. calcd for C25H23N5O2 0.5H2O: C,
69.11; H, 5.57; N, 16.12. Found: C, 69.49; H, 5.51; N,
16.18.
4-[2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinyl]-1-cyclohexenyl]butyric acid (7b). 7b was
prepared according to a method similar to that descri-
bed for 7a, using 7a as the starting material instead of
3a. White solid (2 steps, 80%); mp 145±147 ꢀC (EtOAc±
n-hexane); IR (Nujol) 1700, 1655, 1625, 1585,
Using the same procedure 8a, 8b, 8f, 8j and 8l were also
prepared as described for 7d from the appropriate
amine hydrochloride.
N-Methyl-2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-
3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetamide (8a).
Amorphous powder (43%). FT-IR (KBr) 1659, 1587,
1
1510 cm 1; H NMR (DMSO-d6) d 1.20±1.90 (m, 8H),
2.00±2.50 (m, 6H), 6.90 (d, 1H, J=9.7 Hz), 7.07 (t, 1H,
J=6.9 Hz), 7.13 (d, 1H, J=9.7 Hz), 7.38±7.65 (m, 6H),
7.80 (d, 1H, J=8.8 Hz), 8.82 (d, 1H, J=6.9 Hz), 11.92
(s, 1H); MS m/z 455 (M+H)+. Anal. calcd for C27H26
N4O3: C, 71.35; H, 5.77; N, 12.33. Found: C, 70.93; H,
5.76; N, 12.23.
1
1529 cm
;
1H NMR (CDCl3) d 1.80±2.00 (m, 4H),
2.15±2.50 (m, 4H), 2.60±2.70 (m, 1H), 2.74 (d, 3H,
J=4.7 Hz), 3.12 (d, 1H, J=14.8 Hz), 6.84 (t, 1H,
J=9.6 Hz), 6.90±7.00 (m, 1H), 7.11 (d, 1H, J=9.6 Hz),
7.29±7.65 (m, 6H), 7.86 (d, 1H, J=9.0 Hz), 8.54 (d, 1H,
J=6.9 Hz); MS m/z 440 (M+H)+. Anal. calcd for
.
2-[2-(2-Hydroxyethyl)-1-cyclohexenyl]-6-(2-phenylpyr-
azolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone (7c). To a
solution of 3a (2.07 g, 4.86 mmol) in THF (40 mL) was
C26H25N5O2 0.5H2O: C, 69.63; H, 5.84; N, 15.61.
Found: C, 69.80; H, 5.83; N, 15.89.
.
added dropwise a solution of BH3 THF complex in
N,N-Dimethyl-2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyr-
idin-3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetamide
THF (1 M solution, 9.72 mL, 9.72 mmol) at 10 ꢀC over
a period of 10 min under a nitrogen atmosphere. The
reaction mixture was allowed to warm to ambient tem-
perature and stirred for an additional 5.5 h, then cooled
to 10 ꢀC and treated with 1 N aqueous HCl. The organic
layer was separated and the aqueous layer was extracted
with CH2Cl2. The combined organic layers were washed
in turn with 1 N aqueous NaOH solution, water and
brine, dried over MgSO4, evaporated, and puri®ed by
column chromatography on silica gel (toluene:MeOH
10:1 elution) to give 7c (1.41 g, 70%) as a white solid.
Recrystallization a€orded an analytical sample; mp
(8b). White solid (40%); mp 178±179 ꢀC (EtOH); FT-
1
IR (KBr) 1666, 1639, 1591, 1527 cm
;
1H NMR
(DMSO-d6) d 1.60±1.85 (m, 4H), 2.05±2.40 (m, 4H),
2.69 (s, 3H), 2.80 (s, 3H), 2.93 (s, 2H), 6.89 (d, 1H,
J=9.7 Hz), 7.07±7.11 (m, 2H), 7.37±7.66 (m, 6H), 7.90
(d, 1H, J=8.9 Hz), 8.81 (d, 1H, J=6.9 Hz); MS m/z 454
+
.
(M+H) . Anal. calcd for C27H27N5O2 0.5H2O: C, 70.11;
H, 6.10; N, 15.14. Found: C, 70.33; H, 5.97; N, 15.20.
Ethyl [methyl-[2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyr-
idin-3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetyl]amino]-
acetate (8f). Yellow solid (80%); mp 56±60 ꢀC (EtOAc±
n-hexane); FT-IR (KBr) 1743, 1670, 1639, 1591,
198±199 ꢀC (aq EtOH); IR (Nujol) 3375, 1650, 1630,
1
1585, 1520 cm
;
1H NMR (CDCl3) d 1.66±1.97 (m,
1
4H), 2.01±2.24 (m, 2H), 2.33±2.47 (m, 4H), 3.23 (t, 1H,
J=5.4 Hz), 3.63±3.83 (m, 2H), 6.85 (t, 1H, J=9.7 Hz),
6.92 (t, 1H, J=6.9 Hz), 7.08 (d, 1H, J=9.7 Hz), 7.32
(dd, 1H, J=8.9, 6.9 Hz), 7.45±7.50 (m, 3H), 7.60±7.65
(m, 2H), 7.91 (d, 1H, J=8.9 Hz), 8.53 (d, 1H, J=
6.9 Hz); MS m/z 413 (M+H)+. Anal. calcd for
C25H24N4O2: C, 72.79; H, 5.86; N, 13.58. Found: C,
72.52; H, 5.97; N, 13.56.
1527 cm 1; H NMR (DMSO-d6) d 0.96±1.67 (m, 3H),
1.60±1.85 (m, 4H), 2.05±2.40 (m, 4H), 2.70±3.00 (m,
5H), 3.90±4.10 (m, 4H), 6.80±7.13 (m, 3H), 7.35±7.63
(m, 6H), 7.89 (d, 1H, J=8.9 Hz), 8.81 (d, 1H, J=
6.9 Hz); MS m/z 526 (M+H)+. Anal. calcd for C30H31
N5O4: C, 68.55; H, 5.94; N, 13.32. Found: C, 68.32; H,
6.06; N, 12.86.
Ethyl 3-[methyl-[2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]-
pyridin-3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetyl]-
amino]propionate (8j). Amorphous foam (75%). FT-IR
2-[2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinyl]-1-cyclohexenyl]acetamide (7d). To a solu-
tion of 3a (432 mg, 1 mmol) in a mixture of CH2Cl2
(20 mL) and THF (10 mL) was added successively HOBt
(162 mg, 1.2 mmol), EDC (0.28 mL, 1.5 mmol) and
NH4Cl (65 mg, 1.2 mmol) at ambient temperature under
a nitrogen atmosphere and stirred overnight. The reac-
tion mixture was partitioned between EtOAc and water,
and the organic layer was separated, washed in turn
with water, saturated aqueous NaHCO3 solution and
brine, dried over MgSO4, evaporated, and puri®ed by
column chromatography on silica gel (EtOAc elution) to
give 7d (185mg, 43%) as a beige solid. Recrystallization
1
(KBr) 1736, 1666, 1645, 1593, 1529 cm
;
1H NMR
(DMSO-d6) d 0.95±1.16 (m, 3H), 1.55±1.80 (m, 4H),
2.10±2.50 (m, 6H), 2.65±3.20 (m, 7H), 3.80±4.05 (m,
2H), 6.88 (d, 1H, J=9.7 Hz), 7.00±7.12 (m, 2H), 7.30±
7.65 (m, 6H), 7.90 (d, 1H, J=8.9 Hz), 8.81 (d, 1H,
J=6.9 Hz); MS m/z 540 (M+H)+.
Methyl 4-[2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-
3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetylamino]-
butyrate (8l). Amorphous foam (61%). FT-IR (KBr)
1
1736, 1668, 1589, 1529 cm 1; H NMR (DMSO-d6) d

62
S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
1.45±1.85 (m, 6H), 2.16±2.35 (m, 6H), 2.65±3.10 (m,
4H), 3.53 (s, 3H), 6.95 (d, 1H, J=9.7 Hz), 7.03±7.12 (m,
1H), 7.15 (d, 1H, J=9.7 Hz), 7.35±7.67 (m, 7H), 7.91 (d,
1H, J=8.9 Hz), 8.82 (d, 1H, J=6.9 Hz); MS m/z 526
(M+H)+.
3H), 7.60±7.65 (m, 2H), 8.11 (d, 1H, J=8.9 Hz), 8.53 (d,
1H, J=6.9 Hz); MS m/z 480 (M+H)+. Anal. calcd for
C29H29N5O2: C, 72.63; H, 6.10; N, 14.60. Found: C,
72.61; H, 6.19; N, 14.50.
2-[2-[2-Oxo-2-(piperidin-1-yl)ethyl]-1-cyclohexenyl]-6-(2-
phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone
[2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinyl]-1-cyclohexenyl]acetonitrile (7e). To a stirred
mixture of 7d (2.15 g, 5 mmol) and pyridine (2.1 mL,
25 mmol) in CH2Cl2 (25 mL) was added dropwise
TFAA (1.1 mL, 7.5 mmol) at 0 ꢀC, followed by warming
to ambient temperature and stirring an additional 3 h
under a nitrogen atmosphere. The reaction mixture was
partitioned between EtOAc and water, and the organic
layer separated, washed in turn with water, saturated
aqueous NaHCO3 solution, 1 N aqueous HCl, brine,
saturated aqueous NaHCO3 solution, water and brine,
dried over MgSO4, evaporated, and puri®ed by column
chromatography on silica gel (CH2Cl2:EtOAc 100:3 and
100:5 elution) to give 7e (1.28 g, 62%) as a beige solid.
Recrystallization a€orded an analytical sample; mp
163±165 ꢀC (EtOH); FT-IR (KBr) 2251, 1734, 1670,
1633, 1595, 1529 cm 1; 1H NMR (DMSO-d6) d 1.60±1.85
(m, 4H), 2.05±2.40 (m, 4H), 3.26 (s, 2H), 6.94 (d, 1H,
J=9.7 Hz), 7.04±7.17 (m, 2H), 7.38±7.69 (m, 6H), 7.87
(8o). White solid (76%); mp 184±185 ꢀC (EtOAc±Et2O);
1
IR (Nujol) 1660, 1635, 1585, 1530 cm
;
1H NMR
(CDCl3) d 1.28±1.60 (m, 6H), 1.70±1.97 (m, 4H), 2.20±
2.58 (m, 4H), 3.09 (s, 2H), 3.20±3.43 (m, 4H), 6.78 (d,
1H, J=9.7 Hz), 6.93 (dd, 1H, J=8.9, 6.7 Hz), 7.03 (d,
1H, J=9.7 Hz), 7.35 (dd, 1H, J=8.9, 6.7 Hz), 7.46±7.49
(m, 3H), 7.60±7.63 (m, 2H), 8.05 (d, 1H, J=8.9 Hz),
8.52 (d, 1H, J=6.7 Hz); MS m/z 494 (M+H)+. Anal.
.
calcd for C30H31N5O2 0.5H2O: C, 71.69; H, 6.42; N,
13.96. Found: C, 71.71; H, 6.31; N, 13.76.
2-[2-[2-(Morpholin-4-yl)-2-oxoethyl]-1-cyclohexenyl]-6-
(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone
(8p). White solid (89%); mp 202±204 ꢀC (EtOAc±
1
Et2O); IR (Nujol) 1655, 1590, 1530 cm
;
1H NMR
(CDCl3) d 1.70±2.00 (m, 4H), 2.10±2.60 (m, 4H), 3.10 (s,
2H), 3.30±3.70 (m, 8H), 6.76 (d, 1H, J=9.8 Hz), 6.92 (t,
1H, J=6.9 Hz), 7.05 (d, 1H, J=9.8 Hz), 7.34 (t, 1H,
J=6.9 Hz), 7.45±7.49 (m, 3H), 7.59±7.63 (m, 2H), 7.99
(d, 1H, J=9.0 Hz), 8.52 (d, 1H, J=6.9 Hz); MS m/z 496
(M+H)+. Anal. calcd for C29H29N5O3: C, 70.29; H,
5.90; N, 14.13. Found: C, 70.19; H, 5.84; N, 13.72.
(d, 1H, J=8.9 Hz), 8.83 (d, 1H, J=6.9 Hz); MS m/z 408
+
.
(M+H) . Anal. calcd for C25H21N5O 0.5H2O: C, 72.10;
H, 5.32; N, 16.82. Found: C, 72.03; H, 5.13; N, 16.54.
N-(2-Hydroxyethyl)-2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-
a]pyridin-3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]aceta-
mide (8c). To a solution of 3a (800 mg, 1.88 mmol) in a
mixture of CH2Cl2 (10 mL) and THF (10 mL) was
added successively HOBt (305 mg, 2.26 mmol),
2-[2-[2-Oxo-2-(thiomorpholin-4-yl)ethyl]-1-cyclohexenyl]-
6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone
(8q). White solid (92%); mp 181±183 ꢀC (EtOAc); FT-
1
IR (KBr) 1660, 1587, 1529 cm 1; H NMR (CDCl3) d
.
EDC HCl (541 mg, 2.82 mmol) and 2-aminoethanol
1.60±2.00 (m, 4H), 2.10±2.70 (m, 8H), 3.10 (m, 2H),
3.60±4.00 (m, 4H), 6.78 (d, 1H, J=9.7 Hz), 6.94 (t, 1H,
J=6.9 Hz), 7.05 (d, 1H, J=9.7 Hz), 7.26±8.05 (m, 6H),
8.00 (d, 1H, J=9.0 Hz), 8.55 (d, 1H, J=6.9 Hz); MS m/z
512 (M+H)+. Anal. calcd for C29H29N5O2S: C, 68.08; H,
5.71; N, 13.69. Found: C, 67.96; H, 5.66; N, 13.42.
(0.14 mL, 2.26 mmol) at ambient temperature under a
nitrogen atmosphere and stirred for 3 h. The reaction
mixture was partitioned between EtOAc and water, and
organic layer was separated, washed in turn with 1 N
HCl, water, saturated aqueous NaHCO3 solution and
brine, dried over MgSO4, evaporated, and puri®ed by
column chromatography on silica gel (EtOAc elution)
to give 8c (500 mg, 57%) as a solid. Recrystallization
a€orded an analytical sample; mp 204±205 ꢀC (EtOAc);
tert-Butyl [2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-
3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetylamino]ace-
tate (8d). To a solution of 3a (222 mg, 0.52 mmol) and
DMF (1 drop) in CH2Cl2 (4 mL) was added dropwise
(COCl)2 (99 mg, 0.78 mmol) at 5 ꢀC under a nitrogen
atmosphere. After stirring for 20 min at the same tem-
perature, the reaction mixture was allowed to warm to
ambient temperature, stirred for an additional 2 h and
concentrated under reduced pressure to give a residue.
To an ice-cooled mixture of glycine tert-butyl ester hy-
drochloride (95.8 mg, 0.57 mmol) and Et3N (158 mg,
1.56 mmol) in CH2Cl2 (5 mL) was added a solution of
the above residue in CH2Cl2 (2 mL). After the addition,
the reaction mixture was allowed to warm to ambient
temperature and stirred for an additional 5.5 h. The
reaction mixture was washed in turn with 1 N aqueous
HCl, saturated aqueous NaHCO3 solution and brine,
dried over MgSO4, evaporated, and puri®ed by col-
umn chromatography on silica gel (CH2Cl2:EtOAc
10:1 elution) to give 8d (152.9 mg, 55%) as a foam. IR
1
FT-IR (KBr) 3315, 1657, 1585, 1529 cm 1; H NMR
(CDCl3) d 1.75±2.00 (m, 4H), 2.10±2.50 (m, 4H), 2.60±
2.80 (m, 1H), 3.10±3.30 (m, 2H), 3.50±3.90 (m, 4H), 6.83
(d, 1H, J=9.7 Hz), 6.90±7.00 (m, 1H), 7.14 (d, 1H,
J=9.7 Hz), 7.30±7.40 (m, 1H), 7.45±7.70 (m, 6H), 7.87
(d, 1H, J=8.9 Hz), 8.54 (d, 1H, J=6.9 Hz); MS m/z 470
+
.
(M+H) . Anal. calcd for C27H27N5O3 H2O: C, 66.51;
H, 6.00; N, 14.36. Found: C, 66.19; H, 5.82; N, 14.18.
Using a similar procedure 8n±q were also prepared as
described for 8c, from the appropriate amine.
2-[2-[2-Oxo-2-(pyrrolidin-1-yl)ethyl]-1-cyclohexenyl]-6-
(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone
(8n). White solid (75%); mp 178±179 ꢀC (EtOAc); IR
(Nujol) 1660, 1635, 1585, 1525 cm 1; 1H NMR (CDCl3)
d 1.51±2.76 (m, 12H), 2.86±3.55 (m, 6H), 6.77 (d, 1H,
J=9.7 Hz), 6.92 (t, 1H, J=6.9 Hz), 7.03 (d, 1H, J=
9.7 Hz), 7.36 (dd, 1H, J=8.9, 6.9 Hz), 7.45±7.48 (m,
1
(CH2Cl2) 3280, 1735, 1650, 1585, 1525 cm 1; H NMR
(CDCl3) d 1.44 (s, 9H), 1.75±1.92 (m, 4H), 2.11±2.52 (m,

S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
63
4H), 2.78 (d, 1H, J=14.7 Hz), 3.16 (d, 1H, J=14.7 Hz),
3.74 (dd, 1H, J=17.6, 5.5 Hz), 3.91 (dd, 1H, J=17.6,
6.0 Hz, 6.84 (d, 1H, J=9.7 Hz), 6.93 (td, 1H, J=6.9,
1.4 Hz), 7.10 (d, 1H, J=9.7 Hz), 7.32 (ddd, 1H, J=8.9,
6.9, 1.1 Hz), 7.46±7.51 (m, 3H), 7.60±7.68 (m, 3H), 7.88
(d, 1H, J=8.9 Hz), 8.53 (d, 1H, J=6.9 Hz); MS m/z 540
(M+H)+.
[Methyl-[2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-
yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetyl]amino]acetic
acid (8g). To a solution of 8f (710 mg, 1.35 mmol) in a
mixture of 1,4-dioxane (7 mL) and water (4 mL) was
added 1 N aqueous NaOH solution (3.4 mL, 3.38 mmol)
at ambient temperature and the mixture was stirred for
2 h. The reaction mixture was partitioned between
EtOAc and water, and the aqueous layer was separated.
The pH was adjusted to 1.5 with 6 N aqueous HCl,
extracted with EtOAc, washed in turn with water and
brine, dried over MgSO4, evaporated, and puri®ed by
column chromatography on silica gel (CH2Cl2:MeOH
20:1, 10:1 and 6:1 elution) to give 8g (300 mg, 46%) as a
white solid. Recrystallization a€orded an analytical sam-
ple; mp 135±138 ꢀC (aq EtOH); FT-IR (KBr) 1730, 1657,
tert-Butyl 3-[2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyr-
idin-3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetylamino]-
propionate (8h). 8h was obtained from 3a by the same
procedure described for 8d using tert-butyl 3-amino-
propionate instead of tert-butyl 2-aminoacetate. Foam
1
(43%). IR (CH2Cl2) 3280, 1720, 1655, 1585, 1525 cm
;
1H NMR (CDCl3) d 1.45 (s, 9H), 1.75±1.96 (m, 4H),
2.15±2.30 (m, 2H), 2.43 (t, 4H, J=6.9 Hz), 2.71 (d, 1H,
J=14.7 Hz), 3.09 (d, 1H, J=14.7 Hz), 3.35±3.49 (m,
2H), 6.82 (d, 1H, J=9.7 Hz), 6.93 (td, 1H, J=7.0,
1.3 Hz), 7.09 (d, 1H, J=9.7 Hz), 7.32 (dd, 1H, J=8.9,
7.0 Hz), 7.46±7.49 (m, 4H), 7.60±7.65 (m, 2H), 7.87 (d,
1H, J=8.9 Hz), 8.53 (d, 1H, J=7.0 Hz); MS m/z 554
(M+H)+.
1
1585, 1529 cm 1; H NMR (DMSO-d6) d 1.60±1.85 (m,
4H), 2.05±2.40 (m, 4H), 2.70±3.00 (m, 5H), 3.80±3.90 (m,
2H), 6.83±7.12 (m, 3H), 7.35±7.65 (m, 6H), 7.85±7.96 (m,
1H), 8.81 (d, 1H, J=6.9 Hz); MS m/z 498 (M+H)+.
.
Anal. calcd for C28H27N5O4 0.25H2O: C, 69.99; H, 5.52;
N, 13.95. Found: C, 69.98; H, 5.85; N, 14.06.
Using a similar procedure 8k and 8m were prepared as
described for 8g.
2-[2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinyl]-1-cyclohexenyl]acetylamino]acetic acid (8e).
To a solution of 8d (141 mg, 0.262 mmol) in CH2Cl2
(0.28 mL) was added dropwise TFA (299 mg,
2.62 mmol) at 5 ꢀC under a nitrogen atmosphere. After
the addition, the reaction mixture was allowed to warm
to ambient temperature and stirred for 14 h. Evapora-
tion of the solvent gave a residue which was partitioned
between CH2Cl2 and saturated aqueous NaHCO3 solu-
tion. The organic layer was separated and the aqueous
layer was acidi®ed with 1 N aqueous HCl, extracted
with CH2Cl2. The extracts were combined, washed with
brine, dried over MgSO4, and evaporated to give 8e
(106.0 mg, 83%) as a pale yellow solid. Recrystallization
a€orded an analytical sample; mp 190±192ꢀC (EtOAc);
3-[Methyl-[2-[2-[6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-
3-yl)-1(6H)-pyridazinyl]-1-cyclohexenyl]acetyl]amino]-
propionic acid (8k). White solid (80%); mp 138±140 ꢀC
(aq EtOH); FT-IR (KBr) 1720, 1660, 1633, 1587,
1
1529 cm 1; H NMR (DMSO-d6) d 1.60±1.85 (m, 4H),
2.05±2.40 (m, 6H), 2.60±3.20 (m, 7H), 6.82±6.91 (m,
1H), 7.00±7.11 (m, 2H), 7.40±7.65 (m, 6H), 7.85±7.95
(m, 1H), 8.80 (d, 1H, J=6.8 Hz); MS m/z 512 (M+H)+.
Anal. calcd for C29H29N5O4: C, 68.09; H, 5.71; N,
13.69. Found: C, 67.87; H, 5.67; N, 13.60.
4-[2-[2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
1(6H) - pyridazinyl] - 1 - cyclohexenyl]acetylamino]butyric
acid (8m). White solid (80%); mp 206±207 ꢀC (EtOH);
1
IR (Nujol) 3280, 1727, 1675, 1650, 1585, 1530 cm 1; H
1
NMR (CDCl3) d 1.72±2.63 (m, 8H), 2.79 (d, 1H,
J=14.9 Hz), 3.15 (d, 1H, J=14.9 Hz), 3.96 (t, 2H,
J=5.7 Hz), 6.92 (td, 1H, J=7.0, 1.3 Hz), 6.94 (d, 1H,
J=9.7 Hz), 7.11 (d, 1H, J=9.7 Hz), 7.35 (dd, 1H,
J=8.9, 7.0 Hz), 7.45±7.50 (m,3H), 7.57±7.63 (m, 2H),
7.72 (t, 1H, J=5.8 Hz), 7.88 (d, 1H, J=8.9 Hz), 8.55 (d,
1H, J=7.0 Hz); MS m/z 484 (M+H)+. Anal. calcd for
C27H25N5O4: C, 67.07; H, 5.21; N, 14.48. Found: C,
66.80; H, 5.38; N, 14.10.
FT-IR (KBr) 1726, 1659, 1585, 1529 cm 1; H NMR
(DMSO-d6) d 1.43±1.85 (m, 6H), 2.09±2.35 (m, 6H),
2.65±3.10 (m, 4H), 6.95 (d, 1H, J=9.7 Hz), 7.04±7.11
(m, 1H), 7.15 (d, 1H, J=9.7Hz), 7.35±7.70 (m, 7H), 7.91
(d, 1H, J=8.8 Hz), 8.82 (d, 1H, J=6.8Hz); MS m/z 512
(M+H)+. Anal. calcd for C29H29N5O4: C, 68.09; H,
5.71; N, 13.69. Found: C, 67.90; H, 5.78; N, 13.69.
2-[2-[2-(4-Methylpiperazin-1-yl)-2-oxoethyl]-1-cyclohex-
enyl]-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyr-
idazinone hydrochloride (8r). A mixture of 3a (500 mg,
1.2 mmol), 1-methylpiperazine (0.14 mL, 1.3 mmol),
Et3N (0.2 mL, 1.4 mmol), HOBt (180 mg, 1.3 mmol) and
3-[2-[2-[6-Oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-
1(6H)-pyridazinyl]-1-cyclohexenyl]acetylamino]propionic
acid (8i). 8i was obtained from 8h by the same proce-
dure described for 8e. Pale yellow solid (86%). Mp 183±
.
EDC HCl (250 mg, 1.3 mmol) in DMF (10 mL) was
184 ꢀC (EtOAc); IR (Nujol) 3275, 1720, 1670, 1650,
stirred for 16 h at ambient temperature under a nitrogen
atmosphere. The reaction mixture was partitioned
between EtOAc and water, and the organic layer was
separated, washed with water, dried over MgSO4 and
evaporated to give a residue. This residue was treated
with 4 N HCl in EtOAc and the resultant solid was
collected by ®ltration to give 8r (380 mg, 59%) as a
beige solid. Recrystallization a€orded an analytical
sample; mp 188±191 ꢀC (H2O); IR (Nujol) 2675, 2600,
1
1580, 1530 cm
;
1H NMR (CDCl3) d 1.69±2.01 (m,
4H), 2.09±2.48 (m, 4H), 2.54 (t, 2H, J=6.5 Hz), 2.74 (d,
1H, J=15.0 Hz), 3.09 (d, 1H, J=15.0 Hz), 3.41±3.52 (m,
2H), 6.89 (d, 1H, J=9.7 Hz), 6.93 (td, 1H, J=7.0,
1.3 Hz), 7.11 (d, 1H, J=9.7 Hz), 7.34 (dd, 1H, J=8.9,
7.0 Hz), 7.45±7.50 (m, 3H), 7.57±7.64 (m, 3H), 7.88 (d,
1H, J=8.9 Hz), 8.55 (d, 1H, J=7.0 Hz); MS m/z 498
(M+H)+. Anal. calcd for C28H27N5O4: C, 67.59; H,
5.47; N, 14.08. Found: C, 67.25; H, 5.57; N, 13.77.
1
1650, 1630, 1580, 1530 cm 1; H NMR (DMSO-d6) d

64
S. Kuroda et al. / Bioorg. Med. Chem. 8 (2000) 55±64
1.55±190 (m, 3H), 2.00±2.50 (m, 3H), 2.60±3.60 (m,
10H), 2.66 (s, 3H), 3.87 (s, 1H), 4.34 (s, 1H), 6.90 (d,
1H, J=9.7 Hz), 7.05±7.13 (m, 1H), 7.10 (d, 1H,
J=9.7 Hz), 7.40±7.60 (m, 4H), 7.61±7.70 (m, 2H), 7.89
(d, 1H, J=8.8 Hz), 8.83 (d, 1H, J=6.9 Hz), 11.01 (s,
1H); MS m/z 509 (M+H)+. Anal. calcd for C30H33
and Dr. David Barrett for helpful discussions and for
critical evaluation of this paper.
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.
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1
FT-IR (KBr) 1662, 1637, 1587, 1529 cm 1; H NMR
(CDCl3) d 1.70±2.00 (m, 4H), 2.20±2.60 (m, 4H), 2.70±
2.80 (m, 4H), 3.10 (s, 2H), 3.30±3.70 (m, 4H), 6.77 (d,
1H, J=9.7 Hz), 6.89±6.96 (m, 1H), 7.04 (d, 1H,
J=9.7 Hz), 7.30±7.40 (m, 1H), 7.45±7.70 (m, 5H), 8.00
(d, 1H, J=8.9 Hz), 8.52 (d, 1H, J=6.9 Hz); MS m/z 495
+
.
(M+H) . Anal. calcd for C29H30N6O2 0.5H2O: C,
69.17; H, 6.20; N, 16.69. Found: C, 69.41; H, 6.35; N,
16.41. To a solution of the above compound in HCO2H
(10 mL) was added concd HCl (1.43 mL) at ambient
temperature and the mixture was stirred for 2 h. The
reaction mixture was partitioned between EtOAc and
water, and the aqueous layer was separated and washed
with EtOAc. The pH of the solution was adjusted to 12
with 4 N aqueous NaOH solution and then extracted
twice with CH2Cl2. The extracts were combined, washed
with brine, dried over MgSO4, evaporated and puri®ed
by column chromatography on silica gel (CH2Cl2:
MeOH 20:1 and 10:1 elution) to give 8s (2.65 g, 76%) as
beige solid. Recrystallization a€orded an analytical
sample; mp 208±209 ꢀC (EtOAc); FT-IR (KBr) 1662,
1
1637, 1587, 1529 cm 1; H NMR (CDCl3) d 1.70±2.00
(m, 4H), 2.20±2.60 (m, 4H), 2.70±2.80 (m, 4H), 3.10 (s,
2H), 3.30±3.70 (m, 4H), 6.77 (d, 1H, J=9.7 Hz), 6.89±
6.96 (m, 1H), 7.04 (d, 1H, J=9.7 Hz), 7.30±7.40 (m,
1H), 7.45±7.70 (m, 5H), 8.00 (d, 1H, J=8.9 Hz), 8.52 (d,
1H, J=6.9 Hz); MS m/z 495 (M+H)+. Anal. calcd
.
for C29H30N6O2 0.5H2O: C, 69.17; H, 6.20; N, 16.69.
Found: C, 69.41; H, 6.35; N, 16.41.
Acknowledgements
The authors would like to acknowledge Dr. Hisashi
Takasugi and Dr. Kiyoshi Taniguchi for encouragement,