7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridines as novel inhibitors of human eosinophil phosphodiesterase

Article abstract of DOI:10.1021/jm9800090

High-throughput file screening against inhibition of human lung PDE4 led to the discovery of 3-ethyl-1-(4-fluorophenyl)-6-phenyl-7-oxo-4,5,6,7,- tetrahydro-1H-pyrazolo[3,4-c]pyridine (11) as a novel PDE4 inhibitor. Subsequent SAR development, using an eosinophil PDE assay, led to analogues up to 50-fold more potent than 11 with IC₅₀ values of 0.03-1.6 μM. One such compound, CP-220,629 (22) (IC₅₀ = 0.44 μM), was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED₅₀ 2.0 mg/kg, po) and demonstrated a significant reduction in eosinophil (55%), neutrophil (65%), and IL-1β (82%) responses to antigen challenge in atopic monkeys (10 mg/kg, po).

Full text of DOI:10.1021/jm9800090

2268
J . Med. Chem. 1998, 41, 2268-2277
7-Oxo-4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in es a s Novel In h ibitor s of
Hu m a n Eosin op h il P h osp h od iester a se
Allen J . Duplantier,* Catharine J . Andresen, J ohn B. Cheng, Victoria L. Cohan, Christian Decker,
Frank M. DiCapua, Kenneth G. Kraus, Kerry L. J ohnson, Claudia R. Turner, J ohn P. UmLand,
J ohn W. Watson, Ronald T. Wester, Alison S. Williams, and J ohn A. Williams
Central Research Division, Pfizer Inc, Groton, Connecticut 06340
Received J anuary 6, 1998
High-throughput file screening against inhibition of human lung PDE4 led to the discovery of
3-ethyl-1-(4-fluorophenyl)-6-phenyl-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine (11) as
a novel PDE4 inhibitor. Subsequent SAR development, using an eosinophil PDE assay, led to
analogues up to 50-fold more potent than 11 with IC50 values of 0.03-1.6 µM. One such
compound, CP-220,629 (22) (IC50 ) 0.44 µM), was efficacious in the guinea pig aerosolized
antigen induced airway obstruction assay (ED50 2.0 mg/kg, po) and demonstrated a significant
reduction in eosinophil (55%), neutrophil (65%), and IL-1â (82%) responses to antigen challenge
in atopic monkeys (10 mg/kg, po).
In tr od u ction
Phosphodiesterases (PDEs) have been classified into
seven major families (PDE1-7) according to their
substrate sensitivity, Ca2+/calmodulin requirement, and
inhibitor selectivity.1-5 PDE4, a cAMP-specific and
Ca2+-independent enzyme, is a key isozyme in the
hydrolysis of cAMP in mast cells, basophils, eosinophils,
monocytes and lymphocytes.1-5 As a result, inhibitors
of PDE4 block the release of various mediators from
these cells and may be useful antiinflammatory drugs.
Recently, four human cDNA isoforms of PDE4 (PDE4-
A, -B, -C, and -D) were identified; mRNA for all four
isoforms was expressed in human lung; and PDE4-A,
-B, and -D were expressed in eosinophils.6
Over the past decade, many pharmaceutical compa-
nies have focused on the inhibition of PDE4 for the
treatment of asthma.1-5 The biology of the PDE4
isozyme and the SAR of its reported inhibitors have
recently been reviewed in the literature.1-5 In general
cyclopentoxy moieties of rolipram, respectively (Figure
the therapeutic utility of selective PDE4 inhibitors, such
1), in binding to the active site of PDE4. In this paper
as the prototypical agent rolipram, have been hampered
we report the evolution of the SAR of 11 and its possible
by nausea and emesis limiting their therapeutic poten-
relationship to rolipram. Since eosinophils are believed
tial.7,8 We and several other pharmaceutical companies
to be a critical proinflammatory target cell for asthma,1,2
have developed SAR around the structure of rolipram
the SAR developed around 11 was evaluated against
human eosinophil PDE.
in efforts to decrease emetic potency and retain ef-
ficacy.5,9 These efforts have led to second generation
PDE4 inhibitors derived from rolipram, and some of
Ch em istr y
these compounds have entered clinical trials for asthma
(e.g., RP 73401,10 SB 207499, and CDP 84011).2,5 How-
A procedure for the preparation of 11 has been
reported in the patent literature.12 We have modified
the synthesis in order to optimize yields and provide
for structural modifications in the latter steps of the
synthesis at the 1-, 3-, and 6-positions of the pyrazol-
opyridine bicycle (Scheme 1). The substituent at the
lactam nitrogen (6-position of the pyrazolopyridines)
was introduced in the first step by a Goldberg reaction
between an aryl iodide and 2-pyrrolidinone in the
presence of copper and K2CO3 at 130-170 °C to form
the N-arylpyrrolidinones 1a -g. This substituent could
also be incorporated in the last step of the synthesis as
ever, selective PDE4 inhibitors have yet to provide
significant efficacy in the clinic.
As an alternative to designing analogues derived from
the structure of rolipram, random file screening was
conducted against human lung PDE4 in the hope of
finding a novel PDE4 inhibitor. This exercise led to the
discovery of the pyrazolopyridine, 11, a selective PDE4
inhibitor that is structurally unrelated to rolipram.
Despite this structural dissimilarity, SAR optimization
of 11 led to the hypothesis that the ethyl and 4-fluo-
rophenyl moieties of 11 could mimic the methoxy and
S0022-2623(98)00009-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/30/1998

Inhibitors of Human Eosinophil Phosphodiesterase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2269
F igu r e 1. Hypothesis: the ethyl and 4-fluorophenyl moieties of 11 overlap with the methoxy and cyclopentoxy moieties of rolipram,
respectively.
Sch em e 1a
a
Reagents and conditions: (i) Cu, K2CO3, 130-170 °C [general procedure A]; (ii) R2MgBr, ether, 0 °C [general procedure B]; (iii) ethyl
oxalyl chloride, NaOH, benzene [general procedure C]; (iv) NaOEt, EtOH; (v) methyl-4-tolyltriazine, dichloroethane, reflux [general
procedure D]; (vi) R1NHNH2‚HCl, NaOMe, EtOH, reflux [general procedure E]; (vii) R1NHNH2‚HCl; 120 °C; N2 stream [general procedure
F].
described below. The pyrazolopyridine 3-substituent
was introduced by treating 1a -g with a Grignard
reagent to give amino ketones 2-5. The amino ketone
intermediates were found to be unstable and were
therefore acylated with ethyl oxalyl chloride immedi-
ately after purification and subsequently cyclized to
3-hydroxy-2-oxo-1,2,5,6-tetrahydropyridines 6-9 by heat-
ing at reflux with NaOMe/EtOH. Esterification of 6a -g
with methyl-4-methylphenyltriazine13 provided the cor-
responding vinylogous methyl esters, 10a -g. The vi-
nylogous acids 6-9 or esters 10a -g were treated with
an alkyl or aryl hydrazine hydrochloride and either
fused at 120 °C or heated at reflux in NaOMe/EtOH to
give a mixture of the desired 7-oxo-4,5,6,7-tetrahydro-
1H-pyrazolo[3,4-c]pyridines (11, 12, 14-28) and the
corresponding regioisomers (e.g., 35 and 36). The
correct regiochemical assignment to 11 was confirmed
by single-crystal X-ray diffraction.
To prepare a variety of substituents at the lactam
nitrogen of the pyrazolopyridines, the 4-methoxyphenyl
analogue, 12, was treated with aqueous ceric am-
monium nitrate in acetonitrile14 to give the unsubsti-
tuted lactam 13 (Scheme 2). The lactam nitrogen of 13
was either coupled with an aryl- or heteroaryl halide
in the presence of copper and K2CO3 at 130-170 °C to
give compounds 29-33 or alkylated with benzyl bro-
mide to give compound 34.
Biology
For the purpose of high throughput screening, a PDE4
isozyme mixture was used, which was isolated from
human lung tissue15 with slight modifications to the
published procedure.9 Rolipram-sensitive PDE4 frac-
tions had 2 mL of ethylene glycol added for storage at
-25 °C. Subsequent SAR was developed from a human
eosinophil PDE assay, also utilizing a PDE4 isozyme

2270 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Duplantier et al.
Sch em e 2a
offered no additional improvement. Modification of R2
gave dramatic results as replacement of the ethyl group
with methyl, n-propyl, or n-butyl significantly reduced
the eosinophil PDE potency (26-28).
With the 1- and 3-positions of the pyrazolpyridine
bicycle optimized as cyclopentyl and ethyl, respectively,
we returned to modification of R3, incorporating various
heterocyclic and substituted phenyl groups. Replace-
ment of the phenyl group of 14 with 2-thiophene (29)
improved eosinophil PDE activity (IC50 ) 0.063 vs 0.16
µM), replacement with either the 3-thiophene group (30)
or the 2-pyridyl group (32) slightly decreased activity
(IC50 ) 0.66 and 1.64 µM, respectively), and replacement
with the 3-pyridyl group (33) was equipotent. The
3-chlorophenyl analogue 31 was equipotent to the
3-methoxyphenyl analogue 21. The benzyl analogue 34
(IC50 ) 0.42 µM) showed evidence that an aryl group is
not necessary at R3. Notably, the eosinophil PDE
potencies of compounds such as 11, 14, 16-25, and 29-
34 are comparable to that of PDE4 inhibitors which
have progressed into clinical development (rolipram, RP
73401, SB 207499, and CDP 840).
a
Reagents and conditions: (i) ceric ammonium nitrate, CH3CN/
water; 0 °C [general procedure G]; (ii) aryl iodide or bromide, Cu,
K2CO3, 130-170 °C; (iii) NaH, THF, benzylbromide [general
procedure H].
mixture. Selected compounds were evaluated for PDE4
isozyme selectivity against isolated soluble PDE1 (hu-
man cardiac ventricle), PDE3 (human heart), and PDE5
(human corpus cavernosal tissue). Compounds were
also evaluated for their ability to elevate intracellular
levels of cAMP in human U-937 cells.
The anti-anaphylactic activity of PDE4 inhibitors was
determined by their ability to block the airway obstruc-
tive response of anti-OA-IgG1 passively sensitized guinea
pigs challenged with aerosolized ovalbumin.9 Selected
active compounds were subsequently evaluated in mon-
keys using a crossover experimental design that allows
each monkey to serve as its own negative control.
Previous studies demonstrated that treatment with the
PDE4 inhibitor, rolipram, prior to antigen challenge,
could reduce the magnitude of leukocyte infiltration into
the lungs of atopic monkeys and also reduce levels of
TNF-R and IL-8 in their bronchoalveolar (BAL) lavage
fluid.16 Chronic treatment with rolipram also prevented
the development of airway hyperresponsiveness and
eosinophil influx after multiple antigen challenge in
atopic monkeys.16 Because the efficacy during chronic
studies may reflect the acute efficacy of a compound,
we evaluated selected PDE4 inhibitors for their ability
to inhibit cellular and biochemical responses 4 h after
aerosolized antigen challenge.
With the above SAR in hand we considered whether
11 had any structural overlaps with either rolipram or
cAMP. Initial studies using classical molecular models
led to a possible similarity between the ethyl and
4-fluorophenyl moieties of 11 and the methoxy and
cyclopentoxy moieties of rolipram, respectively (Figure
1). Subsequently, low-energy conformers for each com-
pound were determined using the SYBYL package.
Since the molecular shape and atom types of both
rolipram and 11 allow for a number of possible overlaps,
we chose to characterize the electrostatic field around
both compounds by using the GRID suite of programs.17
This method calculates the steric and electrostatic field
of a compound by systematically moving a particle of
defined volume and charge around the molecule of
interest. These compounds were analyzed with several
different GRID probes (CH2, CO, NH, OH, CO-, and
NH3). While the CH2 probe was useful in defining the
boundaries of the molecules, the remaining probes
indicated areas predisposed to either accepting or
donating a hydrogen bond. On the basis of the electro-
static map garnered through the use of the NH3 probe,
we found that the interaction potential around the
pyrazolo nitrogens of 11 closely approximates the same
potential around the ether oxygens of rolipram. This
suggested an overlap of these two compounds based on
their interaction potentials as shown in Figure 2. We
reasoned that if this hypothesis were correct, it would
explain the improved affinity of the cyclopentyl ana-
logues such as compound 21 for PDE4. The overlap of
compound 21 with rolipram based on their GRID
interaction potential is shown in Figure 3.
Str u ctu r e-Activity Rela tion sh ip s
Since the evaluation of compound 11 (IC50 ) 0.44 µM)
and its regioisomer, 35 (7% inhibition, 100 µM), in the
human lung PDE4 assay revealed that the 2-substituted
pyrazolopyridines (35) were poor PDE4 inhibitors, only
the 1-substituted isomers were subsequently evaluated
in the human eosinophil PDE assay.
As shown in Table 1, replacement of the 4-fluorophen-
yl group (R1) in compound 11 with cyclopentyl to give
14 improved the human eosinophil PDE inhibitory
potency by 10-fold. Insertion of either a methyl or
methoxy group at the 2- or 3-position of the phenyl ring
of 11 (R3) enhanced the eosinophil PDE inhibitory
activity (16, 17, 19, and 20), whereas placing these
groups in the 4-position slightly reduced the activity (15
and 18). Combining the cyclopentyl group at R1 with
either the 3-methoxyphenyl or 2-methylphenyl group at
R3 (Table 1) led to compounds 21 (eosinophil PDE IC50
) 34 nM) and 22, respectively. Further modifications
of R1 replacing the cyclopentyl group in compound 21
with cyclohexyl (23), cyclobutyl (24), or 3-sulfolanyl (25)
To test if the above compounds could inhibit PDE4
in whole cells, selected compounds were evaluated for
their ability to elevate intracellular levels of cAMP in
the human monocytic U-937 cell line (Table 1). Note
that with the exception of the very weak eosinophil PDE
inhibitors, 27 and 28, all analogues were active. This
assay was only used as a means to evaluate a com-
pound’s ability to penetrate cells and was not used as a
tool to develop SAR. It is unclear why some compounds

Inhibitors of Human Eosinophil Phosphodiesterase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2271
Ta ble 1. Structure-Activity Relationships of Close-in Analogues of 11 and Inhibition of Eosinophil PDE
HEOS PDE
IC50 (µM) (
SE (n)
U-937 cells
cAMP elev EC50
(µM) ( SE (n)
compd
11
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
R1
4-fluorophenyl ethyl
cyclopentyl ethyl
4-fluorophenyl ethyl
4-fluorophenyl ethyl
4-fluorophenyl ethyl
4-fluorophenyl ethyl
4-fluorophenyl ethyl
4-fluorophenyl ethyl
R2
R3
formula
analysisa mp (°C)
phenyl
phenyl
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
20H18FN3O
19H23N3O
21H20FN3O2 C, H, N
21H20FN3O2 C, H, N
21H20FN3O2 C,b H, N
21H20FN3O
21H20FN3O
21H20FN3O
20H25N3O2
20H25N3O
C, H, N
C, H, N
112-5
68-9
1.64 ( 0.86 (3)
0.16 ( 0.07 (3)
6.04 ( 1.96 (3)
0.49 ( 0.29 (4)
1.08 ( 0.58 (3)
2.24 ( 0.44 (3)
0.27 ( 0.08 (3)
0.89 ( 0.49 (3)
0.88 ( 0.39 (3)
0.07 ( 0.04 (2)
6.25 ( 1.31 (3)
4-methoxyphenyl
3-methoxyphenyl
2-methoxyphenyl
4-methylphenyl
3-methylphenyl
2-methylphenyl
3-methoxyphenyl
2-methylphenyl
3-methoxyphenyl
3-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
108-9
139-40
103-4
134-6
94-5
3.21 (1)
0.50 (1)
0.78 (1)
C, H, N
C, H, Nc
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Nd
141-2
63-4
cyclopentyl
cyclopentyl
cyclohexyl
cyclobutyl
3-sulfolanyl
ethyl
ethyl
ethyl
ethyl
ethyl
0.034 ( 0.010 (3) 0.33 ( 0.07 (5)
0.44 ( 0.23 (4) 0.33 ( 0.09 (4)
130-1
21H27N3O2
19H23N3O2
0.048 ( 0.010 (3) 0.98 ( 0.66 (3)
0.15 ( 0.10 (4)
0.46 ( 0.25 (3)
0.52 ( 0.36 (2)
5.27 (1)
19H23N3O4S C, H, Ne
20H18FN3O2 C, H, N
22H22FN3O2 C, H, N
4-fluorophenyl methyl
4-fluorophenyl n-propyl 4-methoxyphenyl
4-fluorophenyl n-butyl
140-2 15.7 ( 1.9 (3)
97-8 84.9 ( 10.5 (2)
95-6 84.7 (1)
11% at 1.0 (1)
13% at 0.1 (1)
phenyl
22H22FN3O
17H21N3OS
17H21N3OS
C, H, N
C, H, N
(HRMS)f
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
2-thiophene
3-thiophene
3-chlorophenyl
2-pyridyl
59-60
0.063 ( 0.030 (5) 0.001 ( 0.001 (5)
0.66 ( 0.24 (4)
0.039 ( 0.02 (3)
1.64 ( 0.17 (3)
0.16 ( 0.02 (3)
0.42 ( 0.07 (3)
3.34 ( 0.79 (16)
0.036 ( 0.020 (4)
0.041 ( 0.02 (3)
19H22ClN3O C, H, N
72-3
18H22N4O
18H22N4O
20H25N3O
(HRMS)f
(HRMS)f
C, H, N
3-pyridyl
0.017 (1)
0.036 (1)
1.23 ( 0.22 (8)
benzyl
35-6
rolipram
RP 73401
SB 207499
CDP 840
0.009 ( 0.001 (8) 0.04 ( 0.01 (5)
0.17 ( 0.05 (5)
0.67 ( 0.33 (3)
3.63 ( 2.35 (3)
0.29 ( 0.12 (3)
a
b
d
Unless indicated, all values were within 0.4%. C: calcd, 69.03; found, 69.70. c N: calcd, 12.03; found, 11.52. N: calcd, 12.91; found,
12.34. e N: calcd, 10.79; found, 9.82. f High-resolution mass spectra showed peak for [M + 1].
F igu r e 3. Overlap of rolipram with 21. The interaction
potential of the rolipram ether oxygens is shown in red, and
F igu r e 2. Overlap of rolipram with 11. The interaction
the interaction potential of the pyrazol nitrogens for 21 is
potential of the rolipram ether oxygens is shown in red, and
shown in orange. Rolipram and 21 are respectively colored
the interaction potential of the pyrazol nitrogens for 11 is
magenta and yellow.
shown in blue. Rolipram and 11 are respectively colored
magenta and green.
Ta ble 2. Inhibitory Activity of 11 and 22 against Other
Phoshodiesterases
(11, 14, 18, 29, 30, 33, and 34) are more potent in the
compd
PDE1
PDE3
PDE5
U-937 cell assay than in the human eosinophil assay.
Two compounds were chosen as representatives to
evaluate PDE4 selectivity. As shown in Table 2, 11 and
22 were weak inhibitors of human PDE1 (cardiac
ventricle), PDE3 (heart), and PDE5 (corpus cavernosal
tissue).
In vivo, 11 and 22 were active in the guinea pig
aerosolized antigen-induced airway obstruction assay
(AAIAO) (ED50 ) 4.3 and 2.0 mg/kg, po, respectively)
11
2% at 10 µM
33% at 100 µM
50% at 10 µM
1% at 1 µM
21% at 10 µM
7% at 1 µM
22
8% at 10 µM
9% at 100 µM
at doses comparable to rolipram (ED50 0.6 mg/kg, po)
(Table 3). In atopic monkeys challenged with antigen,
22 (10 mg/kg, po) inhibited eosinophil increases by 55%,
neutrophil increases by 65%, and reduced BAL levels

2272 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Duplantier et al.
Ta ble 3. Effects of 11, 22, and Rolipram in the Guinea Pig Aerosolized Antigen Induced Airway Obstruction (AAIAO) Assay and on
Acute Cellular and Biochemical Responses to Antigen Challenge in Atopic Monkey
monkeys dosed at 10 mg/kg, po
AAIAO assay
compd
11
22
rolipram
ED50 (mg/kg, po)
% eosinophil inh
% cytokine inhb
plasma levelsc
a
4.3
2.0
0.6
not tested
55.2 ( 8.71
49.4 ( 17.6
not tested
not tested
0.28 ( 0.015 µM
0.91 ( 0.15 µM
82.1 ( 12.6 (IL-1â)
84.0 ( 4.9 (IL-8)
33.4 ( 3.3 (TNF)
a
ED50 based on log linear regression on a minimum of three point dose response curve. Each point is generated by n ) 5 animals.
Data only reported for cytokine levels that were significantly lower after treatment compared to controls. c Plasma samples were taken
b
at the time of antigen challenge and evaluated by HPLC.
was collected in a microtiter plate for quantitation. A 50 µL
aliquot of each eluant was spotted on Mylar sheet, allowed to
air-dry completely, and 3H counted using a Packard Matrix
96 counter. This method provided the capacity of screening
approximately 3000-6000 compounds per week. Each com-
pound was tested at a final concentration of ∼40 µM. Potential
inhibitors identified in the primary HTS screen were subse-
quently confirmed using traditional test tube assays to deter-
mine IC50’s.
Eosin op h il P h osp h od iester a se Activity. Human pe-
ripheral blood was collected in ethylenediaminetetraacetic acid
(EDTA), diluted 1:2 in piperazine-N, N′-bis-2-ethanesulfonic
acid (PIPES) buffer, and then layered over 60% Percoll
solution. Gradients were formed by centrifugation for 30 min
at 2000 rpm at 4 °C. The remainder of the isolation procedure,
which was based on the procedure of Kita et al.,18 was carried
out at 4 °C. The neutrophil/eosinophil layer was collected from
the Percoll gradient, and the red blood cells were lysed.
Remaining cells were washed in PIPES (1% fetal calf serum),
incubated with anti-CD16 microbeads (MACS) for 1 h, and
passed over a magnetic column to remove the neutrophils.
Eosinophils were collected in the eluate and analyzed for
viability by trypan blue and purity by Diff-Quick (Baxter)
stain. Eosinophil purity was routinely >98% using this
method.
of IL-1â by 82%; 22 had no statistically significant effect
on IL-8 responses, and TNF-R could not be measured
in this experiment. Rolipram (10 mg/kg, po) had a
similar effect on eosinophil (49% inhibition) and neu-
trophil increase (62%). However, unlike 22, rolipram
reduced BAL levels of IL-8 and TNF by 84% and 33%,
respectively, and had no statistically significant effect
on IL-1â. Neither compound reduced IL-6 levels after
antigen challenge. Although the implications of dif-
ferential effects on cytokine responses are currently
unclear, it is fair to say that both compounds are capable
of inhibiting biochemical responses in addition to chemo-
tactic responses of inflammatory leukocytes.
Finally, ferrets9 dosed with 22 (1 mg/kg, sc, five
animals) showed no adverse effects, whereas five out of
five ferrets dosed with rolipram (1 mg/kg, sc) displayed
a gagging behavior.
Con clu sion s
In conclusion, we have discovered a novel PDE4
inhibitor (11) by high-throughput file screening. Mo-
lecular modeling of 11 overlapped with rolipram pro-
vided hypothetical similarities that helped rationalize
SAR results. Optimization of substituents at the 1-, 3-,
and 6-position of the pyrazolopyridine bicycle of 11 led
to CP-220,629 (22), which shows potent inhibition of
PDE4 activity both in vitro and in vivo, and a reduced
emetic liability compared to rolipram.
Purified eosinophils were resuspended in 750 µL of PDE
lysis buffer (20 mM triethylamine, 1 mM EDTA, 100 µg/mL
bacitracin, 2 mM benzamidine, 50 µM leupeptin, 50 µM
phenylmethyl sulfonyl fluoride, and 10 µg/mL soybean trypsin
inhibitor) and quick frozen in liquid nitrogen. Cells were
thawed slowly and sonicated, and disruption was confirmed
by Trypan blue stain. Disrupted cells were centrifuged at
105000g for 30 min at 4 °C to isolate membranes. Cytosol was
decanted, and the membrane was resuspended to 500 µg/mL
for use as PDE source in the hydrolysis assay.
Exp er im en ta l Section
Compounds were dissolved in DMSO at 0.01 M and then
diluted 1:25 in water to 0.40 mM. This suspension was serially
diluted 1:10 in 4% DMSO, for a final DMSO concentration in
the assay of 1%.
Biology. High -Th r ou gh p u t P DE4 Activity. The PDE4
assay9 was adapted to a 96-well microtiter plate format for
high throughput screening. This adaptation required reduced
volumes and the use of Millipore MultiScreen plates to provide
the equivalent of 96 miniature Affi-Gel 601 columns for
separation of [3H]cAMP from the product [3H]AMP. Reaction
components were added to 96-well microtiter plates, in se-
quence (using a Soken SigmaPet 96 liquid dispenser): 5 µL of
compound (or 0.3% DMSO), 5 µL of 4X assay buffer (50 mM
Tris, 10 mM MgCl2, pH 7.5), 5 µL of [3H]cAMP/cAMP (1 µM
[3H]cAMP/cAMP), 5 µL of PDE4 enzyme (for blank, enzyme
was deactivated by boiling for 10 min). Components were
mixed in microtiter plates. Plates were incubated at 37 °C
for 20 min. The reaction was stopped by adding 100 µL of hot
wash buffer per well (0.1 M N-(2-hydroxyethyl)piperazine-N′-
2-ethanesulfonic acid (HEPES), 0.1 M NaCl, pH 8.5). From
each well of the reaction plate, 75 µL of the reaction mix was
applied to a 200 µL bed volume/well Affi-Gel 601 (boronate
affinity gel from Bio-Rad, equilibrated with washing buffer)
cAMP hydrolysis was assesed by adding equal volumes of
Tris/MgCl2 assay buffer, 4 nM cAMP, test compound, and PDE
source to duplicate 12 × 75 mm glass tubes and incubating
for 25-30 min in a 37 °C shaking water bath. Reaction was
stopped by boiling samples 5 min. Samples were applied to
Affi-gel columns (1 mL bed volume) previously equilibrated
with 0.25 M acetic acid followed by 0.1 mM HEPES/0.1 mM
NaCl wash buffer (pH 8.5). cAMP was washed off column with
HEPES/NaCl, and 5′-AMP was eluted with 4 mL of 0.25 M
actetic acid. One milliliter of eluate was counted in 3 mL of
Ready-Safe for 1 min ([3H]).
Substrate conversion ) (cpm positive control × 4)/total
activity. Conversion rate must be between 3 and 15% for
experiment to be valid.
% inhibition ) 1 - (eluted cpm - bkgd cpm/control cpm -
bkgd cpm) × 100. IC50s were generated by linear regression
of inhibition titer curve (linear portion) and were expressed
in µM.
in
a Millipore MultiScreen 0.65 µm membrane-bottomed
plates. The unbound [3H]cAMP was washed from the resin
with 2.0 mL/well wash buffer, using the Millipore MultiScreen
plates in conjuction with a Pall Biosupport Silent Monitor
vaccum manifold and a custom 96-port elution buffer reservoir.
[3H]AMP was subsequently eluted with 0.25 M acetic acid. The
first 300 µL of eluent was discarded. The next 150 µL of eluent
Hu m a n U-937 cAMP Eleva tion Assa y. Human U937
cells grown in continuous culture were obtained and centri-
fuged at 419g at 22 °C for 5 min. The supernatant was
decanted, and the cell pellet was resuspended in RPMI 1640

Inhibitors of Human Eosinophil Phosphodiesterase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2273
cell culture medium plus 2% fetal bovine serum (FBS). Cells
were counted and viability was checked using a hemocytom-
eter. An appropriate volume of RPMI 1640 + 2% FBS was
added to the cell suspension to make the cell concentration
106 cells/mL. A 500 µL aliquot (5 × 105 cells) of the suspension
was added to a 12 × 75 mm glass tube containing 5 µL of either
DMSO or compound, in duplicate or triplicate. This mixture
was allowed to incubate at 37 °C for 15 min. PGE1 (1 µL)
was added, and the incubation was allowed to proceed for
another 15 min, at which time the tubes were placed in a
boiling water bath for 10 min.
Tubes were centrifuged for 10 min at 2930g. The samples
were either assayed fresh or stored at -20 °C. A cAMP
radioimmunoassay kit was used to quantitate cAMP levels.
The EC50 value of a compound is that concentration of
compound which produced 50% of an arbitrary maximal
response (e.g., that concentration of cAMP produced by 10 µM
rolipram) after subtraction of a baseline cAMP level.
Melting points were determined using a Mel-Temp II capillary
melting point apparatus and are uncorrected. Elemental
analyses were performed by Schwarzkopf Microanalytical Lab.,
Woodside, NY. Reference compounds were prepared using
modified literature procedures.19
Gen er a l P r oced u r e A. N-Ar yla tion of 2-P yr olid in on e.
1-(2-Meth oxyp h en yl)-2-p yr r olid in on e (1d ). A mixture of
2-pyrrolidinone (15.0 g, 176 mmol), 2-iodoanisole (7.6 mL, 59
mmol), copper powder (7.5 g, 117 mmol), and K2CO3 (8.1 g, 59
mmol) was stirred under nitrogen at 150 °C. After 18 h the
reaction mixture was allowed to cool to room temperature and
was suspended in THF (50 mL). The mixture was filtered
through Celite, concentrated to a brown oil, and chromato-
graphed (1:1 ethyl acetate/hexane) to give 9.3 g (82%) of a pale
yellow oil: 1H NMR (300 MHz, CDCl3) δ 2.20 (pentet, 2H),
2.55 (t, J ) 8.0 Hz, 2H), 3.75 (t, J ) 6.0 Hz, 2H), 3.82 (s, 3H),
6.93-7.02 (m, 2H), 7.25-7.30 (m, 2H); MS m/z 191.
Gen er al P r ocedu r e B. Gr ign ar d Reaction with N-Ar yl-
2-p yr r olid in on e. N-(3-Met h ylp h en yl)-6-a m in oh exa n -3-
on e (2f). To a stirred suspension of Mg turnings (1.9 g, 79
mmol) in 30 mL of anhydrous ether was added dropwise
bromoethane (5.9 mL, 79 mmol). A mild reflux was initiated
after ∼1 mL was added. After all of the Mg was consumed,
the reaction mixture was cooled to 0 °C, and 1-(3-methylphen-
yl)-2-pyrrolidinone (8.7 g, 50 mmol) was added at once. After
being warmed to room temperature and stirred for 2 h, the
reaction mixture was poured over ice and extracted with ethyl
acetate. The combined organics were washed with water and
brine, dried over Na2SO4, filtered, and concentrated to afford
8.8 g (86%) of an unstable white solid which was used
immediately in the next step without purification: 1H NMR
(300 MHz, CDCl3) δ 1.00-1.10 (m, 3H), 1.83-1.96 (m, 2H),
2.22 (s, 3H), 2.39-2.46 (m, 2H), 2.50-2.58 (m, 2H), 3.06-3.15
(m, 2H), 3.59 (broad s, 1H), 6.35-6.56 (m, 2H), 6.99-7.09 (m,
1H), 7.22-7.28 (m, 1H).
Aer osolized An t igen In d u ced Air w a y Ob st r u ct ion
Assa y. This assay was used to evaluate the ability of a
compound to block the airway obstruction resulting from an
aerosolized antigen induced pulmonary anaphylaxis and has
been described.9
Mon k ey Aller gic In fla m m a tion Mod el. Using a cross-
over design, six atopic, cynomolgus monkeys were treated
orally with vehicle (controls, n ) 3) or drug (n ) 3). One hour
later, each monkey was anesthetized (10 mg/kg Ketamine, 1.0
mg/kg Rompun, intramuscularly) and intubated with a cuffed
endotracheal tube. Bronchoalveolar lavage (BAL) was per-
formed using one 15 mL wash of phosphate buffered saline
(PBS) delivered through a pediatric fiberoptic bronchoscope
(Olympus, model BF 3C20, J apan) inserted through the
endotracheal tube and wedged into a third to fifth generation
bronchus. Lavage fluid was gently aspirated and collected in
a syringe. After BAL was complete, each animal received a 2
min exposure to a concentration of Ascaris suum aerosol which
doubled respiratory system resistance (Rrs) in previous experi-
ments. Each monkey was returned to its cage, and 4 h later,
a second BAL was performed, using 15 mL of PBS, on the
opposite side of the lung. One week after the first trial, control
and treated monkeys were reversed and the experiment was
repeated. This design yielded an n ) 6 with each monkey
serving as its own control. Compound 22 was dissolved in 2%
Tween80 and delivered orally.
To determine the percent composition of each leukocyte type,
two slides from each monkey BAL sample were obtained by
centrifuging 2 × 150 µL of the lavage fluid for 2 min at 500
rpm in a Cytospin centrifuge (Shandon Instruments, Sewickly,
PA). The slides were stained in Diff-Quick for differential cell
count. Two hundred leukocytes were counted on each slide.
The percent composition of each cell type was averaged for
the two slides. Total leukocyte count per milliliter of BAL fluid
was determined by diluting 20 µL of sample in 20 mL of Isoton
(Coulter Diagnostics, Hialeah FL), adding 3 drops of Zapoglo-
bin (Coulter Diagnostics) to lyse erythrocytes, and reading the
sample using a Coulter Counter (Coulter Electronics, Hialeah,
FL).
After total and differential leukocyte counts were deter-
mined, BAL samples were centrifuged at 1800 rpm for 20 min.
The supernatant was collected in Centriprep-3 concentrator
tubes (Amicon, Inc. Beverly, MA) and centrifuged at 3000 rpm
until a 2 mL concentrate remained (approximately 95 min,
then 45 min). Commercially available ELISA kits for human
IL-1â, IL-6, IL-8, and TNFR (R & D Systems, Minneapolis,
MN) were used to analyze the concentrated BAL sample for
the levels of these cytokines.
Ch em istr y. Gen er a l Meth od s. Anhydrous THF and
ether were distilled over Na under a N2 atmosphere. Other
solvents and reagents were of reagent grade and were used
as supplied by the manufacturer. All reactions were run under
a N2 atmosphere. Organic extracts were routinely dried over
anhydrous Na2SO4. Concentration refers to rotory evapora-
ration under reduced pressure. Chromatography refers to
“flash chromatography” on EM Science silica gel (40-63 µm).
N-P h en yl-6-a m in oh exa n -3-on e (2a ): no purification; un-
stable pale yellow solid; 98% yield; 1H NMR (300 MHz, CDCl3)
δ 1.05 (t, J ) 7.3 Hz, 3H), 1.84-1.94 (m, 2H), 2.45 (q, J ) 7.3
Hz, 2H), 2.56 (t, J ) 7.2 Hz, 2H), 3.15 (t, J ) 6.8 Hz, 2H), 3.66
(broad s, 1H), 6.58-6.72 (m, 3H), 7.16-7.23 (m, 2H).
N-(4-Meth oxyp h en yl)-6-a m in oh exa n -3-on e (2b): recrys-
tallized from isopropyl ether; unstable off-white solid; 90%
1
yield; H NMR (300 MHz, CDCl3) δ 1.03 (t, J ) 7.3 Hz, 3H),
1.84-1.95 (m, 2H), 2.38-2.46 (m, 2H), 2.54 (t, J ) 7.0 Hz, 2H),
3.07 (t, J ) 6.0 Hz, 2H), 3.74 (s, 3H), 6.58 (d, J ) 6.0 Hz, 2H),
6.67 (d, J ) 6.0 Hz, 2H).
N-(3-Meth oxyp h en yl)-6-a m in oh exa n -3-on e (2c): no pu-
rification; unstable pale yellow solid; 93% yield; 1H NMR (300
MHz, CDCl3) δ 1.07 (t, J ) 7.3 Hz, 3H), 1.84-1.93 (m, 2H),
2.40-2.49 (m, 2H), 2.55 (t, J ) 7.0 Hz, 2H), 3.10 (t, J ) 7.0
Hz, 2H), 3.75 (s, 3H), 6.12-6.27 (m, 3H), 7.02 (d, J ) 7.5 Hz,
1H).
N-(2-Meth oxyp h en yl)-6-a m in oh exa n -3-on e (2d ): no pu-
1
rification; unstable yellow oil; 68% yield; H NMR (300 MHz,
CDCl3) δ 1.05 (t, J ) 7.3 Hz, 3H), 1.90-1.97 (m, 2H), 2.42 (q,
J ) 7.3 Hz, 2H), 2.53 (t, J ) 7.2 Hz, 2H), 3.13 (t, J ) 6.8 Hz,
2H), 3.83 (s, 3H), 4.19 (broad s, 1H), 6.58-6.88 (m, 3H), 7.23-
7.29 (m, 1H).
N-(4-Meth ylp h en yl)-6-a m in oh exa n -3-on e (2e): no puri-
fication; unstable off-white solid; 96% yield; 1H NMR (300
MHz, CDCl3) δ 1.06 (t, J ) 7.3 Hz, 3H), 1.84-1.96 (m, 2H),
2.25 (s, 3H), 2.49-2.58 (m, 2H), 2.55 (t, J ) 7.0 Hz, 2H), 3.11
(t, J ) 7.0 Hz, 2H), 3.53 (broad s, 1H), 6.54 (d, J ) 8.0 Hz,
2H), 6.98 (d, J ) 8.0 Hz, 2H).
N-(2-Meth ylp h en yl)-6-a m in oh exa n -3-on e (2g): no puri-
fication; unstable yellow oil; 53% yield; 1H NMR (300 MHz,
CDCl3) δ 1.08 (t, J ) 7.3 Hz, 3H), 1.91-2.03 (m, 2H), 2.14 (s,
3H), 2.42-2.50 (m, 2H), 2.59 (t, J ) 7.5 Hz, 2H), 3.18 (t, J )
8.0 Hz, 2H), 6.56-6.68 (m, 2H), 7.03-7.18 (m, 2H).
N-(4-Meth oxyp h en yl)-5-a m in op en ta n -2-on e (3b): no pu-
1
rification; unstable pale yellow oil; 99% yield; H NMR (250
MHz, CDCl3) δ 1.88 (m, 2H), 2.13 (s, 3H), 2.56 (t, J ) 8.0 Hz,
2H), 3.07 (t, J ) 8.0 Hz, 2H), 3.74 (s, 3H), 6.56 (d, J ) 9.0 Hz,
2H), 6.78 (d, J ) 9.0 Hz, 2H).

2274 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Duplantier et al.
1
N-(4-Meth oxyp h en yl)-7-a m in oh ep ta n -4-on e (4b): no pu-
rification; unstable pale green oil; 71% yield; 1H NMR (250
MHz, CDCl3) δ 0.92 (t, J ) 7.5 Hz, 3H), 1.62 (m, 2H), 1.88 (m,
2H), 2.40 (t, J ) 8.0 Hz, 2H), 2.53 (t, J ) 8.0 Hz, 2H), 3.08 (t,
J ) 7.5 Hz, 2H), 3.74 (s, 3H), 6.58 (d, J ) 7.5 Hz, 2H), 6.79 (d,
J ) 7.5 Hz, 2H).
from 3b; H NMR (250 MHz, CDCl3) δ 2.46 (s, 3H), 2.76 (t, J
) 7.2 Hz, 2H), 3.78-3.84 (m, 5H), 6.93 (d, J ) 8.9 Hz, 2H),
7.23 (d, J ) 8.9 Hz, 2H); MS m/z 262.
4-Bu tyr yl-3-h ydr oxy-1-(4-m eth oxyph en yl)-2-oxo-1,2,5,6-
tetr a h yd r op yr id in e (8b): recrystallized from 1:9 ethyl
acetate/ether; yellow needles; 13% yield from 4b (not opti-
mized); mp 125-6° C; 1H NMR (250 MHz, CDCl3) δ 1.01 (t, J
) 7.2 Hz, 3H), 1.71 (q, J ) 7.2 Hz, 2H), 2.68-2.80 (m, 4H),
3.80 (s, 3H), 3.79-3.86 (m, 2H), 6.93 (d, J ) 9.0 Hz, 2H), 7.24
(d, J ) 9.0 Hz, 2H); MS m/z 290.
3-Hyd r oxy-2-oxo-1-p h en yl-4-va ler yl-1,2,5,6-tetr a h yd r o-
p yr id in e (9a ): white solid; 44% yield from 5a ; 1H NMR (300
MHz, CDCl3) δ 0.95 (t, J ) 7.3 Hz, 3H), 1.32-1.46 (m, 2H),
1.60-1.71 (m, 2H), 2.72-2.81 (m, 4H), 3.89 (t, J ) 6.5 Hz, 2H),
7.22-7.45 (m, 5H), 11.97 (broad s, 1H); MS m/z 273.
Gen er a l P r oced u r e D. Ester ifica tion of Vin ylogou s
Acid s 6a -g. 3-Meth oxy-1-(4-m eth ylp h en yl)-2-oxo-4-p r o-
p ion yl-1,2,5,6-tetr a h yd r op yr id in e (10e). A solution of 6e
(5.9 g, 23 mmol) and 3-methyl-1-p-methylphenyltriazene (5.1
g, 34 mmol) in 1,2-dichloroethane (25 mL) was heated at reflux
for 45 min. The mixture was allowed to cool to room temper-
ature and was poured into water and acidified with 6 N HCl.
The aqueous layer was extracted 3 times with CH2Cl2, and
the combined organics were washed with 1 N HCl followed by
water and brine, dried over MgSO4, filtered, and concentrated
to give 6.2 g (quantitative yield) of a brown oil. Note that the
aqueous layer contained p-toluidine as a byproduct and was
isolated for disposal. No impurities were detected by TLC or
1H NMR and the product was used without purification: 1H
NMR (300 MHz, CDCl3) δ 1.12 (t, J ) 7.2 Hz, 3H), 2.34 (s,
3H), 2.71 (t, J ) 6.7 Hz, 2H), 2.93 (q, J ) 7.2 Hz, 2H), 3.77 (t,
J ) 6.8 Hz, 2H), 3.94 (s, 3H), 7.20 (s, 4H); MS m/z 273.
3-Meth oxy-2-oxo-1-p h en yl-4-p r op ion yl-1,2,5,6-tetr a h y-
d r op yr id in e (10a ): dark brown oil; quantitative yield; 1H
NMR (300 MHz, CDCl3) δ 1.13 (t, J ) 7.2 Hz, 3H), 2.73 (t, J
) 6.7 Hz, 2H), 2.93 (q, J ) 7.2 Hz, 2H), 3.81 (t, J ) 6.7 Hz,
2H), 3.94 (s, 3H), 7.23-7.62 (m, 5H); MS m/z 260.
N-P h en yl-8-a m in oocta n -5-on e (5a ): recrystallized from
1
isopropyl ether; unstable white crystals; 54% yield; H NMR
(300 MHz, CDCl3) δ 0.92 (t, J ) 7.8 Hz, 3H), 1.25-1.38 (m,
2H), 1.52-1.64 (m, 2H), 1.88-1.96 (m, 2H), 2.42 (t, J ) 7.0
Hz, 2H), 2.55 (t, J ) 7.0 Hz, 2H), 3.14 (t, J ) 7.0 Hz, 2H), 3.60
(broad s, 1H), 6.59-6.71 (m, 3H), 7.14-7.20 (m, 2H).
Gen er a l P r oced u r e C. Acyla tion w ith Eth yl Oxa lyl
Ch lor id e F ollow ed by Dieck m a n n Con d en sa tion . 3-Hy-
d r oxy-2-oxo-4-p r op ion yl-1-(3-m et h ylp h en yl)-1,2,5,6-t et -
r a h yd r op yr id in e (6f). N-(3-Methylphenyl)-6-aminohexan-
3-one (2f) (8.8 g, 43 mmol) was dispersed in a mixture of 40
mL of benzene and 86 mL of 1 N NaOH, and with vigorous
mechanical stirring ethyl oxalyl chloride (7.2 mL, 64 mmol)
was added. After the mixture was stirred at reflux for 1.5 h,
the layers were separated and the aqueous layer was extracted
with ethyl acetate. The combined organics were washed with
water and brine, dried over MgSO4, filtered, and concentrated
to give an amber oil. The oil was dissolved in 20 mL of
anhydrous ethanol and treated with a solution of NaOMe in
MeOH (prepared from the careful addition of Na (1.0 g) to 10
mL of anhydrous MeOH). After being stirred at reflux over
1.5 h, the mixture was concentrated and 100 mL of water was
added. The resulting mixture was acidified to pH 3 with 6 N
HCl, and the dull yellow precipitate was filtered and washed
with water. Recrystallization from 75 mL of isopropyl ether
afforded 6.8 g (61%) of pale yellow crystals: mp 115-116 °C;
1H NMR (300 MHz, CDCl3) δ 1.16 (t, J ) 7.2 Hz, 3H), 2.37 (s,
3H), 2.74-2.82 (m, 4H), 3.85 (t, J ) 6.8 Hz, 2H), 7.08-7.14
(m, 3H), 7.30 (t, J ) 7.7 Hz, 1H); MS m/z 259.
3-Hyd r oxy-2-oxo-1-p h en yl-4-p r op ion yl-1,2,5,6-tetr a h y-
d r op yr id in e (6a ): pale yellow solid; 72% yield from 2a ; mp
141-3 °C (lit.12 144-6 °C); 1H NMR (300 MHz, CDCl3) δ 1.17
(t, J ) 7.2 Hz, 3H), 2.74-2.81 (m, 4H), 3.82 (t, J ) 6.81 Hz,
3-Me t h oxy-1-(4-m e t h oxyp h e n yl)-2-oxo-4-p r op ion yl-
1,2,5,6-tetr a h yd r op yr id in e (10b): brown solid; quantitative
yield; mp 81-2° C; H NMR (300 MHz, CDCl3) δ 1.13 (t, J )
7.2 Hz, 3H), 2.71 (t, J ) 6.8 Hz, 2H), 2.93 (q, J ) 7.2 Hz, 2H),
3.75 (t, J ) 6.8 Hz, 2H), 3.81 (s, 3H), 3.94 (s, 3H), 6.91 (d, J )
9.0 Hz, 2H), 7.23 (d, J ) 9.0 Hz, 2H). Anal. (C16H19NO4) C,
H, N.
3-Me t h oxy-1-(3-m e t h oxyp h e n yl)-2-oxo-4-p r op ion yl-
1,2,5,6-tetr a h yd r op yr id in e (10c): red brown solid; 95%
1
2H), 7.24-7.44 (m, 5H), 11.72 (broad s, 1H). Anal. (C14H15
NO3) C, H, N.
-
3-H yd r oxy-1-(4-m e t h oxyp h e n yl)-2-oxo-4-p r op ion yl-
1,2,5,6-tetr a h yd r op yr id in e (6b): recrystallized from 1:9
ethyl acetate/ether; pale yellow crystals; 65% yield from 2b;
1
mp 121-2 °C; H NMR (300 MHz, CDCl3) δ 1.16 (t, J ) 7.2
Hz, 3H), 2.75-2.82 (m, 4H), 3.82 (t, J ) 6.8 Hz, 2H), 3.80 (s,
3H), 6.93 (d, J ) 9.0 Hz, 2H), 7.22 (d, J ) 9.0 Hz, 2H); MS m/z
276.
1
yield; mp 54-5° C; H NMR (300 MHz, CDCl3) δ 1.13 (t, J )
7.2 Hz, 3H), 2.72 (t, J ) 6.7 Hz, 2H), 2.94 (q, J ) 7.2 Hz, 2H),
3.79 (t, J ) 6.7 Hz, 2H), 3.81 (s, 3H), 3.94 (s, 3H), 6.78-6.92
(m, 3H), 7.27-7.33 (m, 1H), MS m/z 290.
3-H yd r oxy-1-(3-m e t h oxyp h e n yl)-2-oxo-4-p r op ion yl-
1,2,5,6-tetr a h yd r op yr id in e (6c): recrystallized from isopro-
pyl ether; white crystals; 90% yield from 2c; mp 130-2 °C; 1H
NMR (300 MHz, CDCl3) δ 1.17 (t, J ) 7.2 Hz, 3H), 2.74-2.81
(m, 4H), 3.81 (s, 3H), 3.86 (t, J ) 6.8 Hz, 2H), 6.81-6.90 (m,
3H), 7.29-7.34 (m, 1H); MS m/z 275. Anal. (C15H17NO4) C,
H, N.
3-H yd r oxy-1-(2-m e t h oxyp h e n yl)-2-oxo-4-p r op ion yl-
1,2,5,6-tetr a h yd r op yr id in e (6d ): recrystallized from iso-
propyl ether; white crystals; 49% yield from 2d ; mp 119-20
°C; 1H NMR (300 MHz, CDCl3) δ 1.17 (t, J ) 7.2 Hz, 3H), 2.74-
2.81 (m, 4H), 3.65-3.72 (m, 2H), 3.84 (s, 3H), 6.97-7.02 (m,
2H), 7.22-7.35 (m, 2H); MS m/z 275.
3-Me t h oxy-1-(2-m e t h oxyp h e n yl)-2-oxo-4-p r op ion yl-
1,2,5,6-tetr a h yd r op yr id in e (10d ): brown oil; quantitative
yield; H NMR (300 MHz, CDCl3) δ 1.15 (t, J ) 7.2 Hz, 3H),
1
2.74 (t, J ) 6.7 Hz, 2H), 2.96 (q, J ) 7.2 Hz, 2H), 3.59-3.67
(m, 2H), 3.89 (s, 3H), 3.97 (s, 3H), 6.96-7.02 (m, 2H), 7.20-
7.33 (m, 2H); MS m/z 289.
3-Meth oxy-2-oxo-4-pr opion yl-1-(3-m eth ylph en yl)-1,2,5,6-
tetr a h yd r op yr id in e (10f): brown oil; 97% yield; 1H NMR
(300 MHz, CDCl3) δ 1.13 (t, J ) 7.2 Hz, 3H), 2.36 (s, 3H), 2.71
(t, J ) 6.7 Hz, 2H), 2.93 (q, J ) 7.2 Hz, 2H), 3.78 (t, J ) 6.7
Hz, 2H), 3.94 (s, 3H), 7.09-7.37 (m, 4H); MS m/z 273.
3-Meth oxy-2-oxo-4-pr opion yl-1-(2-m eth ylph en yl)-1,2,5,6-
3-Hydr oxy-2-oxo-4-pr opion yl-1-(4-m eth ylph en yl)-1,2,5,6-
tetr ah ydr opyr idin e (6e): recrystallized from isopropyl ether;
golden brown solid; 54% yield from 2e; mp 110-12 °C (lit.12
1
tetr a h yd r op yr id in e (10g): brown oil; 95% yield; H NMR
(300 MHz, CDCl3) δ 1.15 (t, J ) 7.2 Hz, 3H), 2.25 (s, 3H), 2.76
(t, J ) 6.7 Hz, 2H), 2.95 (q, J ) 7.2 Hz, 2H), 3.68-3.70 (m,
2H), 3.97 (s, 3H), 7.10-7.30 (m, 4H); MS m/z 273.
1
mp 116-8 °C); H NMR (300 MHz, CDCl3) δ 1.16 (t, J ) 7.2
Hz, 3H), 2.35 (s, 3H), 2.75-2.82 (m, 4H), 3.84 (t, J ) 6.9 Hz,
2H), 7.21 (m, 4H); MS m/z 260.
Gen er a l P r oced u r e E. Con d en sa tion of Vin ylogou s
Acid s 6-9 or Ester 10 w ith a n Alk yl- or Ar ylh yd r a zin e.
3-Eth yl-1-(4-flu or oph en yl)-6-ph en yl-7-oxo-4,5,6,7-tetr ah y-
d r o-1H-p yr a zolo[3,4-c]p yr id in e (11) a n d 3-E t h yl-2-(4-
flu or oph en yl)-6-ph en yl-7-oxo-4,5,6,7-tetr ah ydr o-2H-pyr a-
zolo[3,4-c]p yr id in e (35). A solution of NaOMe in MeOH was
prepared by adding Na (127 mg, 5.5 mmol) to anhydrous
MeOH (3 mL). To this was added a solution of compound 6a
3-Hydr oxy-2-oxo-4-pr opion yl-1-(2-m eth ylph en yl)-1,2,5,6-
tetr a h yd r op yr id in e (6g): pale yellow amorphous solid; 65%
yield from 2g; 1H NMR (300 MHz, CDCl3) δ 1.12-1.23 (m, 3H),
2.25 (s, 3H), 2.39-2.50 (m, 2H), 2.74-2.85 (m, 2H), 3.53-3.91
(m, 2H), 7.07-7.30 (m, 4H); MS m/z 259.
4-Acetyl-3-h yd r oxy-1-(4-m eth oxyp h en yl)-2-oxo-1,2,5,6-
tetr a h yd r op yr id in e (7b): yellow amorphous solid; 87% yield

Inhibitors of Human Eosinophil Phosphodiesterase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2275
(2.7 g, 11.0 mmol) in anhydrous EtOH (90 mL) followed by
solid 4-fluorophenylhydrazine hydrochloride (1.95 g, 12.0
mmol). After being stirred at reflux for 20 h the mixture was
cooled to room temperature, concentrated and purified by
chromatography (2:3 ether/hexane) to provide 1.9 g (54%) of a
oxo-4,5,6, 7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (26):
purified by chromatography (1:3 ethyl acetate/hexane) followed
by recrystallization from ethyl acetate/petroleum ether (bp
range 35-60 °C); light brown solid; 29% yield; mp 140-2° C;
1H NMR (250 MHz, CDCl3) δ 2.33 (s, 3H), 2.90 (t, J ) 6.7 Hz,
2H), 3.79 (s, 3H), 4.04 (t, J ) 6.7 Hz, 2H), 6.85-7.55 (m, 8H).
Anal. (C20H18FN3O2) C, H, N.
1
white solid (11): mp 112-5 °C (lit.12 mp 106-8 °C); H NMR
(300 MHz, CDCl3) δ 1.32 (t, J ) 7.6 Hz, 3H), 2.74 (q, J ) 7.6
Hz, 2H), 2.97 (t, J ) 6.7 Hz, 2H), 4.11 (t, J ) 6.7 Hz, 2H),
7.02-7.57 (m, 9H). Anal. (C20H18FN3O) C, H, N.
Further elution of the column with 1:3 ethyl acetate/hexane
provided a white solid which was recrystallized from isopropyl
ether to give 0.5 g (14%) of a white crystalline solid (35): mp
199-200 °C (lit.12 208-210 °C); 1H NMR (300 MHz, CDCl3) δ
1.14 (t, J ) 7.6 Hz, 3H), 2.69 (q, J ) 7.6 Hz, 2H), 2.97 (t, J )
6.5 Hz, 2H), 4.06 (t, J ) 6.5 Hz, 2H), 7.14-7.47 (m, 9H). Anal.
(C20H18FN3O) C, H, N.
1-(4-F lu or op h en yl)-6-(4-m eth oxyp h en yl)-7-oxo-3-p r o-
p yl-4,5,6,7-t et r a h yd r o-1H -p yr a zolo[3,4-c]p yr id in e (27):
purified by chromatography (1:1 ethyl acetate/hexane) followed
by recrystallization from ethyl acetate/pet ether (bp range 35-
1
60 °C); off-white solid; 30% yield; mp 97-8 °C; H NMR (250
MHz, CDCl3) δ 1.01 (t, J ) 7.3 Hz, 3H), 1.66-1.81 (m, 2H),
2.68 (t, J ) 7.6 Hz, 2H), 2.94 (t, J ) 6.7 Hz, 2H), 3.79 (s, 3H),
4.04 (t, J ) 6.7 Hz, 2H), 6.86-7.57 (m, 8H). Anal. (C22H22
FN3O2) C, H, N.
-
1-Cyclopen tyl-3-eth yl-7-oxo-6-ph en yl-4,5,6,7-tetr ah ydr o-
1H -p yr a zolo[3,4-c]p yr id in e (14) a n d 2-Cyclop en t yl-3-
eth yl-7-oxo-6-p h en yl-4,5,6,7-tetr a h yd r o-2H-p yr a zolo[3,4-
c]p yr id in e (36): using general procedure E, upon cooling a
precipitate formed from the reaction mixture and was filtered
and washed with hexane to give a white crystalline solid (36);
3-Bu t yl-1-(4-flu or op h en yl)-7-oxo-6-p h en yl-4,5,6,7-t et -
r a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (28): purified by chro-
matography (1:3 ether/hexane) followed by recrystallization
from petroleum ether; white needles; 44% yield; mp 95-6 °C;
1H NMR (300 MHz, CDCl3) δ 0.96 (t, J ) 7.3 Hz, 3H), 1.39-
1.47 (m, 2H), 1.64-1.74 (m, 2H), 2.70 (t, J ) 7.7 Hz, 2H), 2.96
(t, J ) 6.6 Hz, 2H), 4.10 (t, J ) 6.6 Hz, 2H), 7.02-7.09 (m,
1
28% yield; mp 145-7 °C; H NMR (250 MHz, CDCl3) δ 1.22
(t, J ) 7.7 Hz, 3H), 1.63-2.30 (m, 8H), 2.69 (q, J ) 7.6 Hz,
2H), 2.86 (t, J ) 6.5 Hz, 2H), 3.98 (t, J ) 6.5 Hz, 2H), 4.59
(pentet, J ) 7.9 Hz, 1H), 7.22-7.44 (m, 5H).
2H), 7.20-7.40 (m, 5H), 7.50-7.55 (m, 2H). Anal. (C22H22
-
FN3O) C, H, N.
Gen er a l P r oced u r e F . F u sion of 10 w ith Alk yl or Ar yl
Hyd r a zin es. 1-Cyclop en tyl-3-eth yl-6-(4-m eth oxyp h en yl)-
7-oxo-4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (12).
A 500 mL round-bottom flask was charged with compound 10b
(35.51 g, 122.7 mmol), anhydrous THF (200 mL), and cyclo-
pentylhydrazine hydrochloride (20.13 g, 147.3 mmol). The
mixture was stirred with heating until homogeneous, and the
THF was then removed by distillation. The mixture was then
purged with N2 to blow off HCl gas (as evidenced by holding
moistened pH paper in fume trail). The resulting dark orange
oil was fused at 120 °C. After 2 h the oil was allowed to cool
to ambient temperature at which time it solidified to a brown
solid lump. Purification by chromatography (1:3 ethyl acetate/
hexane) provided 38.9 g (93%) of a white solid: mp 103-4 °C;
1H NMR (400 MHz, CDCl3) δ 1.23 (t, J ) 7.7 Hz, 3H), 1.59-
1.63 (m, 2H), 1.85-1.91 (m, 2H), 2.01-2.07 (m, 4H), 2.64 (q,
J ) 7.7 Hz, 2H), 2.85 (t, J ) 6.7 Hz, 2H), 3.80 (s, 3H), 3.92 (t,
J ) 6.7 Hz, 2H), 5.65 (pentet, J ) 7.9 Hz, 1H), 6.92 (d, J ) 8.7
Hz, 2H), 7.22 (d, J ) 8.7 Hz, 2H). Anal. (C20H25N3O2) C, H,
N.
The filtrate was purified by chromatography (1:1 ethyl
acetate/hexane); white crystalline solid (14); 52% yield; mp
68-9 °C; 1H NMR (250 MHz, CDCl3) δ 1.25 (t, J ) 7.7 Hz,
3H), 1.59-2.17 (m, 8H), 2.69 (q, J ) 7.6 Hz, 2H), 2.89 (t, J )
6.6 Hz, 2H), 4.00 (t, J ) 6.7 Hz, 2H), 5.69 (pentet, J ) 7.9 Hz,
1H), 7.22-7.44 (m, 5H). Anal. (C19H23N3O) C, H, N.
3-Eth yl-1-(4-flu or op h en yl)-6-(3-m eth oxyp h en yl)-7-oxo-
4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (16): puri-
fied by chromatography (1:1 ethyl acetate/hexane) followed by
recrystallization from ethyl acetate/pentane; white crystals;
1
68% yield; mp 139-40 °C; H NMR (300 MHz, CDCl3) δ 1.31
(t, 7.8 Hz, 3H), 2.74 (q, J ) 7.6 Hz, 2H), 2.96 (t, J ) 6.7 Hz,
2H), 3.78 (s, 3H), 4.09 (t, J ) 6.7 Hz, 2H), 6.76-7.56 (m, 8H).
Anal. (C21H20FN3O2) C, H, N.
3-Eth yl-1-(4-flu or op h en yl)-6-(2-m eth oxyp h en yl)-7-oxo-
4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (17): puri-
fied by chromatography (1:3 ethyl acetate/hexane) followed by
recrystallization from ethyl acetate/pentane; pale yellow crys-
talline solid; 31% yield; mp 103-4 °C; 1H NMR (300 MHz,
CDCl3) δ 1.32 (t, J ) 7.7 Hz, 3H), 2.74 (q, J ) 7.6 Hz, 2H),
2.95 (t, J ) 6.7 Hz, 2H), 3.83 (s, 3H), 3.80-4.25 (m, 2H), 6.94-
7.59 (m, 8H). Anal. (C21H20FN3O2) H, N; C: calcd, 69.03;
found, 69.70.
3-Eth yl-1-(4-flu or op h en yl)-6-(4-m eth oxyp h en yl)-7-oxo-
4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (15): puri-
fied by chromatography (1:4 ethyl acetate/hexane) followed by
recrystallization from ether/pentane; white needles; 80% yield;
1
mp 108-9° C; H NMR (300 MHz, CDCl3) δ 1.32 (t, J ) 7.6
3-E t h yl-1-(4-flu or op h en yl)-6-(4-m et h ylp h en yl)-7-oxo-
4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (18): puri-
fied by chromatography (1:3 ethyl acetate/hexane); yellow solid;
Hz, 3H), 2.75 (q, J ) 7.6 Hz, 2H), 2.96 (t, J ) 6.7 Hz, 2H),
3.79 (s, 3H), 4.05 (t, J ) 6.7 Hz, 2H), 6.89 (d, J ) 9.0 Hz, 2H),
7.06 (t, J ) 8.7 Hz, 2H), 7.21 (d, J ) 9.0 Hz, 2H), 7.53 (dd, J
) 4.9 and 9.0 Hz, 2H). Anal. (C21H20FN3O2) C, H, N.
1-Cyclopen tyl-3-eth yl-6-(3-m eth oxyph en yl)-7-oxo-4,5,6,7-
tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (21): purified by
chromatography (1:3 ethyl acetate/hexane) followed by recrys-
tallization from ether/pentane; white crystals; 71% yield; mp
63-4 °C; 1H NMR (300 MHz, CDCl3) δ 1.24 (t, J ) 7.6 Hz,
3H), 1.61-2.11 (m, 8H), 2.67 (q, J ) 7.6 Hz, 2H), 2.87 (t, J )
6.7 Hz, 2H), 3.82 (s, 3H), 3.97 (t, J ) 6.7 Hz, 2H), 5.61-5.71
(pentet, 1H), 6.79-7.34 (m, 4H). Anal. (C20H25N3O2) C, H,
N.
1
23% yield; mp 134-6 °C; H NMR (300 MHz, CDCl3) δ 1.31
(t, J ) 7.6 Hz, 3H), 2.33 (s, 3H), 2.73 (q, J ) 7.6 Hz, 2H), 2.95
(t, J ) 6.7 Hz, 2H), 4.07 (t, J ) 6.7 Hz, 2H), 7.02-7.08 (m,
2H), 7.18 (s, 4H), 7.50-7.55 (m, 2H). Anal. (C21H20FN3O) C,
H, N.
3-E t h yl-1-(4-flu or op h en yl)-6-(3-m et h ylp h en yl)-7-oxo-
4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (19): puri-
fied by chromatography (1:3 ether/hexane) followed by recrys-
tallization from ether/pentane; yellow solid; 52% yield; mp
94-5 °C; 1H NMR (300 MHz, CDCl3) δ 1.31 (t, J ) 7.6 Hz,
3H), 2.34 (s, 3H), 2.74 (q, J ) 7.6 Hz, 2H), 2.96 (t, J ) 6.7 Hz,
2H), 4.08 (t, J ) 6.7 Hz, 2H), 7.03-7.57 (m, 8H). Anal.
(C21H20FN3O) C, H; N: calcd, 12.02; found, 11.52.
1-C y c lo p e n t y l-3-e t h y l-6-(2-m e t h y lp h e n y l)-7-o x o -
4,5,6,7- tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (22): puri-
fied by chromatography (1:9 ethyl acetate/hexane) followed by
recrystallization from ether/pentane; white crystals; 41% yield;
3-E t h yl-1-(4-flu or op h en yl)-6-(2-m et h ylp h en yl)-7-oxo-
4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (20): puri-
fied by chromatography (1:4 ethyl acetate/hexane) followed by
recrystallization from isopropyl ether; pale orange crystals;
1
mp 134-5 °C; H NMR (300 MHz, CDCl3) δ 1.25 (t, J ) 7.6
Hz, 3H), 1.61-2.10 (m, 8H), 2.28 (s, 3H), 2.67 (q, J ) 7.6 Hz,
2H), 2.79-3.00 (m, 2H), 3.69-3.77 (m, 1H), 3.91-4.00 (m, 1H),
5.59-5.70 (pentet, 1H), 7.17-7.31 (m, 4H). Anal. (C20H25N3O)
C, H, N.
1
33% yield; mp 141-2 °C; H NMR (300 MHz, CDCl3) δ 1.33
(t, J ) 7.7 Hz, 3H), 2.26 (s, 3H), 2.75 (q, J ) 7.6 Hz, 2H), 2.88-
3.08 (m, 2H), 3.80-3.86 (m, 1H), 4.02-4.11 (m, 1H), 7.02-
7.59 (m, 8H). Anal. (C21H20FN3O) C, H, N.
1-Cycloh exyl-3-eth yl-6-(3-m eth oxyph en yl)-7-oxo-4,5,6,7-
tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (23): purified by
1-(4-F lu or op h en yl)-6-(4-m et h oxyp h en yl)-3-m et h yl-7-

2276 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Duplantier et al.
chromatography (1:1 ethyl acetate/hexane); 48% yield; colorless
oil; 1H NMR (250 MHz, CDCl3) δ 1.25 (t, J ) 7.8 Hz, 3H), 1.25-
2.05 (m, 10H), 2.70 (q, J ) 7.6 Hz, 2H), 2.90 (t, J ) 7.2 Hz,
2H), 3.80 (s, 3H), 3.95 (t, J ) 7.2 Hz, 2H), 5.05-5.20 (m, 1H),
6.75-7.33 (m, 4H). Anal. (C21H27N3O2) C, H, N.
1-Cyclobu tyl-3-eth yl-6-(3-m eth oxyph en yl)-7-oxo-4,5,6,7-
tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (24): purified by
chromatography (1:4 ethyl acetate/hexane); 47% yield; colorless
oil; 1H NMR (300 MHz, CDCl3) δ 1.25 (t, J ) 7.7 Hz, 3H), 1.69-
2.04 (m, 2H), 2.33-2.44 (m, 2H), 2.69 (q, J ) 7.6 Hz, 2H), 2.60-
2.73 (m, 2H), 2.87 (t, J ) 6.7 Hz, 2H), 3.82 (s, 3H), 3.96 (t, J
) 6.7 Hz, 2H), 5.73-5.79 (m, 1H), 6.79-7.33 (m, 4H). Anal.
(C19H23N3O2) C, H; N: calcd, 12.91; found, 12.34.
1-Cyclop en t yl-3-et h yl-7-oxo-6-(2-p yr id yl)-4,5,6,7-t et -
r a h yd r o-1H -p yr a zolo[3,4-c]p yr id in e (32): purified by chro-
matography (1:9 ethyl acetate/hexane); gold oil; 79% yield; 1H
NMR (300 MHz, CDCl3) δ 1.23 (t, J ) 7.6 Hz, 3H), 1.61-2.11
(m, 8H), 2.66 (q, J ) 7.6 Hz, 2H), 2.84 (t, J ) 6.6 Hz, 2H),
4.29 (t, J ) 6.6 Hz, 2H), 5.64 (pentet, J ) 7.8 Hz, 1H), 7.09 (t,
J ) 6.1 Hz, 1H), 7.66-7.80 (m, 2H), 8.44 (broad s, 1H); HRMS
calcd for C18H23N4O 311.187 17, found 311.187 52.
1-Cyclop en t yl-3-et h yl-7-oxo-6-(3-p yr id yl)-4,5,6,7-t et -
r a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (33): purified by chro-
matography (1:1 ethyl acetate/hexane); pale yellow oil; 95%
1
yield; H NMR (300 MHz, CDCl3) δ 1.23 (t, J ) 7.6 Hz, 3H),
1.60-2.09 (m, 8H), 2.65 (q, J ) 7.6 Hz, 2H), 2.90 (t, J ) 6.7
Hz, 2H), 4.02 (t, J ) 6.6 Hz, 2H), 5.60 (pentet, J ) 7.8 Hz,
1H), 7.33-7.38 (m, 1H), 7.73-7.76 (m, 1H), 8.47 (broad s, 1H),
8.62 (broad s, 1H); HRMS calcd for C18H23N4O 311.18717,
found 311.1872.
3-E t h yl-6-(3-m et h oxyp h en yl)-7-oxo-1-(3-su lfola n yl)-
4,5,6,7-tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (25): puri-
fied by chromatography (1:3 ethyl acetate/hexane); 40% yield;
1
pale yellow oil; H NMR (300 MHz, CDCl3) δ 1.25 (t, J ) 7.6
Hz, 3H), 2.60-2.77 (m, 4H), 2.89 (t, J ) 6.7 Hz, 2H), 3.07-
3.18 (m, 1H), 3.46-3.74 (m, 3H), 3.82 (s, 3H), 4.00 (t, J ) 6.6
Hz, 2H), 6.07-6.18 (m, 1H), 6.70-6.91 (m, 3H), 7.30-7.37 (m,
1H). Anal. (C19H23N3O4S) C, H; N: calcd, 10.79; found, 9.82.
6-(3-Ben zyl)-1-cyclopen tyl-3-eth yl-7-oxo-4,5,6,7-tetr ah y-
d r o-1H-p yr a zolo[3,4-c]p yr id in e (34). To a stirred solution
of compound 13 (0.30 g, 1.29 mmol) in anhydrous THF (5 mL)
was added 60% NaH in mineral oil (77 mg, 1.93 mmol). The
mixture was heated at reflux for 1h and then cooled to room
temperature. Benzyl bromide was added and the resulting
mixture was heated at reflux for 2 h, then cooled to room
temperature and MeOH (few drops) was added. Concentration
followed by chromatography (1:4 ethyl acetate/hexane) pro-
vided 385 mg (92%) of a colorless opaque oil: 1H NMR (250
MHz, CDCl3) δ 1.25 (t, J ) 7.5 Hz, 3H), 1.60-2.15 (m, 8H),
2.55-2.70 (m, 4H), 3.50 (t, J ) 7.2 Hz, 2H), 4.70 (s, 2H), 5.70
(pentet, J ) 7.8 Hz, 1H), 7.25-7.40 (m, 5H). Anal. (C20H25N3O)
C, H, N.
Gen er a l P r oced u r e G. Dep r ot ect ion of t h e La ct a m
Nitr ogen . 1-Cyclopen tyl-3-eth yl-7-oxo-4,5,6,7-tetr ah ydr o-
1H-p yr a zolo[3,4-c]p yr id in e (13). A 2 L flask was charged
with compound 12 (30.85 g, 90.0 mmol) and CH3CN (750 mL).
The pale yellow solution was cooled to 0 °C, and a solution of
ammonium cerium nitrate (150 g) in water (500 mL) was
slowly added over 15 min. The solution turned red/brown
during the addition. After 1 h of stirring, the solution was
diluted with cold water (500 mL) and extracted 4× with cold
ethyl acetate. The combined organics were washed 2× with
cold 50% saturated NaHCO3 at which time an emulsion
formed. The emulsion was broken up by adding solid NaCl
and was then washed with 10% NaSO3 and brine, dried over
Na2SO4, and concentrated to a dark brown oil. Purification
by chromatography (gradient of 1:1 ethyl acetate/hexane to
neat ethyl acetate) provided 11.6 g (55%) of a yellow crystalline
solid: mp 145-6 °C; 1H NMR (300 MHz, CDCl3) δ 1.21 (t, J )
7.6 Hz, 3H), 1.62-1.71 (m, 2H), 1.83-1.87 (m, 2H), 1.88-2.12
(m, 4H), 2.62 (q, J ) 7.6 Hz, 2H), 2.72 (t, J ) 6.8 Hz, 2H),
3.51 (dt, J ) 2.8 and 6.8 Hz, 2H), 5.61 (pentet, J ) 7.8 Hz,
1H), 5.66 (broad s, 1H). Anal. (C13H19N3O) C, H, N.
Gen er a l P r oced u r e H. Ar yla tion of 13. 1-Cyclop en tyl-
3-eth yl-7-oxo-6-(2-th iop h en e)-4,5,6,7-tetr a h yd r o-1H-p yr a -
zolo[3,4-c]p yr id in e (29). A mixture of compound 13 (0.15
g, 0.64 mmol), copper powder (82 mg, 1.29 mmol), 2-iodo-
thiophene (0.135 mg, 0.64 mmol), and K2CO3 (89 mg, 0.64
mmol) were combined in a vial and stirred at 150 °C. After
18 h the mixture was allowed to cool to ambient temperature
and was purified by chromatography (1:3 ether/hexane) fol-
lowed by recrystallization from pentane to give 157 mg (49%)
of white needles: mp 59-60 °C; 1H NMR (300 MHz, CDCl3) δ
1.24 (t, J ) 7.6 Hz, 3H), 1.66-2.11 (m, 8H), 2.66 (q, J ) 7.6
Hz, 2H), 2.92 (t, J ) 6.8 Hz, 2H), 4.14 (t, J ) 6.8 Hz, 2H), 5.68
(pentet, J ) 7.8 Hz, 1H), 6.77 (dd, J ) 1.4 and 4.0 Hz, 1H),
6.93 (dd, J ) 3.8 and 5.6 Hz, 1H), 7.02 (dd, J ) 1.3 and 5.6
Hz, 1H). Anal. (C17H21N3OS) C, H, N.
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1
yield; H NMR (300 MHz, CDCl3) δ 1.23 (t, J ) 7.6 Hz, 3H),
1.59-2.11 (m, 8H), 2.66 (q, J ) 7.6 Hz, 2H), 2.87 (t, J ) 6.7
Hz, 2H), 4.03 (t, J ) 6.7 Hz, 2H), 5.67 (pentet, J ) 7.8 Hz,
1H), 7.17 (dd, J ) 1.6 and 3.0 Hz, 1H), 7.26-7.31 (m, 2H);
HRMS calcd for C17H22N3OS 316.1483, found 316.1475.
6-(3-Ch lor op h en yl)-1-cyclop en tyl-3-eth yl-7-oxo-4,5,6,7-
tetr a h yd r o-1H-p yr a zolo[3,4-c]p yr id in e (31): purified by
chromatography (1:19 ethyl acetate/hexane) followed by re-
crystallization from ether/pentane; colorless crystals; 52%
1
yield; mp 71-2 °C; H NMR (300 MHz, CDCl3) δ 1.24 (t, J )
7.6 Hz, 3H), 1.55-2.10 (m, 8H), 2.65 (q, J ) 7.6 Hz, 2H), 2.88
(t, J ) 6.6 Hz, 2H), 3.97 (t, J ) 6.6 Hz, 2H), 5.62 (pentet, J )
7.8 Hz, 1H), 7.21-7.35 (m, 4H). Anal. (C19H22ClN3O) C, H,
N.

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