Palladium-catalyzed cross-coupling reactions for the synthesis of 6,8- disubstituted 1,7-naphthyridines: A novel class of potent and selective phosphodiesterase type 4D inhibitors

Article abstract of DOI:10.1021/jm991094u

Recently, four subtypes of the human phosphodiesterase type 4 (PDE4A-D) enzyme have been described. So far, only very few PDE4 subtype-selective inhibitors are known. Herein, we describe the synthesis of 6,8-disubstituted 1,7-naphthyridines and their characterization as potent and selective inhibitors of PDE4D which suppress the oxidative burst in human eosinophils with IC₅₀ values as low as 0.7 nM. SAR development and the extended use of palladium-catalyzed cross-coupling reactions led to compound 11 which inhibited human PDE4D with an IC₅₀ value of 1 nM. Thus, compound 11 was 55, 175, and 1000 times more potent in inhibiting PDE4D over PDE4B, PDE4A, and PDE4C. In a Brown Norway rat model of allergic asthma, compound 11 when given by the oral route (1 mg/kg) reduced by more than 50% the influx of eosinophils, T-cells, and neutrophils into bronchoalveolar lavage fluid (BALF) samples obtained from antigen-challenged animals. Thus, PDE4D- selective inhibitors of the 1,7-naphthyridine class have the potential as an oral therapy for treating asthma.

Full text of DOI:10.1021/jm991094u

J . Med. Chem. 2000, 43, 675-682
675
P a lla d iu m -Ca ta lyzed Cr oss-Cou p lin g Rea ction s for th e Syn th esis of
6,8-Disu bstitu ted 1,7-Na p h th yr id in es: A Novel Cla ss of P oten t a n d Selective
P h osp h od iester a se Typ e 4D In h ibitor s
Rene Hersperger,* Katharine Bray-French,§ Lazzaro Mazzoni, and Thomas Mu¨ller
Novartis Pharma AG, Research, CH-4002 Basel, Switzerland
Received J une 4, 1999
Recently, four subtypes of the human phosphodiesterase type 4 (PDE4A-D) enzyme have been
described. So far, only very few PDE4 subtype-selective inhibitors are known. Herein, we
describe the synthesis of 6,8-disubstituted 1,7-naphthyridines and their characterization as
potent and selective inhibitors of PDE4D which suppress the oxidative burst in human
eosinophils with IC50 values as low as 0.7 nM. SAR development and the extended use of
palladium-catalyzed cross-coupling reactions led to compound 11 which inhibited human PDE4D
with an IC50 value of 1 nM. Thus, compound 11 was 55, 175, and 1000 times more potent in
inhibiting PDE4D over PDE4B, PDE4A, and PDE4C. In a Brown Norway rat model of allergic
asthma, compound 11 when given by the oral route (1 mg/kg) reduced by more than 50% the
influx of eosinophils, T-cells, and neutrophils into bronchoalveolar lavage fluid (BALF) samples
obtained from antigen-challenged animals. Thus, PDE4D-selective inhibitors of the 1,7-
naphthyridine class have the potential as an oral therapy for treating asthma.
In tr od u ction
diverse cellular distribution of PDE4 subtypes remains
to be established. The most prominent side effects
related to the use of PDE4 inhibitors are nausea and
emesis.1,6 In addition, there is still concern about other
potential untoward effects such as immunosuppression,
metabolic disturbances, increased gastric acid secretion,
and effects on the central nervous system.1 Although
the mechanism related to the induction of nausea and
emesis is not fully understood, one hypothesis is that
binding of inhibitors to the so-called rolipram high-
affinity binding site is responsible for these effects so
that potent PDE4 inhibitors with relatively low affinity
for the rolipram high-affinity binding site should display
good antiinflammatory activity with a decreased poten-
tial for side effects. Apparently, SB207499 (Ariflo; Chart
1), a PDE4 inhibitor currently in phase III clinical trials
for the indications asthma and COPD, was selected
based on an optimized ratio of PDE4 catalytic activity
relative to affinity to the rolipram high-affinity binding
site.7 However, in clinical trials SB207499 still induced
emesis at the first and/or second doses, albeit this effect
apparently disappeared with continued treatment.8
Thus, prediction of emesis in humans based on a
relatively low ratio of PDE4 inhibition versus rolipram
binding remains unsatisfactory.
Modulation of the intracellular second messenger
cyclic 3′,5′-adenosine monophosphate (cAMP) by phos-
phodiesterase type 4 (PDE4) inhibitors represents a
promising new approach for the treatment of chronic
inflammatory diseases such as asthma, COPD, and
rheumatoid arthritis.1,2 Originally fuelled mainly by
preclinical findings, this approach has received sub-
stantial support from recent clinical studies, and the
search for novel PDE4 inhibitors is currently one of the
most active fields in the pharmaceutical industry.3
PDE4 belongs to a family of at least seven structurally,
biochemically, and pharmacologically distinct isoen-
zymes that catalyze the hydrolysis of cAMP or 3′,5′-
guanosine monophosphate (cGMP) to the corresponding
inactive 5′-nucleotide products.4 Predominantly located
in lymphocytes, monocytes, macrophages, neutrophils,
eosinophils, and mast cells, but also in endothelial cells
and epithelial cells, PDE4 seems to be the most impor-
tant member of the PDE family in regulating immune
and inflammatory responses.1,5 Recently, different iso-
genes and splice variants of human PDE4 have been
described.4 The four gene products (PDE4A-D) are
characterized by their selective, high-affinity hydrolysis
of cAMP and sensitivity to inhibition by rolipram.
According to an analysis of a number of human periph-
eral blood cells and cell lines, mRNAs of PDE4A,B,D
are expressed in the majority of cell populations of the
immune system.5 In particular, PDE4D was promi-
nently expressed in eosinophils, whereas PDE4C mRNA
was not detected in any of the tested inflammatory cell
lines. Nevertheless, the functional significance of the
Our research efforts in finding novel PDE4 inhibitors
were based upon a different approach. We wanted to
find orally active, second-generation PDE4 inhibitors
displaying selectivity for one of the subtypes of human
PDE4, in our case PDE4D, and thereby answer the
question if such compounds retain the broad antiin-
flammatory properties of ‘universal’, non-PDE4 subtype-
selective inhibitors. Furthermore, one might argue that,
at least conceptually, PDE4 subtype-specific inhibitors
may have a more favorable side effect profile than the
current ‘universal’ PDE4 isozyme-selective compounds.9
Indeed, SB207499, the most advanced PDE4 inhibitor
* To whom correspondence should be addressed. Tel: +41 61 324
31 86. Fax: +41 61 324 30 36. E-mail: rene.hersperger@pharma.
novartis.com.
§ Current address: RCC Registration and Consulting Co. Ltd., CH-
4452 Itingen, Switzerland.
10.1021/jm991094u CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/01/2000

676 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Hersperger et al.
Ch a r t 1
Sch em e 1a
in clinical trials, is one of the very few compounds which
is claimed to be moderately selective toward inhibition
of PDE4D (relative to PDE4A-C).2,7 PDE4 subtype-
selective compounds could also serve as useful tools to
answer the still open question of the functional signifi-
cance of the diverse cellular distribution of PDE4
subtypes.
The majority of the currently described PDE4 inhibi-
tors are structural variants of rolipram and contain the
characteristic 3,4-dialkoxybenzyl substructure (Chart 1).
Xanthine-type molecules form another class of PDE4
inhibitors. Alternatively, nitrogen-containing bicyclic
aromatic ring systems, e.g. quinolines, heteroquinolines,
or nitraquazones, emerged as very interesting ‘tem-
plates’ for PDE4 inhibitors. Isoquinolines represent
another novel potent type of PDE4-selective inhibitor.10
Recent reviews summarize the currently known differ-
ent chemical classes of selective PDE4 inhibitors.3
Herein, we describe the discovery of 1,7-naphthyri-
dines as a novel class of potent and PDE4D subtype-
selective inhibitors. Palladium-catalyzed cross-coupling
reactions11 were used as powerful synthetic tools for the
preparation of a variety of 6,8-disubstituted 1,7-naph-
thyridines starting from a common, appropriately func-
tionalized intermediate. We report the SAR of these
compounds with respect to PDE4A-D inhibition. Since
eosinophils are believed to be key effector cells in the
pathogenesis of asthma,12 inhibition of the fMLP-
induced oxidative burst in human eosinophils was used
to evaluate the cellular activity of these PDE4D-selec-
tive compounds. Finally, we report data for the most
potent compounds in an in vivo model of allergic
asthma.
a
Reagents and conditions: (a) (CH3)3SiCN, then diethylcar-
bamyl chloride, toluene, 60 °C; (b) HBr in acetic acid (33%), 25
°C; (c) Pd(dba)2, (C6H5)3P, K2CO3, DMF/H2O/toluene, 100 °C; (d)
NaNO2, CF3SO3H, DMF, 25 °C; (e) Pd(dba)2, (C6H5)3P, 2 N Na2CO3
or 2 N K2CO3, THF or DMF, 80 °C; (f) Pd(dba)2, (C6H5)3P, LiCl,
DMF, 80-100 °C; (g) (C2H5)3SiH, [1,1′-bis(diphenylphosphino)-
ferrocene]dichloropalladium(ll), DMF, 60 °C; (h) 1-methylpipera-
zine, CO, (C2H5)3N, Pd(dba)2, DMF, 80 °C.
bromide, gave intermediate 3. Subsequent attachment
of 3-substitued phenyl rings to position 8 of the 1,7-
naphthyridine by Suzuki cross-coupling reaction led to
compounds 4a -c. Endeavors to convert the amino group
into a leaving group at position 6 of the 1,7-naphthyri-
dine which would allow us to apply further palladium-
catalyzed cross-coupling reactions were less straight-
forward. All attempts to replace the 6-amino group by
an iodo, bromo, or chloro atom via a Sandmeyer-type
reaction failed. However, using similar reaction condi-
tions and trifluoromethanesulfonic acid as a cosolvent
allowed us to directly transform amines 4a -c into the
corresponding triflates 5a -c. Subsequent carbon-
carbon bond formation either via Suzuki or Stille-type
reactions then led to the final products 6-13. The
nonsubstituted compound 14 was prepared via pal-
ladium-catalyzed reduction of triflate 5a using triethyl-
silane as reagent.16 Finally, palladium-catalyzed car-
bonylation of triflate 5a in the presence of 4-methyl-
piperidine as a nucleophile led to the amide 15.17
Str u ctu r e-Activity Rela tion sh ip s a n d Biology.
Most of the SAR around position 6 of the 1,7-naphthyri-
dine was established with the 3-nitrophenyl moiety at
position 8 (Table 1), since we quickly realized that an
appropriately meta-substituted phenyl ring at this
position was a prerequisite for obtaining good PDEA-D
inhibitory activity. Other groups at position 8 such as
phenyl, benzyl, naphthalene, heteroaromatic rings (e.g.
furan-2-yl, thiophen-2-yl or benzothiophen-2-yl), or non-
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of the key intermediate 3
is shown in Scheme 1. 2-Cyano-3-pyridylacetonitrile (2)
was obtained from the N-oxide 1 by a modified Reis-
sert-Heinze reaction.13 This procedure turned out to
be superior to the previously described synthesis14 and
led to regioselective cyanation at position 2 of the
pyridine ring. Cyclization of dinitrile 2 following a
modified procedure15 with hydrogen bromide in acetic
acid, i.e. circumventing the use of gaseous hydrogen

Synthesis of 6,8-Disubstituted 1,7-Naphthyridines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 677
Ta ble 1. Inhibition of PDE4A-D and Subtype Selectivity Profile
IC50 (µM)a
compd
4a
5a
5b
5c
6
7
8
9
10
11
12
13
14
15
R
E
PDE4A
PDE4B
PDE4C
PDE4D
1.6((0.8)
ratiob 4B/4D
NH2
NO2 >10
NO2
Cl
4.6((1.2)
0.31((0.08)
0.46
0.42((0.12)
0.11((0.03)
0.081((0.012)
0.51
>10
2.9
28
9.6
5.8
55
27
64
4.9
21
49
23
21
2.1
4.7
3.9
3.6c
OSO2CF3
OSO2CF3
OSO2CF3
phenyl
3-pyridyl
2-furanyl
2-tolyl
1.8((0.6)
1.4
0.77((0.31)
0.35
0.12((0.09)
9.5
3.8((0.8)
3.2((0.9)
4.3
6.4((2.5)
2.0
0.011((0.003)
0.048
CN
0.072((0.046)
0.002((0.001)
0.003((0.001)
0.008((0.0002)
0.070((0.012)
0.007
NO2
NO2
NO2
NO2
NO2
NO2
Cl
0.61((0.17)
>10
>10
0.89((0.16)
0.34((0.07)
allyl
0.279((0.007) 0.15((0.06)
0.088((0.014) 0.049((0.007)
0.059((0.005) 0.045((0.004)
4-carboxyphenyl
4-carboxyphenyl
4-carboxyphenyl
H
0.068((0.009) 0.001((0.0002)
0.19((0.045)
0.57((0.02)
0.002((0.0002)
0.004((0.001)
4.1((2.3)
CN
NO2 >10
0.14((0.034)
0.085((0.007)
8.8((0.5)
9.3
0.31((0.11)
2.7((0.6)
>10
>10
0.84((0.18)
7.0((0.3)
4-methylpiperazin-1-ylcarbonyl NO2 >10
2.0
SB207499
0.41((0.10)
1.4((0.2)
0.079((0.028)
0.39((0.10)
rolipramd
a
b
Data indicated as IC50 or mean IC50 ( SEM (n ) 2-5). Selectivity ratio indicated as IC50 value in inhibiting PDE4B or c PDE4A
d
divided by IC50 value in inhibiting PDE4D. Racemic. No satisfactory analysis obtained for compounds 4c, 13, and 14 but products were
sufficiently pure according to 1H NMR, HRMS, and HPLC.
aromatic cyclic groups (e.g. morpholin-4-yl) gave sig-
nificantly less active compounds, regardless of the
nature of the 6-substituent (IC50 > 1 µM for PDE4A-
D, data not shown). Despite the optimized 8-(3-nitro-
phenyl)-1,7-naphthyridine scaffold, compounds contain-
ing a small group at position 6 such as 4a and 14 were
only active in the micromolar range in the PDE4A-D
assays. As soon as larger groups were attached to this
position, potency toward inhibition of PDE4B and,
particularly, PDE4D increased dramatically. For in-
stance, replacing the 6-hydrogen atom of compound 14
(PDE4D IC50 ) 4100 nM) by a phenyl ring (compound
6, PDE4D IC50 ) 2 nM) increased the potency against
PDE4D by 2000-fold. Remarkably, compound 6 was
selective toward PDE4D being 55, 175, and 1000 times
more potent on PDE4D than on PDE4B, PDE4A, and
PDE4C, respectively. Aromatic substitution at position
6 was not a prerequisite for achieving potent and
selective PDE4D inhibition. Thus, the allylic compound
10 (PDE4D IC50 ) 7 nM) was only 3.5 times less active
in the PDE4D assay than compound 6, albeit the
selectivity toward PDE4B was decreased by 21-fold.
Even the triflate 5a was still very potent (PDE4D IC50
) 11 nM), indicating that there is some degree of steric
tolerance at position 6. On the other hand, the amide
15 was only weakly active (PDE4D IC50 ) 2000 nM)
demonstrating the subtlety of the SAR around position
6. Heteroaromatic substitution at this position also gave
very potent compounds (7 and 8) with similar selectivity
profiles. A comparison of the IC50 values of compounds
9 and 6 shows that the methyl group at the ortho
position of the phenyl ring not only diminished inhibi-
tory potency toward PDE4D (IC50 ) 70 nM versus 2 nM)
but also almost completely abolished PDE4D/PDE4B
selectivity (5-fold versus 55-fold). The benzoic acid-
substituted 1,7-naphthyridine 11 was the most active
compound found in our series. It selectively inhibited
PDE4D (IC50 ) 1 nM) and was 49-, 68-, and 88-fold less
potent on PDE4B, PDE4A, and PDE4C, respectively.
Thus, compound 11 when compared to SB207499 was
80-fold more potent in inhibiting PDE4D and, particu-
larly, had a significantly better selectivity profile toward
the other PDE4 subtypes. As mentioned above, meta-
substituted phenyl rings at position 8 of the 1,7-
naphthyridine were a prerequisite for achieving potent
PDE4D inhibitory activity. Particularly, electron-with-
drawing substituents such as the nitro, cyano, or chloro
group were best suited. A comparison of the 3′-nitro-
phenyl-substituted compounds 5a and 11 with the
corresponding 3′-chlorophenyl analogues (5b, 12) and
3′-cyanophenyl analogues (5c, 13) shows that 8-(3′-
nitrophenyl)-substituted 1,7-naphthyridines were most
potent in inhibiting PDE4D followed by the 8-(3′-
chlorophenyl) and 8-(3′-cyanophenyl) analogues. The
same rank order could be found with respect to their
selectivity profile toward inhibition of PDE4A-D.
A selection of compounds was also tested against
phosphodiesterase type 3 (PDE3) and for their ability
to compete with the (rat) high-affinity [3H]rolipram
binding site (Table 2).7,18 All molecules were completely
inactive against PDE3. Against rolipram binding, the
compounds measured were about 2-fold more potent in
the rolipram binding assay than in the PDE4D enzyme
assay. Thus, in terms of their ability to inhibit PDE4D
catalytic activity versus their ability to compete for the
high-affinity [3H]rolipram binding site compounds 5a ,
6, and 11-13 were not selective. Nevertheless, they had
a very similar profile to SB207499 which, in our hands,
was also about twice as potent in inhibiting [3H]rolipram
binding versus inhibiting PDE4D catalytic activity. In
contrast and consistent with earlier findings,7 the
reference compound rolipram was significantly more
potent in displacing [3H]rolipram from its binding site
than in inhibiting PDE4A-D (>125-fold).

678 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Hersperger et al.
Ta ble 2. Inhibition of [3H]Rolipram Binding, of fMLP-Induced
Oxidative Burst in Human Eosinophils, and of PDE3
neutrophils in BALF samples obtained from these
animals. In addition, BALF samples also contained
increased levels of both proteins and eosinophil granule-
derived eosinophil peroxidase (EPO). These two proteins
are markers for increased airway microvascular perme-
ability21 and for eosinophil activation at the site of
inflammation, respectively. Whereas the triflate 5a and
the phenyl-substituted compound 6 showed no oral
activity at all, the benzoic acid-substituted 1,7-naph-
thyridines 11-13 when given twice, 1 h before and 24
h after OA challenge (1 mg/kg), potently inhibited
eosinophil influx and production of EPO. Although drug
plasma levels were not measured, this finding indicates
that the benzoic acid moiety was important to convey
oral effect bioavailability. The reference compound
SB207499 was also active in this model, albeit at a
higher concentration and to a lesser extent. Consistent
with its in vitro profile compound 11 showed the
greatest activity and completely blocked eosinophil
influx and EPO production. A deeper analysis of the
BALF samples of animals treated with 11-13 revealed
that additional markers of airway inflammation such
as infiltration of T-lymphocytes and neutrophils and the
release of proteins were also inhibited although to a
smaller extent. Again, compound 11 showed the greatest
effect on these parameters. Thus, the selective PDE4D
inhibitors 11-13 showed potent antiinflammatory ac-
tivity in an in vivo model of allergic asthma.
IC50
compd
4a
5a
6
7
8
9
10
11
12
13
14
rolipram (nM)a
HUEOS (nM)b
PDE3 (µM)c
-
7.4
4.0
41((11)
4((4)
10((10)
3((3)
11((12)
42((13)
3((3)
0.7((0.4)
-
-
>100
>100
-
-
-
-
-
-
-
-
0.6((0.2)
0.8
>100
>100
>100
-
2.4((0.8)
58
-
548((344)
-
15
-
>100
>100
>100
SB207499
40((13)
3.1((0.3)
-
-
rolipramd
a
b
Data indicated as IC50 or mean IC50 ( SEM (n ) 3-4). Data
indicated as mean IC50 ( SEM. For each compound three
independent measurements were done using eosinophils from
three different donors. c n ) 1-2. Racemic.
d
Ta ble 3. Inhibition of OA-Induced Leukocyte Infiltration,
Eosinophil Activation, and Protein Release in BALF from
Actively Sensitized Brown Norway Rats
dosea
compd (mg/kg) eosinophb EPOc T-lymphb neutrophb proteind
5a
6
11
12
13
2 × 1
2 × 1
2 × 1
2 × 1
2 × 1
0
0
0
2
-
-
-
-
-
-
86 ( 4
92 ( 3 59 ( 8
71 ( 4
76 ( 4
49 ( 7
51 ( 14 55 ( 13 23 ( 15 55 ( 6
44 ( 13 92 ( 1 40 ( 16 21 ( 21 33 ( 14
Con clu sion
SB207499 2 × 10 56 ( 10 41 ( 15 40 ( 9
64 ( 8
47 ( 5
a
We have used palladium-catalyzed cross-coupling
reactions to synthesize 6,8-disubstituted 1,7-naphthyri-
dines which constitute a novel class of potent and
selective inhibitors of the PDE4D. In vitro these com-
pounds potently inhibited fMLP-induced oxidative burst
in human eosinophils, a key effector cell in the patho-
genesis of asthma. In contrast to the archetype PDE4
inhibitor rolipram, these PDE4D-selective compounds
showed no preference for competing for the [3H]rolipram
binding site versus PDE4D catalytic activity and, in this
respect, had a similar profile as the reference compound
SB207499. It remains to be shown whether selectivity
for PDE4D over PDE4A-C translates into a reduced
risk of emesis in man. The benzoic acid-substituted 1,7-
naphthyridines 11-13 were orally active in an animal
model of asthma and potently inhibited inflammatory
cell influx, eosinophil activation, and protein release into
BALF from ovalbumin-challenged actively sensitized
Brown Norway rats. Thus, PDE4D subtype-selective
inhibitors from the 1,7-naphthyridine class have the
potential as a novel oral therapy for the treatment of
asthma.
Since BALF is collected only 48 h after allergen challenge, all
compounds were given twice (po), 1 h prior to and 24 h after
allergen exposure. For more details, see Experimental Section.
b
Percentage inhibition of the cell numbers measured in BALF
from allergen-challenged, drug-treated versus allergen-challenged,
vehicle-treated (control) animals. The results obtained are the
mean ( SEM of 5-10 animals/group. c Percentage inhibition of
eosinophil activation measured as EPO activity recovered in BALF
from allergen-challenged, drug-treated versus control animals.
d
Percentage inhibition on protein concentration recovered in
BALF from allergen-challenged, drug-treated versus control ani-
mals.
A panel of compounds was tested for their inhibitory
effect on the fMLP-induced oxidative burst in human
peripheral blood eosinophils19 (Table 2). This assay was
used both to evaluate the propensity of the compounds
to modulate a key effector cell involved in the pathology
of asthma12 and as a means to check the compounds’
ability to penetrate cells. The assay was not used as a
tool to develop SAR. The IC50 values obtained had a
certain degree of variation since each compound was
independently tested in eosinophils derived from blood
from three different donors. This did not allow us to
make a clear potency rank order in this cellular assay.
Nevertheless, all compounds tested potently inhibited
the oxidative burst of human eosinophils, and the
Exp er im en ta l Section
Gen er a l. Melting points were measured with a Buechi
B-540 and are uncorrected. 1H NMR spectra were recorded
on a Bruker DPX-400 (400 MHz) or AM-360 (360 MHz) or a
Varian Gemini-200 (200 MHz) instrument, using residual
solvent protons as references. Mass spectra were recorded on
a VG 70-SE instrument or a MAT 900 mass spectrometer (ESI)
at nominal 6000 resolution. Flash chromatography was carried
out using silica gel (Merck; Kieselgel 60 F254, 230-400 mesh).
Preparative thin-layer chromatography was done using silica
gel 60 F254 PLC plates (Merck; 20 × 20, 0.5 mm) and analytical
TLC was performed using precoated glass plates. AR grade
solvents (Fluka) and commercial reagents (Fluka, Aldrich,
benzoic acid-substituted 1,7-naphthyridine 11 (IC50
)
0.7 nM) was again among the most potent compounds.
To address the question as to whether our PDE4D-
selective compounds had antiinflammatory properties
in vivo, we tested the more promising molecules in the
antigen-induced pulmonary eosinophilia rat model, an
animal model of allergic asthma20 (Table 3). Briefly,
ovalbumin (OA)-sensitized Brown Norway rats were
challenged with aerosolized antigen (OA). This resulted
in an accumulation of eosinophils, T-lymphocytes, and

Synthesis of 6,8-Disubstituted 1,7-Naphthyridines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 679
Lancaster) were used without further purification in all cases.
Brine refers to a saturated aqueous NaCl solution and
concentration procedures were carried out using a rotary
evaporator at water aspirator pressure. Organic extracts were
routinely dried over anhydrous Na2SO4. Pd-catalyzed reactions
were run under an argon atmosphere in degassed solvents.
3-Cya n om eth ylp yr id in e-2-ca r bon itr ile (2). To a stirred
suspension of 3-cyanomethylpyridine N-oxide (10 g, 74.6 mmol)
in toluene (60 mL) was added trimethylsilanecarbonitrile (9.0
g, 90.7 mmol). To this suspension was added dimethylcarbamyl
chloride (8.0 g, 74.4 mmol) and the mixture was stirred for 20
h at 60 °C. The mixture was cooled and diluted with EtOAc
and 1 N K2CO3. The layers were separated and the aqueous
phase was extracted twice with EtOAc. The combined organic
phases were dried and concentrated. Recrystallization of the
crude product from ethanol gave 7.2 g (67%) of product 2: mp
61-63 °C; 1H NMR (200 MHz, CDCl3) δ 4.08 (s, 2H), 7.62 (dd,
J ) 5.0, 9.0 Hz, 1H), 8.05 (d, J ) 9.0 Hz, 1H), 8.73 (d, J ) 5.0
Hz, 1H). Anal. (C8H5N3) C, H, N.
in a 2:1 mixture of DMF/trifluoromethanesulfonic acid (30 mL)
was carefully added in several portions sodium nitrite (2.07
g, 30.0 mmol). The solution was stirred for 3 h at 25 °C. The
reaction mixture was diluted with EtOAc and washed with
water, 2 N NaOH, and water again. The organic phase was
dried and concentrated. The solid was triturated in ether (120
mL) and filtered and the filtrate concentrated. The remaining
residue was purified by chromatography (toluene) to afford 3.6
g of product (60%): mp 106-108 °C; 1H NMR (360 MHz,
DMSO-d6) δ 7.91 (dd, J ) 8.4, 8.4 Hz, 1H), 7.99 (dd, J ) 5.4,
7.2 Hz, 1H), 8.35 (s, 1H), 8.44 (d, broad, J ) 8.4 Hz, 1H), 8.65
(d, J ) 7.2 Hz, 1H), 8.69 (d, J ) 8.4 Hz, 1H), 9.06 (s, broad,
1H), 9.25 (d, J ) 5.4 Hz, 1H); MS (m/e) [M + H]+ 400. Anal.
(C15H8F3N3O5S) C, H, F, N, O, S.
Tr iflu or om eth a n esu lfon ic Acid 8-(3-Ch lor op h en yl)-
[1,7]n a p h th yr id in -6-yl Ester (5b). Purified by chromatog-
raphy (10:0.5 toluene/acetone): pale brown solid, 34% yield.
Analytical sample recrystallized from 2-propanol: mp 96-97
1
°C; H NMR (360 MHz, CDCl3) δ 7.45-7.51 (m, 2H), 7.55 (s,
1H), 7.70 (dd, J ) 5.4, 9.0 Hz, 1H), 8.16-8.19 (m, 1H), 8.27
(d, broad, J ) 9.0 Hz, 1H), 8.31 (s, broad, 1H), 9.13 (dd, J )
0.3, 5.4 Hz, 1H); HRMS [M + H]+ found 388.9980, calcd for
8-Br om o-[1,7]n a p h th yr id in -6-yla m in e (3). To a solution
of 33% HBr in acetic acid (120 mL) was added in portions
3-cyanomethylpyridine-2-carbonitrile (2) (20 g, 140 mmol) so
that the reaction temperature did not exceed 30-35 °C. The
reaction mixture was stirred at ambient temperature for 1 h.
The suspension was cooled in an ice bath, filtered, and washed
with EtOAc. The solid was triturated in boiling MeOH (400
mL), cooled to ambient temperature, and filtered. The finely
powdered material was taken up in water (400 mL) and
saturated Na2CO3 added until pH 8 was reached. The suspen-
sion was vigorously stirred for 30 min and filtered to afford
23.1 g (74%) of the title compound 3: mp 187 °C dec; 1H NMR
(360 MHz, DMSO-d6) δ 6.45 (s, broad, 2H), 6.62 (s, 1H), 7.50
(dd, J ) 5.4, 8.4 Hz, 1H), 8.04 (d, J ) 8.4 Hz, 1H), 8.62 (d,
broad, J ) 5.4 Hz, 1H). Anal. (C8H6BrN3) C, H, Br, N.
Gen er a l P r oced u r e A. Su zu k i Cou p lin gs of 8-Br om o-
[1,7]n a p h th yr id in -6-yla m in e (3): 8-(3-Nitr op h en yl)-[1,7]-
n a p h th yr id in -6-yla m in e (4a ). To a solution of 3 (5.0 g, 22.3
mmol) in toluene/DMF (1:1) (60 mL) were added bis(diben-
zylideneacetone)palladium (256 mg, 0.44 mmol), triphenylphos-
phine (460 mg, 1.75 mmol), 3-nitrophenylboronic acid (3.91 g,
23.4 mmol), and K2CO3 (6.2 g, 44.9 mmol), dissolved in 15 mL
of water. The mixture was stirred under reflux for 4 h. The
reaction mixture was diluted with EtOAc, filtered through
Celite, and washed with water. The organic layer was dried
and concentrated. The solid was triturated in ether and filtered
to afford 5.4 g of product (90%). An analytical sample was
recrystallized from 2-propanol: mp 228-229 °C; 1H NMR (360
MHz, DMSO-d6) δ 6.36 (s, 2H), 6.74 (s, 1H), 7.49 (dd, J ) 4.2,
6.6 Hz, 1H), 7.80 (dd, J ) 8.4, 8.4 Hz, 1H), 8.10 (d, J ) 8.4 Hz,
1H), 8.33 (dd, J ) 2.4, 8.4 Hz, 1H), 8.59 (d, J ) 6.6 Hz, 1H),
8.62 (d, J ) 4.2 Hz, 1H), 8.93 (d, J ) 2.4 Hz, 1H); HRMS [M
+ H]+ found 267.0881, calcd for C14H11N4O2 267.0882. Anal.
(C14H10N4O2) C, H, N, O.
C
15H9ClF3N2O3S 388.9981. Anal. (C15H8ClF3N2O3S) C, H, Cl,
F, N, O, S.
Tr iflu or om et h a n esu lfon ic Acid 8-(3-Cya n op h en yl)-
[1,7]n a p h th yr id in -6-yl Ester (5c). Purified by chromatog-
raphy (10:0.5 toluene/acetone): white solid, 50% yield; mp
1
102-104 °C; H NMR (360 MHz, CDCl3) δ 7.61 (s, 1H), 7.66
(dd, J ) 7.2, 7.2 Hz, 1H), 7.75 (dd, J ) 5.4, 8.4 Hz, 1H), 7.81
(d, J ) 7.2 Hz, 1H), 8.32 (d, broad, J ) 8.4 Hz, 1H), 8.54 (d, J
) 7.2 Hz, 1H), 7.74 (s, broad, 1H), 9.15 (dd, J ) 1.8, 5.4 Hz,
1H); MS (m/e) [M + H]+ 380. Anal. (C16H8F3N3O3S) C, H, F,
N, O, S.
Gen er a l P r oced u r e C. Stille Cou p lin gs of Tr iflu or o-
m eth a n esu lfon ic Acid 8-(3-Nitr op h en yl)-[1,7]n a p h th yr i-
d in -6-yl Ester (5a ): 8-(3-Nitr op h en yl)-6-p yr id in -3-yl-[1,7]-
n a p h th yr id in e (7). To a degassed solution of 5a (150 mg,
0.375 mmol) in DMF (2 mL) were added bis(dibenzylidene-
acetone)palladium (4.5 mg, 0.008 mmol), triphenylphosphine
(8 mg, 0.031 mmol), LiCl (48 mg, 1.13 mmol), and 3-tributyl-
stannylpyridine (145 mg, 0.394 mmol). The reaction mixture
was stirred at 110 °C for 16 h. The solution was diluted with
EtOAc and water. A part of the product precipitated. The
biphasic mixture was filtered and the residue thoroughly
washed with water and dried at 50 °C under reduced pressure
to give 64 mg of pure product. The organic layer of the filtrate
was separated, washed with water, dried, and concentrated.
The residue was triturated in ether and filtered to give
additional 28 mg of pure product (75%, total yield). An
analytical sample was recrystallized from acetonitrile: mp
1
247-248 °C; H NMR (400 MHz, DMSO-d6) δ 7.64 (dd, J )
5.4, 8.4 Hz, 1H), 7.87-7.94 (m, 2H), 8.42 (d, broad, J ) 7.2
Hz, 1H), 8.60 (d, J ) 7.2 Hz, 1H), 8.64-8.79 (m, 4H), 9.15 (s,
broad, 2H), 9.50 (s, broad, 1H); HRMS [M + H]+ found
329.1042, calcd for C19H13N4O2 329.1039. Anal. (C19H12N4O2)
C, H, N, O.
8-(3-Ch lor op h en yl)-[1,7]n a p h th yr id in -6-yla m in e (4b).
Purified by chromatography (10:1 toluene/acetone): white
solid, 74% yield. Analytical sample recrystallized from 2-pro-
panol: mp 148-149 °C; 1H NMR (400 MHz, DMSO-d6) δ 6.28
(s, broad, 2H), 6.68 (s, 1H), 7.46 (dd, J ) 5.4, 8.4 Hz, 1H), 7.48-
7.55 (m, 2H), 7.99-8.10 (m, 3H), 8.58 (s, broad, 1H); HRMS
[M + H]+ found 256.0639, calcd for C14H11ClN3 256.0642. Anal.
(C14H10ClN3‚0.15H2O) C, H, Cl, N.
6-F u r a n -2-yl-8-(3-n itr op h en yl)-[1,7]n a p h th yr id in e (8).
The reaction was finished after 3 h at 80 °C. Product purified
by trituration in ether: pale yellow solid, 62% yield. Analytical
sample recrystallized from 2-propanol: mp 202-203 °C; 1H
NMR (360 MHz, CDCl3) δ 6.60 (dd, J ) 1.8, 4.8 Hz, 1H), 7.26
(d, J ) 4.8 Hz, 1H), 7.59-7.65 (m, 2H), 7.71 (dd, J ) 7.2, 7.2
Hz, 1H), 8.02 (s, 1H), 8.24 (d, broad, J ) 7.2 Hz, 1H), 8.36 (d,
broad, J ) 7.2 Hz, 1H), 8.64 (d, J ) 7.2 Hz, 1H), 9.00 (dd, J )
1.2, 5.4 Hz, 1H), 9.17 (s, broad, 1H); HRMS [M + H]+ found
318.0883, calcd for C18H12N3O3 318.0879. Anal. (C18H11N3O3)
C, H, N, O.
6-Allyl-8-(3-n itr op h en yl)-[1,7]n a p h th yr id in e (10). The
reaction was finished after 2 h at 80 °C. Product purified by
trituration in ether: pale yellow solid, 52% yield; mp 84-86
°C; 1H NMR (360 MHz, CDCl3) δ 3.80 (d, J ) 6.6 Hz, 2H),
5.18 (d, J ) 18 Hz, 1H), 5.24 (d, J ) 24 Hz, 1H), 6.15-6.28
(m, 1H), 7.78-7.87 (m, 3H), 8.36 (d, broad, J ) 7.2 Hz, 1H),
8.49 (d, J ) 7.2 Hz, 1H), 8.62 (d, J ) 7.2 Hz, 1H), 8.99 (s,
3-(6-Am in o-[1,7]n aph th yr idin -8-yl)ben zon itr ile (4c). Pu-
rified by trituration in ether: pale yellow solid, 90% yield.
Analytical sample recrystallized from 2-propanol: mp 182-
1
184 °C; H NMR (400 MHz, DMSO-d6) δ 6.33 (s, broad, 2H),
6.72 (s, 1H), 7.48 (dd, J ) 5.4, 8.4 Hz, 1H), 7.72(dd, J ) 7.2,
7.2 Hz, 1H), 7.93 (d, J ) 7.2 Hz, 1H), 8.07 (d, J ) 7.2 Hz, 1H),
8.40 (d, J ) 8.4 Hz, 1H), 8.46 (s, broad, 1H), 8.60 (d, broad, J
) 5.4 Hz, 1H); HRMS [M + H]+ found 247.0978, calcd for
C
15H11N4 247.0984.
Gen er a l P r oced u r e B. Syn th esis of Tr iflu or om eth a n e-
su lfon ic Acid [1,7]Na p h th yr id in -6-yl Ester s 5a -c: Tr i-
flu or om eth a n esu lfon ic Acid 8-(3-Nitr op h en yl)-[1,7]n a p h -
th yr id in -6-yl Ester (5a ). To a solution of 4a (4 g, 15.0 mmol)

680 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Hersperger et al.
broad, 1H), 9.05 (d, broad, J ) 5.4 Hz, 1H); MS (m/e) [M+H]+
290. Anal. (C17H13N3O3) Calcd: C(70.09), H(4.50), N(14.42),
O(10.98). Found: C(69.55), H(4.40), N(14.22), O(10.70).
solved at 80 °C in a 3:1 mixture water-DMF (40 mL) and the
crude product was precipitated by carefully adding 2 N HCl
(8 mL) and water (10 mL). The cold suspension was filtered
and the crude product was purified by trituration in hot THF
to give 1.12 g of pure product (56%). An analytical sample was
recrystallized from formic acid/water: mp > 250 °C; 1H NMR
(360 MHz, DMSO-d6) δ 7.78 (dd, J ) 7.2, 7.2 Hz, 1H), 7.86
(dd, J ) 5.4, 8.4 Hz, 1H), 8.00 (d, J ) 7.2 Hz, 1H), 8.12 (d, J
) 7.8 Hz, 2H), 8.41 (d, J ) 7.8 Hz, 2H), 8.54 (d, J ) 7.2 Hz,
1H), 8.58 (d, J ) 7.2 Hz, 1H), 8.63 (s, 1H), 8.66 (s, 1H), 9.10
(s, broad 1H); HRMS [M + H]+ found 352.1090, calcd for
Gen er a l P r oced u r e D. Su zu k i Cou p lin gs of Tr iflu or o-
m eth a n esu lfon ic Acid 8-(3-Nitr op h en yl)-[1,7]n a p h th yr i-
d in -6-yl Ester (5a ): 8-(3-Nitr op h en yl)-6-p h en yl-[1,7]n a p h -
th yr id in e (6). To a solution of 5a (300 mg, 0.751 mmol) in
THF (5 mL) and 2 N Na2CO3 (1.5 mL) were added bis-
(dibenzylideneacetone)palladium (17.3 mg, 0.030 mmol), tri-
phenylphosphine (15.7 mg, 0.060 mmol), and phenylboronic
acid (118 mg, 0.968 mmol). The reaction mixture was stirred
at 80 °C for 16 h. The solution was diluted with EtOAc, filtered
trough Celite, and washed with 1 N NaOH and brine. The
organic layer was dried and concentrated to give 205 mg of
pure product (84%). An analytical sample was recrystallized
from 2-propanol: mp 177-178 °C; 1H NMR (360 MHz, CDCl3)
δ 7.45-7.59 (m, 3H), 7.65 (dd, J ) 5.4, 8.4 Hz, 1H), 7.72 (dd,
J ) 7.2, 7.2 Hz, 1H), 8.13 (s, 1H), 8.23-8.31 (m, 3H), 8.36 (d,
broad, J ) 7.2 Hz, 1H), 8.70 (d, J ) 7.2 Hz, 1H), 9.04 (d, broad,
J ) 5.4 Hz, 1H), 9.24 (s, broad, 1H); HRMS [M + H]+ found
328.1088, calcd for C20H14N3O2 328.1086; Anal. (C20H13N3O2)
C, H, N, O.
C
22H14N3O2 352.1086.
8-(3-Nit r op h en yl)-[1,7]n a p h t h yr id in e (14). To a de-
gassed solution of 5a (50 mg, 0.125 mmol) in DMF (1 mL) were
added triethylsilane (36.4 mg, 0.313 mmol) and [1,1′-bis-
(diphenylphosphino)ferrocene]dichloropalladium(ll) (2 mg,
0.0024 mmol). The mixture was stirred at 60 °C for 3 h. Then,
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(ll) (2
mg, 0.0024 mmol) was added again and the reaction mixture
was stirred at 60 °C for an additional hour. The solution was
diluted with EtOAc and washed with water. The organic layer
was dried and concentrated. Purification by preparative thin-
layer chromatography (5:1 toluene/acetone) gave 29 mg of pure
product (92%). An analytical sample was recrystallized from
2-propanol: mp 182-183 °C; 1H NMR (360 MHz, CDCl3) δ 7.67
(dd, J ) 5.4, 8.4 Hz, 1H), 7.70 (dd, J ) 7.2, 7.2 Hz, 1H), 7.73
(d, J ) 5.4 Hz, 1H), 8.27 (d, J ) 7.2 Hz, 1H), 8.36 (d, broad, J
) 8.4 Hz, 1H), 8.55 (d, broad, J ) 7.2 Hz, 1H), 8.77 (d, J ) 5.4
Hz, 1H), 9.08-9.12 (m, 2H); HRMS [M + H]+ found 252.0771,
calcd for C14H10N3O2 252.0773.
8-(3-Nitr op h en yl)-6-o-tolyl-[1,7]n a p h th yr id in e (9). Pu-
rified by trituration in ether: pale yellow solid, 54% yield.
Analytical sample recrystallized from 2-propanol: mp 148-
1
149 °C; H NMR (360 MHz, DMSO-d6) δ 2.49 (s, 3H), 7.33-
7.41 (m, 3H), 7.60-7.65 (m, 1H), 7.86 (dd, J ) 7.2, 7.2 Hz,
1H), 7.88 (dd, J ) 5.4, 8.4 Hz, 1H), 8.17 (s, 1H), 8.37 (d, broad,
J ) 8.4 Hz, 1H), 8.60 (d, broad, J ) 8.4 Hz, 1H), 8.69 (d, J )
7.2 Hz, 1H), 9.07 (s, broad, 1H), 9.13 (d, broad, J ) 5.4 Hz,
1H); HRMS [M + H]+ found 342.1240, calcd for C21H16N3O2
342.1243. Anal. (C21H15N3O2‚0.05H2O) C, H, N, O.
(4-Meth ylp ip er a zin -1-yl)[8-(3-n itr op h en yl)-[1,7]n a p h -
th yr id in -6-yl]m eth a n on e (15). To a degassed solution of 5a
(150 mg, 0.376 mmol) in DMF (6 mL) were added bis-
(dibenzylideneacetone)palladium (4.2 mg, 0.007 mmol), tri-
phenylphosphine (7.8 mg, 0.030 mmol), triethylamine (76 mg,
0.750 mmol), and 1-methylpiperazine (41 mg, 0.401 mmol). The
reaction mixture was stirred at 80 °C under a CO atmosphere
for 16 h. The solution was diluted with EtOAc, filtered through
Celite, and washed with water and brine. The organic layer
was dried and concentrated. The solid was triturated in ether
and filtered to give 86 mg of product (61%). An analytical
sample was recrystallized from 2-propanol: mp 149-150 °C;
1H NMR (360 MHz, CDCl3) δ 2.41(s, broad, 3H), 2.62 (s, broad,
4H), 3.83 (s, broad, 2H), 3.95 (s, broad, 2H), 7.68-7.76 (m, 2H),
8.19 (s, 1H), 8.33 (d, J ) 7.2 Hz, 1H), 8.37 (d, J ) 7.2 Hz, 1H),
8.64 (d, J ) 7.2 Hz, 1H), 9.11-9.16 (m, 2H); MS (m/e) [M +
H]+ 378. Anal. (C20H19N5O3) C, H, N, O.
4-[8-(3-Nit r op h en yl)-[1,7]n a p h t h yr id in -6-yl]b en zoic
Acid (11). To a solution of 5a (700 mg, 1.75 mmol) in DMF
(10 mL) and 2 N Na2CO3 (5 mL) were added bis(dibenzylide-
neacetone)palladium (16 mg, 0.028 mmol), triphenylphosphine
(29 mg, 0.111 mmol), and 4-carboxyphenylboronic acid (378
mg, 2.28 mmol). The reaction mixture was stirred at 80 °C for
2 h. The solution was diluted with EtOAc and 2 N HCl. The
precipitated solid was filtered, thoroughly washed with water,
and dried at 50 °C under reduced pressure to give 610 mg of
pure product (94%). An analytical sample was recrystallized
1
from formic acid: mp > 250 °C; H NMR (360 MHz, DMSO-
d6) δ 7.83-7.89 (m, 2H), 8.11 (d, J ) 7.8 Hz, 2H), 8.36-8.42
(m, 3H), 8.58 (d, J ) 7.2 Hz, 1H), 8.68-8.71 (m, 2H), 9.08-
9.12 (m, 2H); HRMS [M + H]+ found 372.0989, calcd for
C
O.
21H14N3O4 372.0984. Anal. (C21H13N3O4‚0.1HCO2H) C, H, N,
In h ibition of Hu m a n cAMP -Sp ecific P DE Isoen zym es.
P DE3: PDE3 was prepared from human platelets by ultra-
sonic homogenization. Platelets were washed once with PBS
and suspended in 10 mL of buffer (sucrose 0.25 M, EDTA 1
mM, Tris 10 mM, dithiothreitol 1 mM, adjusted to pH 7.4 with
HCl) and the following protease inhibitor solutions: 5 mL/mL
of phenylmethanesulfonyl fluoride (7 mg/mL in 2-propanol),
1 mL/mL leupeptin and pepstatin A (1 mg/mL each, in
ethanol). After sonication (15 s at 4 °C; Branson probe
sonicator), homogenates were centrifuged at 2200g. The pellet
was resuspended in the same volume of buffer H and the
sonication repeated. Pooled supernates were stored at -20 °C.
Activity was assayed as described.22
P DE4: Activity was assessed as previously described.22 With
the exception of PDE4B (rat, expressed in S. cerevisiae; human
PDE4B is 97% homologous to rat PDE4B), all isoenzyme
preparations were from human sources; PDE4A,C,D were
expressed in S. cerevisiae.
4-[8-(3-Ch lor op h en yl)-[1,7]n a p h th yr id in -6-yl]ben zoic
Acid (12). To a suspension of 5b (1.66 g, 4.27 mmol) in DMF
(17 mL) and 2 N Na2CO3 (12 mL) were added bis(dibenzyli-
deneacetone)palladium (99 mg, 0.172 mmol), triphenylphos-
phine (73 mg, 0.278 mmol), and 4-carboxyphenylboronic acid
(850 mg, 5.12 mmol). The reaction mixture was stirred at 80
°C for 3 h. The mixture was diluted with EtOAc and 2 N HCl.
The biphasic solution was filtered through Celite; the organic
layer was separated and washed with water and brine. The
solution was concentrated and the precipitated product was
filtered and dried to give 1.43 g of product (93%). An analytical
1
sample was recrystallized from THF: mp > 250 °C; H NMR
(360 MHz, DMSO-d6) δ 7.57-7.64 (m, 2H), 7.85 (dd, J ) 5.4,
9.0 Hz, 1H), 8.12 (d, J ) 7.8 Hz, 2H), 8.13-8.21 (m, 1H), 8.26
(s, broad, 1H), 8.41 (d, J ) 7.8 Hz, 2H), 8.56 (d, J ) 9.0 Hz,
1H), 8.66 (s, 1H), 9.10 (d, broad, J ) 5.4 Hz, 1H); MS (m/e)
[M]+ 360. Anal. (C21H13ClN2O2) C, H, Cl, N, O.
4-[8-(3-Cya n op h en yl)-[1,7]n a p h t h yr id in -6-yl]b en zoic
Acid (13). To a suspension of 5c (2.15 g, 5.67 mmol) in DMF
(22 mL) and 2 N K2CO3 (17 mL) were added bis(dibenzyli-
deneacetone)palladium (131 mg, 0.228 mmol), triphenylphos-
phine (95 mg, 0.363 mmol), and 4-carboxyphenylboronic acid
(1.13 g, 6.81 mmol). The reaction mixture was stirred at 85
°C for 3 h. The hot solution was filtered through Celite and
the filtrate was cooled in an ice bath. The precipitate was
filtered, washed with EtOAc, and dried. The solid was dis-
Rolip r a m Recep tor Bin d in g Assa y Meth od . Rat brains
were suspended in 10 mL/g of ice-cold buffer (Tris 20 mM,
MgCl2 1 mM, dithiothreitol 0.1 mM, pH 7.5), homogenized
using 2 bursts (30 s each) of a Polytron homogenizer (Kine-
matica, Switzerland), and centrifuged (600g, 10 min, 4 °C).
The supernatants were pooled, and centrifuged (24000g, 10
min, 4 °C). The pellets were resuspended in 5 mL/g of buffer.
Protein content was determined using the method of Brad-

Synthesis of 6,8-Disubstituted 1,7-Naphthyridines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 681
ford,23 according to the instructions of the manufacturer
(BioRad). The assay was carried out as described by Schneider
et al.18
of o-phenylenediamine (OPD) by EPO in the presence of
hydrogen peroxide (H2O2). BALF (10 µL) was mixed with 100
µL of substrate (1 mM OPD, 1 mM H2O2, 0.1% Triton-X100,
dissolved in 50 mM Tris-HCl, pH 8.0) in a 96-well flat bottom
microtiter plate and incubated for 30 min at room temperature.
The reaction was stopped by adding 50 µL of H2SO4 (4 M) and
absorbance was measured at 492 nm in a microplate absor-
bance spectrophotometer. The concentration of EPO was
calculated as units/mL according to the activity of serial
dilutions of a standard horseradish peroxidase (Sigma;, 210
U/mg dry wt).
Oxid a tive Bu r st fr om Hu m a n Eosin op h ils. Blood was
obtained from normal individuals. Granulocytes were sepa-
rated from mononuclear cells by Ficoll hypaque gradient
centrifugation. Erythrocytes were lysed by two cycles of
hypotonic lysis and the remaining granulocytes were incubated
with anti-CD16 coated immunomagnetic particles (Miltenyi
Biotec, Bergisch Glasbach, Germany). Magnetically labeled
neutrophils were then depleted by passing the granulocytes
through a MACS (magnetic cell separation) column which
resulted in a more than 98% pure eosinophil preparation.
These purified human eosinophils were diluted in HBSS and
pipetted into 96-well microtiter plates (MTP) at 104 cells/well.
Each well contained a 200-µL sample comprising: 100 µL of
eosinophil suspension, 50 µL of HBSS, 10 µL of lucigenin, 20
µL of activation stimulus, 20 µL of compound of interest. The
samples were incubated with compound (dissolved in DMSO
and thereafter diluted in buffer) or vehicle for between 10 and
30 min prior to addition of fMLP (0.01-10 µM). MTPs were
agitated (Titertek MTP mixer) to facilitate mixing of the cells
and medium and then placed into a Hamamatsu luminometer.
Total chemiluminescence and the temporal profile of each well
was measured simultaneously over 20 min and the results
were expressed as a percentage of fMLP-induced chemilumi-
nescence in the absence of compound. Results were fitted to
the Hill equation and IC50 values calculated. For each com-
pound three independent measurements were done using
eosinophils from three different normal donors.
Ack n ow led gm en t. The authors thank Dr. Paul
Manley for critical review of the manuscript and com-
ments and E. Bacher, B. Glasser, and A. Ursprung for
their excellent technical assistance. We also acknowl-
edge the contributions of Novartis Pharma Research
Analytics for spectral data.
Refer en ces
(1) Teixeira, M. M.; Gristwood, R. W.; Cooper N.; Hellewell, P. G.
Phosphodiesterase (PDE) 4 inhibitors: antiinflammatory drugs
of the future? Trends Pharmacol. Sci. 1997, 18, 164-170.
(2) Phosphodiesterase Inhibitors; Schudt, C., Dent, G., Rabe, K. F.,
Eds.; Academic Press: London, 1996.
(3) Shire, M. G.; Muller, G. W. Exp. Opin. Ther. Patents 1998, 8,
531-544. Norman, P. PDE4 inhibitors 1998. Exp. Opin. Ther.
Patents 1998, 8, 771-784.
Br ow n Nor w a y Ra t Mod el of Air w a y In fla m m a tion .
Sen sitiza tion , ch a llen ge, a n d br on ch oa lveola r la va ge
flu id collection : Male Brown Norway rats (approximately
200 g) were used for the study of cell accumulation. Food and
water were available ad libitum. Ovalbumin (OA) (20 mg/mL)
was mixed in a blender (Polytron, Kinematica Ltd.) with
aluminum hydroxide (20 mg/mL) and injected (sc) concomi-
tantly with B. pertussis vaccine (0.25 mL/animal ip) on days
1, 15, and 21. On day 28, sensitized animals were restrained
in plastic tubes and exposed (1 h) to an aerosol of OA (3.2 mg/
mL) using a nose-only exposure system. Animals were killed
48 h later with pentobarbital (250 mg/kg ip). The lungs were
lavaged using 3 aliquots (4 mL) of Hank’s solution (100 mL
HBSS×10, 100 mL of 0.1 M EDTA solution, 10 mL of 1.0 M
HEPES solution, 1 L of water), the recovered cells were pooled,
and the total volume of recovered fluid was adjusted to 12 mL
by addition of Hank’s solution. Compounds were dissolved in
DMSO (10 mg in 0.1 mL), diluted with neoral placebo (10 mL),
and administered by gavage 1 h prior to and 24 h after antigen
exposure. Control groups of actively sensitized animals re-
ceived saline alone with or without exposure to antigen.
BALF cell cou n tin g: Erythrocytes in lavage fluid were
lysed (Quicklyser QLA-200A, TOA Medical Electronics Ltd.,
J apan). Smears were prepared by diluting the recovered fluid
(to approximately 106 cells/mL) and centrifuging an aliquot.
Smears were air-dried, fixed using a solution of fast green in
methanol (2 mg/mL) for 5 s, and stained with eosin G (5 s)
and thiazin (5 s) in order to differentiate cell types. A total of
500 cells/smear were counted by light microscopy under oil
immersion (×1000).
(4) Beavo, J . A. Cyclic nucleotide phosphodiesterases: functional
implications of multiple isoforms. Physiol. Rev. 1995, 75, 725-
48. See also: Beavo, J . A. Isolation and characterization of a
dual-substrate phosphodiesterase gene family: PDE10A. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 7071-7076.
(5) Mu¨ller, T.; Engels, P.; Fozard, J . R. Subtypes of the type 4 cAMP
phosphodiesterases: structure, regulation and selective inhibi-
tion. Trends Pharmacol. Sci. 1996, 17, 294-298.
(6) Barnette, M. S.; Christensen, S. B.; Underwood, D. C.; Torphy,
T. J . Phosphodiesterase 4: Biological underpinnings for the
design of improved inhibitors. Pharmacol. Rev. Commun. 1996,
8, 65-73.
(7) Christensen, S. B.; Guider, A.; Forster, C. J .; Gleason, J . G.;
Bender, P. E.; Karpinski, J . M.; DeWolf, W. E. J r.; Barnette, M.
S.; Underwood, D. C.; Griswold, D. E.; Cieslinski, L. B.; Burman,
M.; Bochnowicz, S.; Osborn, R. R.; Manning, C. D.; Grous, M.;
Hillegas, L. M.; Bartus, J . O.; Ryan, M. D.; Eggleston, D. S.;
Haltiwanger, R. C.; Torphy, T. J . 4-Cyclohexanecarboxylates:
potent and selective inhibitors of phosophodiesterase 4 for the
treatment of asthma. J . Med. Chem. 1998, 41, 821-835.
(8) Murdoch, R. D.; Cowley, H.; Upward, J .; Webber, D.; Wyld, P.
The safety and tolerability of Ariflo (SB207499), a novel and
selective phospodiesterease 4 inhibitor, in healthy male volun-
teers. Am. J . Respir. Crit. Care Med. 1998, 157, Poster L57.
(9) Alvarez, R.; Sette, C.; Yang, D.; Eglen, R. M.; Wilhelm, R.;
Shelton, E. R.; Conti, M. Activation and selective inhibition of a
cyclic AMP-specific phosphodiesterase, PDE-4D3. Mol. Phar-
macol. 1995, 48, 616-622.
(10) Naef, R. Isoquinolines for asthma therapy. Ger. Offen. DE
4438737 A1 950511.
(11) Stille, J . K. The palladium-catalyzed cross-coupling reactions of
organotin reagents with organic electrophiles. Angew. Chem.,
Int. Ed. Engl. 1986, 25, 508-524. Miyaura, N.; Suzuki, A.
Palladium-catalyzed cross-coupling reactions of organoboron
compounds. Chem. Rev. 1995, 95, 2457-2483.
(12) Robinson, D. S. Eosinophils and asthma. What do measurements
tell us? Clin. Exp. Allergy 1995, 25, 795-798.
(13) Fife, W. K. Regioselective cyanation of pyridine 1-oxides with
trimethylsilanecarbonitrile: a modified Reissert-Henze reaction.
J . Org. Chem. 1983, 48, 1377-1379.
(14) Danieli, R.; Ricci, A. Improvements in the synthesis of 2,7- and
2,6-naphthyridines Synthesis 1973, 46-47.
(15) Tan, R.; Taurins, A. A new synthesis of 1,7-naphthyridine.
Tetrahedron Lett. 1966, 12, 1233-1237.
(16) Kotsuki, H.; Datta, P. K.; Hayakawa, H.; Suenaga, H. An
efficient procedure for palladium-catalyzed reduction of aryl/enol
triflates. Synthesis 1995, 1348-1350.
(17) Cacchi, S.; Ciattini, P. G.; Morera, E.; Ortar, G. Palladium-
catalyzed carbonylation of aryl triflates. Synthesis of arenecar-
boxylic acid derivatives from phenols. Tetrahedron Lett. 1986,
27, 3931-3934.
P r otein d eter m in a tion : The concentration of protein in
BALF supernatants was measured by a colorimetric assay
(Bio-Rad DC protein assay). The assay is based upon the
reaction of a protein with an alkaline copper tartrate solution
and Folin reagent. Briefly, 5 mL of BALF supernatant was
added to a 96-well ELISA plate (NUNC). A mixture containing
alkaline copper tartrate solution (reagent A′; 25 mL) and dilute
Folin reagent (reagent B; 200 mL) was added to each well and
incubated for 15 min at room temperature. The absorbance
was measured at 700 nm. A bovine serum albumin standard
was used to generate a standard curve; the sensitivity of the
assay was 0.04 mg/mL protein.
Deter m in a tion of eosin op h il p er oxid a se (EP O) a ctiv-
ity in BALF : The method utilized is based on the oxidation

682 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Hersperger et al.
(18) Torphy, T. J .; Stadel, J . M.; Burman, M.; Cieslinski, L. B.;
McLaughlin, M. M.; White, J . R.; Livi, G. P. Coexpression of
human cAMP-specific phosphodiesterase activity and high af-
finity rolipram binding in yeast. J . Biol. Chem. 1992, 267, 1798-
1804. Schneider, H. H.; Schmiechen, R.; Brezinski, M.; Seidler,
J . Stereospecific binding of the antidepressant rolipram to brain
protein structures. Eur. J . Pharmacol. 1986, 127, 105-115.
(19) Barnette, M. S.; Manning, C. D.; Cielinski, L. B.; Burman, M.;
Christensen, B.; Torphy, T. J . The ability of phosphodiesterase
IV inhibitors to suppress superoxide production in guinea pig
eosinophils is correlated with inhibition of phosphodiesterase IV
catalytic activity. J . Pharmacol. Exp. Ther. 1995, 273, 674-679.
(20) Schneider, T.; van Velzen, D.; Moqbel, R.; Issekutz, A. C. Kinetics
and quantification of eosinophil and neutrophil recruitment to
allergic lung inflammation in a Brown Norway rat model. Am.
J . Respir. Cell Mol. Biol. 1997, 17, 702-712. Elwood, W.; Lotvall,
J . O.; Barnes, P. J .; Chung, K. F. Characterization of allergen-
induced bronchial hyperresponsiveness and airway inflamma-
tion in actively sensitized brown-Norway rats. Allergy Clin.
Immunol. 1991, 88, 951-60.
(21) Goldie, R. G.; Pedersen, K. E. Mechanisms of increased airway
microvascular permeability: role in airway inflammation and
obstruction. Clin. Exp. Pharmacol. Physiol. 1995, 22, 387-396.
(22) Engels, P.; Sullivan, M.; Mu¨ller, T.; Lu¨bbert, H. Molecular
cloning and functional expression in yeast of a human cAMP-
specific phosphodiesterase subtype. FEBS Lett. 1995, 358, 305-
310.
(23) Bradford, M. M. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
J M991094U