Synthesis and biological evaluation of novel phthalazinone derivatives as topically active phosphodiesterase 4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.014

Inhibitors of phosphodiesterase 4 (PDE4) are an important class of anti-inflammatory drug that act by inhibiting the production of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α). We have synthesized and evaluated a series of 2-substituted phthalazinone derivatives as PDE4 inhibitors. Structure-activity relationship studies led to the identification of benzylamine-substituted phthalazinones as potent PDE4 inhibitors that also suppressed TNF-α production by whole rat blood cells. The most potent of these, when topically administered, were effective in a mouse model of dermatitis.

Full text of DOI:10.1016/j.bmc.2009.08.014

Bioorganic & Medicinal Chemistry 17 (2009) 6959–6970
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel phthalazinone derivatives
as topically active phosphodiesterase 4 inhibitors
*
Kohei Kagayama , Tatsuya Morimoto, Seigo Nagata, Fumitaka Katoh, Xin Zhang, Naoki Inoue,
Asami Hashino, Kiyoto Kageyama, Jiro Shikaura, Tomoko Niwa
Discovery Research Laboratories, Nippon Shinyaku Co., Ltd, 14 Nishinosho-Monguchi-cho, Kisshoin, Minami-ku, Kyoto 601-8550, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of phosphodiesterase 4 (PDE4) are an important class of anti-inflammatory drug that act by
Received 29 June 2009
Revised 5 August 2009
Accepted 5 August 2009
Available online 12 August 2009
inhibiting the production of proinflammatory cytokines, including tumor necrosis factor-a (TNF-a). We
have synthesized and evaluated a series of 2-substituted phthalazinone derivatives as PDE4 inhibitors.
Structure–activity relationship studies led to the identification of benzylamine-substituted phthalazinon-
es as potent PDE4 inhibitors that also suppressed TNF-
potent of these, when topically administered, were effective in a mouse model of dermatitis.
Ó 2009 Elsevier Ltd. All rights reserved.
a production by whole rat blood cells. The most
Keywords:
Phosphodiesterase
PDE4 inhibitor
Phthalazinones
Tumor necrosis factor-
a
Structure–activity relationship
1. Introduction
ever, the therapeutic usefulness of first-generation PDE4 inhibitors,
which are orally taken, is limited by their side effects, including
gastrointestinal side effects such as nausea and vomiting.13 It has
been hypothesized that these side effects could be due either to
binding at a high-affinity allosteric binding site of PDE4 (the so-
called high-affinity rolipram binding site)14–16 or to the activation
of emetic centers within the central nervous system (CNS).13
Although a few PDE4 inhibitors such as roflumilast (Fig. 1) have
been evaluated in clinical trials, most have gastrointestinal side
effects.17,18
Cyclic nucleotide phosphodiesterases (PDEs), which catalyze
the hydrolysis of the second messengers cyclic adenosine mono-
phosphate (cAMP) and cyclic guanosine monophosphate (cGMP)
to the corresponding nucleoside 50-monophosphates, are impor-
tant regulators of intracellular cyclic nucleotide signaling. To date,
eleven families of PDE isoenzyme have been identified in mammals
on the basis of their amino acid sequence, substrate specificity
(cAMP or cGMP), tissue distribution, and other characteristics.1
Among these isoenzymes, PDE4 is a high-affinity cAMP-specific
phosphodiesterase that is expressed in immune and inflammatory
cells such as eosinophils, macrophages, T-lymphocytes and neutro-
phils.2,3 Elevation of cAMP through inhibition of PDE4 results in a
wide range of anti-inflammatory effects in leukocytes.1 Therefore
PDE4 inhibitors are expected to be effective in the treatment of
inflammatory diseases such as asthma,4,5 chronic obstructive pul-
monary disease6,7 and atopic dermatitis.8 Overproduction of tumor
As an alternative to oral administration, topical application of
PDE4 inhibitors is a possible way to maximize their efficacy in
the treatment of inflammatory disease while minimizing their side
F
F
O
F
O
F
O
O
O
O
O
O
necrosis factor-a (TNF-a) is associated with a number of autoim-
mune and inflammatory diseases. PDE4 inhibition leads to sup-
pression of TNF-
a production through the activation of inducible
O
Cl
NH
N
NH
N
NH
cAMP early repressor (ICER),9,10 an effect that plays a key role in
the anti-inflammatory activity of PDE4 inhibitors.11
HN
Cl
O
O
O
The first-generation PDE4 inhibitor rolipram (Fig. 1) has been
N
the starting point for many medicinal chemistry studies.12 How-
rolipram
roflumilast
zardaverine
1
* Corresponding author. Tel.: +81 75 321 9168; fax: +81 75 321 9039.
E-mail address: k.kagayama@po.nippon-shinyaku.co.jp (K. Kagayama).
Figure 1. Chemical structures of PDE4 inhibitors.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.014

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K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
effects. Since topical administration minimizes systemic exposure,
we would not be particularly concerned about binding at the high-
affinity rolipram binding site or penetration into the CNS.19–22
The aim of the present study was to develop potent, topically
active PDE4 inhibitors with minimal gastrointestinal side effects.
We first performed a 3D docking study to identify a lead compound
and to obtain guidance for chemical-modification studies. We then
carried out structure–activity relationship studies on derivatives of
phthalazinone 123 and identified several highly potent PDE4 inhib-
itors that were topically active in a mouse model of dermatitis.
2. Results and discussion
2.1. Design and strategy
We made extensive use of the publicly available X-ray struc-
tures of the complexes of PDE4 with zardaverine (Fig. 1; Protein
Data Bank (PDB) entry 1MKD24) and PDE4D2 with rolipram (PDB
entry 1OYN25). We first computed the shape of the active-site
pocket of PDE4 by using the 3D atomic coordinates of the enzyme,
and then we calculated the surface properties of the active-site
pocket with the molecular modeling suite Molecular Operating
Environment (MOE). In the resulting model (Fig. 2), the white
and red spheres indicate the predicted locations of an inhibitor’s
atoms, with zardaverine shown for reference. The spheres are clas-
sified as either hydrophilic (red) or hydrophobic (white) depending
on whether or not they are in good hydrogen-bonding locations.
Regions of the surface of the active-site pocket are colored accord-
ing to whether nearby interior atoms are hydrophobic (green re-
gions) or hydrophilic (blue regions), while purple indicates
regions where hydrogen bonds are likely to form.
The major part of the surface is shown in green, indicating the
hydrophobic nature of the active-site pocket (Fig. 2). The pocket
is seen to be large, and zardaverine occupies only a part of it. Con-
siderable hydrophobic space is available near zardaverine, as indi-
cated by the white spheres, so extensive chemical modification
should be possible in this region. To identify a promising lead com-
pound, we manually docked known PDE4 inhibitors into the ac-
tive-site pocket by reference to the available cocrystal X-ray
structures 1MKD, 1OYN, 1PTW,26 and 1Q9 M.25 We selected the
racemic phthalazinone 123 for further study because (1) the
Figure 3. (a) X-ray structure of PDE4–zardaverine complex (1MKD24), (b) docked
model of PDE4–1 complex. (4aS,8aR)-4-(3,4-Dimethoxyphenyl)-4a,5,8,8a-tetra-
hydrophthalazin-1(2H)-one (an enantiomer of 1) was used for docking.
cyclohexene ring of one isomer filled the active-site pocket much
better than zardaverine did, and (2) the presence of the nitrogen
atom at the 2-position of the phthalazinone ring permitted exten-
sive chemical modification at that position (Fig. 3). We chose the
cis-isomer of phthalazinone because, according to a previous
study,23 it was likely to have higher activity than the trans-isomer.
The main barrier for topical drug delivery is generally believed
to be the stratum corneum of the skin. Because the stratum cor-
neum is a lipophilic barrier, the lipophilicity of a drug is thought
to be a key parameter controlling its penetration of the skin. Many
approaches have therefore been taken to increase the lipophilicity
of drugs with the aim of enhancing their activity on topical appli-
cation.27,28 Lipophilicity also tends to promote rapid metabolism,29
which could help reduce the systemic side effects of drugs that
happen to be absorbed into the systemic circulation by limiting
their persistence in the circulation. These considerations suggested
to us that chemical modifications directed towards increasing the
lipophilicity of PDE4 inhibitors would be a good way to both en-
hance their topical activity and reduce their side effects. In our
search for PDE4 inhibitors with favorable pharmacokinetic profiles,
therefore, we decided to focus on the introduction of various kinds
of lipophilic moiety at the 2-position of the phthalazinone ring.
2.2. Chemistry
The synthesis of racemic 4-(3,4-dimethoxyphenyl)-5,6,9,10-tet-
rahydrophthalazin-1-one derivatives is shown in Scheme 1. The
keto acid 3, obtained by conventional Friedel–Crafts acylation23
of veratrole (1,2-dimethoxybenzene) by cis-1,2-cyclohexenedi-
carboxylic anhydride 2, was condensed with hydrazine monohy-
drate to produce lead compound 1. N-Alkylation of 1 with ethyl
bromoacetate followed by hydrolysis under basic conditions gave
carboxylic acid 4, which was converted into amide 5 by condensa-
tion with aminopropylimidazole. Treatment of 1 with either of two
alkyl dibromides in the presence of sodium hydride furnished the
N-bromoalkyl-substituted phthalazinones 6a and 6b, which, on
reaction with imidazole, yielded the desired compounds 7a and
7b. Tetrahydrophthalazinone 1 was coupled with the o-, m-, or
Figure 2. Structural properties of the PDE4 active-site pocket. Spheres indicate the
probable locations of ligand atoms and are classified as either hydrophilic (red) or
hydrophobic (white), depending on whether or not they are in good hydrogen-
bonding locations. The green regions on the surface are hydrophobic and the blue
regions are hydrophilic, while purple indicates regions in which hydrogen bonds
are likely to form.

K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
6961
O
O
O
O
O
O
O
O
O
O
a
e
b
c, d
O
O
N
N
N
O
N
N
O
O
OH
NH
OH
N
H
N
N
O
O
O
O
4
5
2
3
1
O
O
O
g
f
N
N
N
N
N
Br
N
X
X
O
O
6a: X =
6b: X =
O
7a: X =
7b: X =
O
Scheme 1. Reagents: (a) veratrole, AlCl3, CH2Cl2; (b) NH2NH2, EtOH; (c) NaH, BrCH2CO2Et, DMF; (d) aqueous NaOH, MeOH; (e) 1-(3-aminopropyl)imidazole, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, Et3N, DMF; (f) bis(2-bromoethyl)ether (for 6a), 1,4-dibromo-2-butene (for 6b), NaH, DMF; (g)
NaH, imidazole, DMF.
Z1
Z2
=
=
N
N
O
O
O
O
O
O
O
N
a
b or c
O
O
4
4
3
N
N
N
Br
Z3
Z4
=
=
N
N
N
N
N
NH
R
3
2
2
O
O
O
1
8: 4-position
9: 3-position
10: 2-position
11a: 4-position, R = Z1
11b: 4-position, R = Z2
11c: 4-position, R = Z3
11d: 4-position, R = Z4
11e: 4-position, R = Z5
11f: 4-position, R = Z6
11g: 4-position, R = Z7
12: 3-position, R = Z7
13: 2-position, R = Z7
Z5
Z6
Z7
=
=
=
NMe
N
N
O
Scheme 2. Reagents: (a) (i) NaH, DMF (ii) a, a, a,
a0-p-dibromoxylene (for 8), a0-m-dibromoxylene (for 9), or a0-o-dibromoxylene (for 10); (b) NaH, imidazole, DMF; (c) various
tertiary amines, K2CO3, THF.
p-isomer of
a
,
a0-dibromoxylene and the resulting benzyl bromides
The optically active benzyl morpholine derivative 11g was
produced as shown in Scheme 4. The racemic keto acid ( )-3 was
8–10 were converted into the desired benzylamine-substituted
phthalazinones 11a–g, 12 and 13 by coupling with various tertiary
amines (Scheme 2).
optically resolved by using the chiral bases (S)-(ꢀ)-
a-methylben-
zylamine and (R)-(+)-a-methylbenzylamine as resolving agents
The synthetic route for the preparation of 6-(3,4-dimethoxy-
phenyl)-4,5-dihydropyridazin-3(2H)-one 17a and the cis-4,5-cyclo-
alkyl-6-(3,4-dimethoxyphenyl)-4,5-dihydropyridazin-3(2H)-ones
17b–d is summarized in Scheme 3. The keto acid 15a was synthe-
sized by Friedel–Crafts acylation of veratrole by succinic anhydride
14a, and condensation of 15a with hydrazine monohydrate yielded
6-(3,4-dimethoxyphenyl)-4,5-dihydropyridazin-3(2H)-one 16a.
The keto acids 15b–d were similarly synthesized by Friedel–Crafts
acylation of veratrole by cis-1,2-cycloalkyldicarboxylic acid
anhydrides 14b–d, and condensation of 15b–d with hydrazine
monohydrate similarly yielded the cis-4,5-cycloalkyl-6-(3,4-dime-
thoxyphenyl)-4,5-dihydropyridazin-3(2H)-ones 16b–d. Transfor-
mation of 16a–d into the desired benzyl morpholines 17a–d was
carried out as shown in Schemes 1 and 2.
as previously described.30 The desired optically active compounds
(+)-11g and (ꢀ)-11g were then prepared in enantiomerically pure
form as outlined in Scheme 3.
2.3. Biological evaluation
The test compounds prepared were evaluated for their ability to
inhibit the enzymatic activity of the PDE4D catalytic domain
cloned from the human acute promyelocytic leukemia cell line
HL-60. The concentration of test compound giving 50% inhibition
of enzyme activity (IC50) was estimated. In addition, the anti-
inflammatory effect of the compounds was assessed by their abil-
ity to suppress the production of TNF-
a by rat whole blood cells
stimulated with lipopolysaccharide (LPS).31

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K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
O
O
O
O
O
O
O
O
R1
R2
a or b
d, e
c
O
R1
R2
R1
R1
R2
O
N
N
N
N
O
OH
NH
O
R2
O
O
14a: R1, R2 = H
15a: R1, R2 = H
16a: R1, R2 = H
17a: R1, R2 = H
14b: R1, R2= -CH2-
15b: R1, R2 = -CH2-
15c: R1, R2 = -(CH2)2-
15d: R1, R2 = -(CH2)4-
16b: R1, R2= -CH2-
16c: R1, R2 = -(CH2)2-
16d: R1, R2= -(CH2)4-
17b: R1, R2 = -CH2-
14c: R1, R2 = -(CH2)2-
14d: R1, R2= -(CH2)4-
17c: R1, R2 = -(CH2)2-
17d: R1, R2 = -(CH2)4-
Scheme 3. Reagents: (a) veratrole, AlCl3, CH2Cl2; (b) 4-bromoveratrole, magnesium turnings, THF; (c) NH2NH2, EtOH; (d) NaH,
a,
a0-p-dibromoxylene, DMF; (e) K2CO3,
morpholine, THF.
e, f, g
e, f, g
a, b
c, d
ring system because the introduction of ionizable substituents is
reported to enhance skin penetration.32 Alkyl amide 5 showed
moderate inhibition of PDE4 (IC50 = 14.3 nM), approximately six-
fold more potent than that of 1, but only relatively weak inhibi-
(+)-3
(-)-3
(+)-11g
(-)-11g
( )-3
tion of TNF-a production by rat whole blood (IC50 = 112.5 nM).
On the one hand, the high PDE4 inhibitory activity of 5 can be
explained by the fact that it fills the active-site pocket of PDE4
in our 3D model. On the other hand, its low activity in the cell-
based assay can be explained by its low lipophilicity (c log P = 0.8).
With a view to identifying a compound with increased lipophilic-
ity, we next varied the substituent at the 2-position of the phtha-
lazinone ring system while keeping the imidazole moiety intact;
that is, we synthesized compounds in which the alkyl amide lin-
Scheme 4. Reagents: (a) (S)-(ꢀ)-
a-methylbenzylamine, AcOEt; (b) aqueous HCl; (c)
(R)-(+)-a-methylbenzylamine, AcOEt; (d) aqueous HCl; (e) NH2NH2, EtOH; (f) NaH,
a,
a0-p-dibromoxylene, DMF; (g) K2CO3, morpholine, THF.
The IC50 values for inhibition of PDE4 activity and TNF-
a pro-
duction for compounds 1 through 11a are shown in Table 1. To
evaluate the effect of filling the space available in the active-site
pocket of PDE4 on the biological activity of PDE4 inhibitors, we
identified the alkyl amide 5 as a starting point for further chem-
ical modification. Alkyl amide 5 was designed to include an imid-
azole moiety at the end of the side chain of the phthalazinone
ker of
5 was replaced with a diethyl ether linker (7a;
c log P = 1.66), a 2-butene linker (7b; c log P = 2.35), or a xylene
linker (11a; c log P = 3.51). The compound with the diethyl ether
linker, 7a, showed approximately threefold greater PDE4 inhibi-
tory activity than 5, while the compound with the 2-butene lin-
ker, 7b, was the most potent, with 36-fold greater PDE4
inhibitory activity than 5 (IC50 = 0.4 nM). The compound with
the xylene linker, 11a, also had good activity (IC50 = 0.7 nM). As
expected, a compound that better filled the available space in
the active-site pocket of PDE4 (with a hydrophobic moiety at
the 2-position of the phthalazinone ring system) was a better
inhibitor. When the ability of the compounds to suppress the pro-
Table 1
Effect of substituents at the 2-position of the phthalazinone ring system on inhibition
of PDE4 and suppression of TNF-
a
production
O
O
R
duction of TNF-a was assessed, 7b and 11a were found to have
over 150-fold greater activity than 5, with IC50 values in the
subnanomolar range. Because 7b and 11a both showed potent
PDE4 inhibitory activity and potent TNF-a-suppressing activity,
we selected the more lipophilic N-benzyl phthalazinone 11a for
further modification in the hope of identifying a compound with
good in vivo efficacy.
N
N
O
Compound
R
PDE4
TNF-a
inhibition
IC50 (nM)
suppression
IC50 (nM)
a
b
We focused on the replacement of the imidazole ring of 11a by
various tertiary amines, mostly including a six-membered hetero-
cyclic ring, because the imidazole ring is reported to inhibit cyto-
chrome P450 and may be generally responsible for unfavorable
1
5
H
85.0
14.3
207.3
112.5
O
drug–drug interactions.33,34 Piperidone derivative 11b (IC50
0.9 nM), 1,4-dioxa-8-azaspiro[4.5]decane derivative 11c (IC50
=
=
N
N
H
N
1.3 nM) and the morpholine compound 11g (IC50 = 0.9 nM) showed
potent inhibition of PDE4 activity comparable to that of 11a, while
piperidine derivative 11d and dimethylamino derivative 11f had
somewhat lower potency (Table 2). A similar profile was observed
N
7a
5.2
0.4
0.7
9.1
0.7
0.5
N
N
O
for the TNF-
ane derivative 11c and morpholine derivative 11g showed excel-
lent inhibition of both PDE4 activity and TNF- production.
a-suppressing activity. 1,4-Dioxa-8-azaspiro[4.5]dec-
N
7b
N
a
These data suggest that the introduction of a suitable amine moi-
ety at the end of the side chain of the phthalazinone ring system
N
11a
a
would improve the potency of both PDE4 inhibition and TNF-
suppression.
a
PDE4 inhibition was assayed with recombinant human PDE4D catalytic domain.
Suppression of TNF-a production by LPS-stimulated rat whole blood cells.
b

K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
6963
Table 2
Table 3
Effect of various heterocyclic rings at the xylene linker on inhibition of PDE4 and
Effect of an extra ring in dihydropyridazinone derivatives on inhibition of PDE4 and
suppression of TNF-
a
production
suppression of TNF-
a
production
O
O
O
O
4
R1
R2
N
N
N
N
N
R
O
3
2
O
O
Compound
R1
R2
H
PDE4 inhibition
IC50 (nM)
TNF-a suppression
b
Compound
Position
R
PDE4
TNF-a
a
IC50 (nM)
442.4
inhibition
suppression
a
b
17a
17b
H
45.9
16.4
IC50 (nM)
IC50 (nM)
118.8
44.8
4.6
N
11a
11b
11c
11d
11e
11f
11g
12
4
4
4
4
4
4
4
3
2
0.7
0.9
1.3
8.2
2.0
9.7
0.9
1.4
1.3
0.5
3.0
N
17cc
17d
4.3
1.0
N
O
N
O
0.9
11g
0.9
0.9
O
a,b
N
N
See corresponding footnotes to Table 1.
The biological activity of the hydrochloride salt was evaluated.
57.1
14.5
34.9
0.9
c
without an extra ring, 17a (IC50 = 442.4 nM). We think that the
cyclohexene ring of 11g plays a dual role because it not only con-
tributed to the PDE4 inhibitory activity of 11g by improving its
binding at the active site but also, by conferring increased lipophil-
icity, enhanced the ability of 11g to penetrate the cell membrane of
NMe
N
N
rat monocytes and lymphocytes in the TNF-a-suppression assay.
We therefore selected 11g for further study.
O
O
O
Next, the optically pure enantiomers of 11g were individually
evaluated (Table 4). In the PDE4 inhibition assay, the cis-(+)-enan-
tiomer (+)-11g displayed an IC50 value of 0.3 nM, corresponding to
29 times the potency of the cis-(ꢀ)-enantiomer (ꢀ)-11g. A similar
N
N
3.0
result was found in the TNF-a-production assay, with (+)-11g dis-
playing 20 times the potency of (ꢀ)-11g.
13
1.0
The effect of topical application of the most potent compounds
identified in the in vitro assays was evaluated in a mouse model of
dermatitis induced by 2,4,6-trinitrochlorobenzene (TNCB) as previ-
ously described.35,36 After topical administration of 20 mg of 1%
test compound in a vaseline ointment, (+)-11g maleate, 11b and
13 reduced the TNCB-induced ear swelling by almost two-thirds
(Table 5), more than the control Protopic ointment (FK506 oint-
ment; Astellas Pharma).
a,b
See corresponding footnotes to Table 1.
Because replacement of the imidazole ring by a morpholine ring
resulted in excellent activity in both in vitro assays, we next inves-
tigated the effect on activity of changing the position of the mor-
pholinomethyl moiety on the phenyl ring (11g, 12 and 13). All
three positions gave almost the same activity in both assays, with
3-substituent derivative 12 showing marginally lower activity in
both assays.
The in vitro activities of dihydropyridazinone derivatives with
an extra ring fused to the dihydropyridazinone ring were evaluated
in 4-(morpholinomethyl)benzyl derivatives 11g and 17a–d (Table
3). All compounds with the fused ring system showed improved
PDE4 inhibitory activity, and the activity increased with increasing
size of the extra ring from three to six members. As expected from
our 3D docking study of 1 and the PDE4 catalytic domain, the cis-
cyclohexene and cyclohexane derivatives 11g and 17d strongly
inhibited PDE4 (IC50 = 0.9 and 1.0 nM, respectively). This suggests
that the cis-cyclohexene and cyclohexane rings of these com-
pounds interact well with the active-site pocket of PDE4. The most
The in vitro metabolic half-life of (+)-11g maleate, expressed as
the half-life of disappearance of the parent compound in the pres-
ence of a human liver microsomal fraction (1 mg protein/mL), was
6 min.
Topical administration of a compound may minimize side ef-
fects by reducing the exposure of the gut and CNS. However, even
though topical doses are generally small, a large proportion of the
dose may be absorbed into the systemic circulation and contribute
to adverse events such as nausea. The low metabolic stability of
Table 4
Effect of 11g stereochemistry on PDE4 inhibition and TNF-
a
suppression
a
b
Compound
PDE4 inhibition IC50 (nM)
TNF-a suppression IC50 (nM)
(+)-11g
(ꢀ)-11g
0.3
8.8
4.2
82.2
potent TNF-a-production-suppressing activity was shown by
cyclohexene derivative 11g (IC50 = 0.9 nM), whose activity was
492-fold stronger than that of the dihydropyridazinone derivative
a,b
See corresponding footnotes to Table 1.

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K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
Table 5
hexene-1,2-dicarboxylic anhydride (55.0 g, 361.5 mmol) was added
Effect of compounds in a mouse dermatitis modela
and the reaction mixture was refluxed for 27 h. The mixture was
then poured into ice water (500 mL) and extracted with CH2Cl2.
The organic layer was washed with brine, dried over MgSO4, and
concentratedinvacuo. TheresiduewascrystallizedfromEt2Otogive
3 as white crystals (25.95 g, 25%). Mp 139.8–141.8 °C. Electrospray
ionization mass spectrometry (ESI-MS) m/z 291.1 [MH]+. IR:
Compound
Reduction of dermatitis (%)
(+)-11gb
11b
13
38.5
36.8
36.8
26.3
Protopic
1702.0, 1671.2, 1513.2, 1417.8, 1260.6, 1147.3, 1020.2 cmꢀ1 1H
.
a
See Section 4 for further details.
The biological activity of the maleate salt was evaluated.
b
NMR (CDCl3) d: 2.30–2.55 (3H, m), 2.79–3.10 (2H, m), 3.92 (3H, s),
3.95 (3H, s), 5.71 (2H, dd, J = 10.6, 29.0 Hz), 6.89 (1H, d, J = 8.4 Hz),
7.49–7.55 (2H, m). Anal. Calcd for C16H18O5: C, 66.19; H, 6.17. Found:
C, 66.19; H, 6.25.
(+)-11g maleate in human liver S9 fractions leads us to expect that,
even if it were largely absorbed after topical administration, it
would disappear rapidly from the systemic circulation. The result-
ing low bioavailability of (+)-11g maleate should ensure a low risk
of contributing to nausea.
4.1.1.2. cis-(+)-6-(3,4-Dimethoxybenzoyl)cyclohex-3-enecarb-
oxylic acid ((+)-3). A stirred solution of the
(267.8 g, 0.922 mol) in ethyl acetate (AcOEt; 4.8 L) was treated
-methylbenzylamine (117.4 mL, 0.922 mol), and
c-keto acid ( )-3
with (S)-(ꢀ)-
a
3. Conclusion
the mixture was stirred for 22 h. The crystalline precipitate that
formed was filtered off, washed with AcOEt, and recrystallized
With the help of 3D docking studies, we have designed and syn-
thesized novel 2-substituted phthalazinone derivatives that po-
from AcOEt to give (+)-3 as the (S)-(ꢀ)-
a-methylbenzylamine salt
(83.38 g, 22%). The diastereomeric salt was dissolved in CHCl3
(1 L) and washed three times with 1 N HCl (300 mL). The organic
layer was washed successively with water and brine, dried over
MgSO4, and concentrated in vacuo to give (+)-3 as a white powder
tently inhibit PDE4 activity and TNF-a production by rat whole
blood cells. Structure–activity relationship studies revealed that
these inhibitory activities were strongly dependent on the nature
of the substituent at the 2-position of the phthalazinone ring sys-
tem, and that a benzyl moiety with an attached six-membered het-
erocyclic ring conferred particularly potent activity. We also
investigated the effects of an extra ring in dihydropyridazinone
derivatives and found that a cis-fused cyclohexene ring conferred
potent activity, consistent with its filling the hydrophobic space
of the active-site pocket of PDE4 as predicted by our 3D docking
studies. The optimized compounds (+)-11g maleate, 11b and 13
were topically active in a mouse dermatitis model, and their effects
were stronger than those of Protopic ointment. Metabolic stability
tests in vitro suggest that (+)-11g would be rapidly metabolized
in vivo, a characteristic that would decrease the risk of side effects
if it were absorbed into the systemic circulation.
(66.7 g, quant.). ½a D25
ꢁ
+20.00 (c 0.50, CHCl3).
4.1.1.3. cis-(ꢀ)-6-(3,4-Dimethoxybenzoyl)cyclohex-3-enecarb-
oxylic acid ((ꢀ)-3). Compound (ꢀ)-3 was prepared from the
c
-
keto acid ( )-3 and purified by crystallization as the (R)-(+)-
a-
methylbenzylamine salt in a manner similar to that described for
(ꢀ)-3. ½a 2D5
ꢀ25.19 (c 0.50, CHCl3).
ꢁ
4.1.1.4. cis-( )-4-(3,4-Dimethoxyphenyl)-4a,5,8,8a-tetrahydr-
ophthalazin-1(2H)-one (1). To a solution of 3 (5.56 g, 19.2 mmol)
in EtOH (28 mL) was added hydrazine monohydrate (1.86 mL,
38.4 mmol) at room temperature. The mixture was stirred at
80 °C for 15 h. On cooling, the product crystallized from the mix-
ture and was filtered off and washed successively with isopropyl
alcohol and Et2O to give 1 as white crystals (3.57 g, 65%). Mp
178.0–179.9 °C. ESI-MS m/z 287.1 [MH]+. IR: 1674.2, 1516.1,
4. Experimental
4.1. Chemistry
1251.9, 1023.2 cmꢀ1 1H NMR (CDCl3) d: 2.18–2.30 (3H, m), 2.85
.
(1H, t, J = 6.0 Hz), 2.95–3.05 (1H, m), 3.43 (1H, dt, J = 8.4 Hz,
5.4 Hz), 3.93 (3H, s), 3.94 (3H, s), 5.65–5.85 (2H, m), 6.87 (1H, d,
J = 8.4 Hz), 7.23 (1H, dd, J = 8.4 Hz, 1.8 Hz), 7.46 (1H, d, J = 2.2 Hz),
8.62 (1H, br s). Anal. Calcd for C16H18N2O3: C, 67.12; H, 6.34; N,
9.78. Found: C, 67.21; H, 6.36; N, 9.88.
Reagents and solvents were used as obtained from the supplier
without further purification. Melting points were determined on a
Büchi B-545 melting-point apparatus, and are uncorrected. Col-
umn chromatography was carried out on a silica gel column (Fuji
Silysia PSQ-100B or Fuji Silysia NH). Thin-layer chromatography
(TLC) was performed on Merck TLC aluminum sheets Silica Gel
60 F254 with detection by UV quenching at 254 nm or spraying
with phosphomolybdic acid. Synthetic yields of compounds were
not optimized. 1H NMR spectra were recorded on a Varian Gem-
ini-200 (200 MHz) spectrometer. Chemical shifts (d) are given in
ppm from the internal standard, tetramethylsilane, and coupling
constants are given in hertz (Hz). Mass spectra were obtained on
a JEOL JMS-HX100 mass spectrometer. Infrared (IR) spectra were
measured on a PerkinElmerÒ Spectrum100 FT-IR Spectrometer.
Optical rotations were determined on a Horiba SEPA-200 high-sen-
sitivity polarimeter with a 5-cm-path-length cell, and elemental
analyses were performed on a J-Science Lab Micro Corder JM 10
elemental analyzer.
4.1.1.5. cis-(+)-4-(3,4-Dimethoxyphenyl)-4a,5,8,8a-tetrahydr-
ophthalazin-1(2H)-one ((+)-1). Compound (+)-1 was prepared
from (+)-3 in a manner similar to that described for 1. Yield 73%,
white crystals. ½a D25
ꢁ
+849.60 (c 0.50, CHCl3).
4.1.1.6. cis-(ꢀ)-4-(3,4-Dimethoxyphenyl)-4a,5,8,8a-tetrahydr-
ophthalazin-1(2H)-one ((ꢀ)-1). Compound (ꢀ)-1 was prepared
from (ꢀ)-3 in a manner similar to that described for 1. ½a D25
ꢀ890.40 (c
ꢁ
0.50, CHCl3).
4.1.1.7. 2-[cis-4-(3,4-Dimethoxyphenyl)-1-oxo-4a,5,8,8a-tetra-
hydrophthalazin-2(1H)-yl]acetic acid (4). Sodium hydride (60%
dispersion in mineral oil; 8.5 mg, 2.11 mmol) was added to a solu-
tion of 1 (700 mg, 2.44 mmol) in dimethylformamide (DMF;
14 mL) at room temperature. After stirring for 30 min, bromoacetic
acid ethyl ester (611 mg, 3.64 mmol) was added and stirring was
continued for 15 h. The reaction mixture was then poured into
water and extracted with AcOEt. The organic layer was washed
successively with water and brine, dried over MgSO4, and
4.1.1. Preparation of 1, 5, 7a and 7b
4.1.1.1. cis-( )-6-(3,4-Dimethoxybenzoyl)cyclohex-3-enecarb-
oxylic acid (3). Veratrole (50.0 g, 361.5 mmol) was added drop-
wise to a suspension of aluminum chloride (53.0 g, 397.7 mmol) in
CH2Cl2 (550 mL) at 0 °C. After stirring at 0 °C for 30 min, cis-4-cyclo-

K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
6965
concentrated in vacuo. The product was purified by flash column
chromatography (SiO2, 12 g; AcOEt/n-hexane = 1:10?AcOEt/n-
hexane = 1:3) to give 2-[cis-4-(3,4-dimethoxyphenyl)-1-oxo-
4a,5,8,8a-tetrahydrophthalazin-2(1H)-yl]acetic acid ethyl ester as
colorless crystals (768 mg, 85%). Mp 113.7–114.8 °C. ESI-MS m/z
373.1 [MH]+. IR: 1747.2, 1671.9, 1515.4, 1257.3, 1206.4, 1140.3,
3.95 (3H, s), 4.20–4.36 (1H, m), 5.60–5.85 (2H, m), 6.87 (1H, d,
J = 8.4 Hz), 7.20 (1H, dd, J = 8.4 Hz, 2.2 Hz), 7.45 (1H, d,
J = 1.8 Hz). Anal. Calcd for C20H25N2O4ꢂ1H2O: C, 52.76; H, 5.98;
N, 6.15. Found: C, 52.47; H, 5.45; N, 5.99.
4.1.1.10. cis-2-[(E)-4-Bromobut-2-enyl]-4-(3,4-dimethoxy-
phenyl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (6b). Com-
pound 6b was prepared from 1 and 1,4-dibromo-2-butene in a
manner similar to that described for 6a. Yield 55%, colorless crys-
tals. Mp 119.7–121.0 °C. ESI-MS m/z 419.1 [MH]+. IR: 1668.0,
1023.8 cmꢀ1 1H NMR (CDCl3) d: 1.26 (3H, t, J = 7.0 Hz), 2.12–2.56
.
(3H, m), 2.89 (1H, t, J = 5.4 Hz), 3.00 (1H, br d, J = 18.2 Hz), 3.38
(1H, dt, J = 11.4 Hz, 5.6 Hz), 3.93 (6H, s), 4.14 (3H, q, J = 7.0 Hz),
4.63 (2H, dd, J = 73.6 Hz, 17.2 Hz), 5.71 (1H, d, J = 14.6 Hz), 5.79
(1H, d, J = 13.2 Hz), 6.87 (1H, d, J = 8.4 Hz), 7.25 (1H, dd,
J = 8.4 Hz, 1.8 Hz), 7.45 (1H, d, J = 1.8 Hz). Anal. Calcd for
C20H24N2O5ꢂ0.3H2O: C, 63.58; H, 6.56; N, 7.41. Found: C. 63.26;
H, 6.11; N, 7.26. To a solution of the ethyl ester (706 mg,
1.90 mmol) in MeOH (14 mL) was added aqueous 1 N NaOH
(2.9 mL). The mixture was stirred at room temperature for 4 h,
neutralized with aqueous 3 N HCl, concentrated in vacuo, and ex-
tracted with CHCl3. The organic layer was washed successively
with water and brine, dried over MgSO4, and concentrated in vacuo
to give 4 as white crystals (641 mg, 98%). Mp 175.2–178.5 °C. ESI-
MS m/z 345.1 [MH]+. IR: 1667.1, 1514.9, 1257.3, 1136.8,
1515.6, 1257.2, 1023.6 cmꢀ1 1H NMR (CDCl3) d: 2.00–2.30 (3H,
.
m), 2.81 (1H, t, J = 5.0 Hz), 2.90–3.05 (1H, m), 3.36 (1H, dt,
J = 11.6 Hz, 6.2 Hz), 3.93 (3H, s), 3.95 (3H, s), 3.80–4.00 (2H, m),
5.65–5.90 (2H, m), 5.90–6.00 (2H, m), 6.87 (1H, d, J = 8.4 Hz),
7.20 (1H, dd, J = 8.4 Hz, 2.8 Hz), 7.48 (1H, d, J = 2.2 Hz). Anal. Calcd
for C20H23BrN2O3ꢂ0.25H2O: C, 56.68; H, 5.59; N, 6.61. Found: C,
56.44; H, 5.45; N, 6.54.
4.1.1.11. cis-2-{2-[2-(1H-Imidazol-1-yl)ethoxy]ethyl}-4-(3,4-
dimethoxyphenyl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one
(7a). To a solution of imidazole (52 mg, 0.75 mmol) in tetrahydro-
furan (THF; 2 mL) was added NaH (60% dispersion in oil; 33 mg,
0.925 mmol) at room temperature, and the mixture was stirred
for 30 min. A solution of 6a (108 mg, 0.25 mmol) in THF (1 mL)
was added dropwise and stirring was continued for 18 h. The
resulting mixture was diluted with ice water and extracted with
AcOEt. The organic layer was washed successively with water
and brine, dried over MgSO4, and concentrated in vacuo. The prod-
uct was purified by flash column chromatography (SiO2, 12 g;
AcOEt?AcOEt/MeOH = 5:1) to give 7a as a colorless oil (40 mg,
1023.9 cmꢀ1 1H NMR (CDCl3) d: 2.10–2.46 (3H, m), 2.89 (2H, t,
.
J = 5.4 Hz), 3.00 (1H, br d, J = 17.6 Hz), 3.38 (1H, dt, J = 11.4 Hz,
5.0 Hz), 3.93 (3H, s), 3.95 (3H, s), 4.67 (2H, dd, J = 47.6 Hz, 18 Hz),
5.53–5.70 (2H, m), 6.86 (1H, d, J = 8.4 Hz), 7.24 (1H, d, J = 6.6 Hz),
7.44 (1H, s). Anal. Calcd for C18H20N2O5ꢂ0.25H2O: C, 61.97; H,
5.92; N, 8.03. Found: C, 62.06; H, 5.74; N, 7.99.
4.1.1.8. N-[3-(1H-Imidazol-1-yl)propyl]-2-[cis-4-(3,4-dimethoxy-
phenyl)-1-oxo-4a,5,8,8a-tetrahydrophthalazin-2(1H)-yl]acetam-
ide (5). To a solution of 4 (200 mg, 0.58 mmol) in DMF (4 mL) was
added 1-(3-aminopropyl)imidazole (87 mg, 0.7 mmol), 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (133 mg,
0.7 mmol), 1-hydroxybenzotriazole (107 mg, 0.7 mmol) and Et3N
(0.24 mL, 1.74 mmol). The mixture was stirred at room temperature
for 15 h, diluted with water and extracted with AcOEt. The organic
layer was washed successively with water and brine, dried over
MgSO4, and concentrated in vacuo. The product was purified by
flash column chromatography (NH–SiO2, 12 g; AcOEt/n-hex-
ane = 1:1?MeOH/AcOEt = 1:5) to give 1 as a colorless powder
(123.8 mg, 47%). ESI-MS m/z 452.3 [MH]+. IR: 1144.7, 1662.5,
38%). ESI-MS m/z 425.0 [MH]+. IR: 1663.9, 1515.6, 1257.5 cmꢀ1
.
1H NMR (CDCl3) d: 2.05–2.30 (3H, m), 2.78 (1H, t, J = 5.4 Hz), 2.98
(1H, br d, J = 17.2 Hz), 3.34 (1H, dt, J = 11.8 Hz, 6.4 Hz), 3.60–3.80
(5H, m), 3.93 (3H, s), 3.95 (3H, s), 4.04 (2H, t, J = 4.8 Hz), 4.14–
4.29 (1H, m), 5.60–5.85 (2H, m), 6.98 (1H, d, J = 8.6 Hz), 6.93 (2H,
s), 7.27 (1H, dd, J = 8.4 Hz, 1.8 Hz), 7.43–7.49 (2H, m). Anal. Calcd
for C23H28N4O4ꢂ0.6H2O: C, 63.46; H, 6.76; N, 12.87. Found: C,
63.30; H, 6.61; N, 13.35.
4.1.1.12. cis-2-[(E)-4-(1H-Imidazol-1-yl)but-2-enyl]-4-(3,4-dime-
thoxyphenyl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (7b). Com-
pound 7b was prepared from 6b in a manner similar to that de-
scribed for 7a except that the crude solid was washed with Et2O
and filtered. Yield 59%, white crystals. Mp 112–115 °C. ESI-MS
m/z 407.3 [MH]+. IR: 3368.2, 1664.2, 1515.3, 1257.8, 1141.1,
1515.3, 1257.9 cmꢀ1 1H NMR (CDCl3) d: 1.99 (2H, t, J = 7.0 Hz),
.
2.14–2.36 (3H, m), 2.84–3.00 (1H, m), 3.00 (1H, br d, J = 18.0 Hz),
3.270 (2H, ddd, J = 12.8 Hz, 6.2 Hz, 2.4 Hz), 3.42 (1H, dt, J = 16.2 Hz,
5.8 Hz), 3.93 (6H, s), 3.96 (2H, t, J = 7.4 Hz), 4.53 (2H, dd, J = 38.8 Hz,
15.4 Hz), 6.75 (2H, br d, J = 24.6 Hz, 10.6 Hz), 6.21 (1H, br t,
J = 7.4 Hz), 6.87 (1H, d, J = 8.6 Hz), 6.92 (1H, s), 7.05 (1H, s), 7.26 (1H,
dd, J = 8.4 Hz, 2.2 Hz), 7.46 (2H, s). Anal. Calcdfor C24H29N5O4ꢂ0.8H2O:
C, 61.87; H, 6.62; N, 15.03. Found: C, 61.87; H, 6.49; N, 15.00.
1023.1 cmꢀ1 1H NMR (CDCl3) d: 2.05–2.30 (3H, m), 2.81 (1H, m),
.
2.90–3.05 (1H, m), 3.35 (1H, dt, J = 11.8 Hz, 5.8 Hz), 3.93 (6H, s),
4.45–4.55 (4H, m), 5.65–5.90 (4H, m), 6.87 (1H, d, J = 8.4 Hz), 6.90
(1H, s), 7.04 (1H, s), 7.24 (1H, dd, J = 8.0 Hz, 2.2 Hz), 7.44 (1H, d,
J = 2.2 Hz). Anal. Calcd for C23H26N4O3ꢂ0.2H2O: C, 67.36; H, 6.49; N,
13.66. Found: C, 67.49; H, 6.70; N, 13.57.
4.1.1.9. cis-2-[2-(2-Bromoethoxy)ethyl]-4-(3,4-dimethoxyphenyl)-
4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (6a). Sodium hydride
(60% dispersion in mineral oil; 8.5 mg, 2.11 mmol) was added to
a solution of 1 (504 mg, 1.76 mmol) in DMF (8 mL) at 0 °C. After
stirring for 10 min, bis(2-bromoethyl) ether (1.23 mL, 8.80 mmol)
was added and stirring was continued for 15 min. The reaction
mixture was poured into water, neutralized with aqueous 1 N
HCl and extracted with AcOEt. The organic layer washed succes-
sively with water and brine, dried over MgSO4, and concentrated
in vacuo. The product was purified by flash column chromatog-
raphy (SiO2, 40 g; CHCl3?CHCl3/MeOH = 100:1) to give 6a as a
colorless oil (823 mg, quant.). ESI-MS m/z 437.2 [MH]+. IR
4.1.2. Preparation of 11a–g, 12 and 13
4.1.2.1. cis-( )-2-[4-(Bromomethyl)benzyl]-4-(3,4-dimethoxy-
phenyl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (8). Com-
pound 8 was prepared from 1 and
a,
a0-p-dibromoxylene in a
manner similar to that described for 6a. Yield 50%, white pow-
der. ESI-MS m/z 469.2 [MH]+. IR: 1667.0, 1515.7, 1257.8,
1137.3, 1023.1 cmꢀ1 1H NMR (CDCl3) d: 1.90–2.35 (3H, m),
.
2.82 (1H, t, J = 5.8 Hz), 3.03 (1H, br d, J = 21.2 Hz), 3.34 (1H, dt,
J = 11.6 Hz, 5.8 Hz), 3.92 (3H, s), 3.94 (3H, s), 4.47 (2H, s), 5.04
(2H, dd, J = 39.8 Hz, 14.6 Hz), 5.60–5.85 (2H, m), 6.85 (1H, d,
J = 8.4 Hz), 7.20–7.40 (5H, m), 7.43 (1H, d, J = 2.2 Hz). Anal. Calcd
for C24H25BrN2O3ꢂ0.25H2O: C, 60.83; H, 5.42; N, 5.91. Found: C,
60.84; H, 5.27; N, 5.71.
2005.4, 1667.5, 1516.1, 1257.2 cmꢀ1 1H NMR (CDCl3) d: 2.10–
.
2.30 (3H, m), 2.81 (1H, t, J = 5.4 Hz), 3.00 (1H, dd, J = 19.6 Hz,
3.0 Hz), 3.30–3.50 (3H, m), 3.75–3.90 (5H, m), 3.93 (3H, s),

6966
K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
4.1.2.2. cis-(+)-2-[4-(Bromomethyl)benzyl]-4-(3,4-dimethoxy-
phenyl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one ((+)-8). Com-
pound (+)-8 was prepared from (+)-1 in a manner similar to that de-
4.1.2.8. cis-2-[4-(1,4-Dioxa-8-azaspiro[4.5]decan-8-ylmethyl)-
benzyl]-4-(3,4-dimethoxyphenyl)-4a,5,8,8a-tetrahydrophthala-
zin-1(2H)-one (11c). Compound 11c was prepared from 8 and
1,4-dioxa-8-azaspiro[4.5]decane in a manner similar to that de-
scribed for 11b. Yield 56%, white powder. ESI-MS m/z 532.3
scribed for 8. ½a D25
ꢁ
+614.80 (c 0.50, CHCl3).
4.1.2.3. cis-(ꢀ)-2-[4-(Bromomethyl)benzyl]-4-(3,4-dimethoxy-
phenyl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one ((ꢀ)-8).
Compound (ꢀ)-8 was prepared from (ꢀ)-1 in a manner simi-
[MH]+. IR: 3346.1, 1666.7, 1514.9, 1257.0 cmꢀ1 1H NMR (CDCl3)
.
d: 1.72 (4H, t, J = 5.8 Hz), 1.90–2.30 (3H, m), 2.50 (4H, t,
J = 6.2 Hz), 2.82 (1H, t, J = 6.2 Hz), 3.02 (1H, br d, J = 18.0 Hz), 3.36
(1H, dt, J = 11.4 Hz, 5.8 Hz), 3.48 (2H, s), 3.92–3.94 (10H, m), 5.02
(2H, dd, J = 40.8 Hz, 14.4 Hz), 5.60–5.85 (2H, m), 6.85 (1H, d,
J = 8.4 Hz), 7.20–7.36 (5H, m), 7.45 (1H, d, J = 2.2 Hz). Anal. Calcd
for C31H37N3O5ꢂ0.5H2O: C, 68.87; H, 7.08; N, 7.77. Found: C,
68.72; H, 6.80; N, 7.73.
lar to that described for 8. ½a D25
ꢁ
-882.79 (c 1.0, CHCl3).
4.1.2.4. cis-2-[3-(Bromomethyl)benzyl]-4-(3,4-dimethoxyphen-
yl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (9). Compound 9
was prepared from 1 and a,
a0-m-dibromoxylene in a manner sim-
ilar to that described for 6a. Yield 53%, white powder. ESI-MS m/z
469.1 [MH]+. IR: 2364.0, 2004.8, 1666.07, 1515.9, 1257.9, 1138.3,
4.1.2.9. cis-4-(3,4-Dimethoxyphenyl)-2-[4-(piperidin-1-ylmeth-
yl)benzyl]-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (11d).
Compound 11d was prepared from 8 and piperidine hydrochloride
in a manner similar to that described for 11b. Yield 74%, colorless
1024.0 cmꢀ1 1H NMR (CDCl3) d: 1.90–2.35 (3H, m), 2.83 (1H, t,
.
J = 5.8 Hz), 3.02 (1H, d, J = 19.4 Hz), 3.35 (1H, dt, J = 11.4 Hz,
5.8 Hz), 3.92 (3H, s), 3.93 (3H, s), 4.47 (2H, s), 5.03 (2H, dd,
J = 50.2 Hz, 11.4 Hz), 5.60–5.85 (2H, m), 6.85 (1H, d, J = 8.4 Hz),
7.20–7.35 (4H, m), 7.40–7.45 (2H, m). Anal. Calcd for
C24H25BrN2O3ꢂ0.4H2O: C, 60.49; H, 5.46; N, 5.88. Found: C, 60.47;
H, 5.18; N, 5.80.
powder. ESI-MS m/z 474.4 [MH]+. IR: 1671.4, 1514.8, 1257.3 cmꢀ1
.
1H NMR (CDCl3) d: 1.40–1.62 (6H, m), 1.85–2.45 (8H, m), 2.82 (1H,
t, J = 5.8 Hz), 3.03 (1H, br d, J = 17.6 Hz), 3.33 (1H, dt, J = 11.8 Hz,
5.8 Hz), 3.45 (2H, s), 3.91 (3H, s), 3.92 (3H, s), 5.02 (2H, dd,
J = 42.2 Hz, 14.2 Hz), 5.60–5.85 (2H, m), 6.85 (1H, d, J = 8.4 Hz),
7.20–7.36 (5H, m), 7.45 (1H, d, J = 1.8 Hz). Anal. Calcd for
C29H35N3O3ꢂ0.5H2O: C, 72.17; H, 7.52; N, 8.71. Found: C, 71.98;
H, 7.56; N, 8.54.
4.1.2.5. cis-2-[2-(Bromomethyl)benzyl]-4-(3,4-dimethoxyphen-
yl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (10). Compound
9 was prepared from 1 and a,
a0-o-dibromoxylene in a manner sim-
ilar to that described for 6a. Yield 62%, pale yellow powder. ESI-MS
m/z 469.1 [MH]+. IR: 1665.2, 1516.6, 1265.5, 1020.5 cmꢀ1. 1H NMR
(CDCl3) d: 1.80–2.30 (3H, m), 2.84 (1H, t, J = 5.8 Hz), 3.05 (1H, d,
J = 18.6 Hz), 3.34 (1H, dt, J = 11.4 Hz, 6.0 Hz), 3.92 (3H, s), 3.93
(3H, s), 4.85 (2H, dd, J = 12.6 Hz, 10.0 Hz), 5.2 (2H, dd, J = 17.4 Hz,
14.4 Hz), 5.73 (2H, m), 6.86 (1H, d, J = 8.4 Hz), 7.20–7.50 (6H, m).
Anal. Calcd for C24H25BrN2O3ꢂ0.25H2O: C, 60.83; H, 5.42; N, 5.91.
Found: C, 60.69; H, 5.28; N, 5.72.
4.1.2.10. cis-4-(3,4-Dimethoxyphenyl)-2-{4-[(4-methylpipera-
zin-1-yl)methyl]benzyl}-4a,5,8,8a-tetrahydrophthalazin-1(2H)-
one (11e). Compound 11e was prepared from 8 and 1-methylpi-
perazine in a manner similar to that described for 11b. Yield
43%, pale yellow oil. ESI-MS m/z 489.3 [MH]+. IR: 2936.7,
1669.5, 1515.5, 1257.7 cmꢀ1 1H NMR (CDCl3) d: 1.90–2.30 (3H,
.
m), 2.28 (3H, s), 2.40–2.55 (8H, m), 2.82 (1H, br t, J = 6.0 Hz),
3.03 (1H, br d, J = 16.0 Hz), 3.25–3.40 (1H, m), 3.46 (2H, s),
3.92 (6H, s), 5.00 (2H, dd, J = 42.0 Hz, 12 Hz), 5.60–5.90 (2H,
m), 6.85 (1H, d, J = 8.4 Hz), 7.24 (2H, d, J = 8.0 Hz), 7.35 (2H, d,
J = 8.0 Hz), 7.45 (1H, d, J = 1.8 Hz). Anal. Calcd for C29H36N4
O3ꢂ0.5H2O: C, 69.99; H, 7.49; N, 11.26. Found: C, 69.97; H,
7.29; N, 11.10.
4.1.2.6. cis-2-{4-[(1H-Imidazol-1-yl)methyl]benzyl}-4-(3,4-di-
methoxyphenyl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (11a).
Compound 11a was prepared from 8 in a manner similar to that
described for 7a. Yield 49%, white powder. ESI-MS m/z 457.3
[MH]+. IR: 3298.1, 1651.0, 1514.6, 1257.7 cmꢀ1 1H NMR (CDCl3)
.
d: 1.80–2.30 (3H, m), 2.81 (1H, t, J = 5.8 Hz), 3.02 (1H, br d,
J = 21.6 Hz), 3.34 (1H, dt, J = 11.8 Hz, 6.2 Hz), 3.89 (3H, s), 3.92
(3H, s), 5.00 (2H, dd, J = 43.0 Hz, 14.4 Hz), 5.08 (2H, s), 5.60–5.90
(2H, m), 6.86 (1H, d, J = 8.0 Hz), 6.88 (1H, s), 7.07–7.11 (3H, m),
7.23 (1H, dd, J = 8.4 Hz, 2.2 Hz), 7.36–7.44 (3H, m), 7.52 (1H, s)
Anal. Calcd for C27H28N4O3ꢂ1.25H2O: C, 67.69; H, 6.42; N, 11.70.
Found: C, 67.52; H, 6.34; N, 11.46.
4.1.2.11. cis-4-(3,4-Dimethoxyphenyl)-2-{4-[(dimethylamino)-
methyl]benzyl}-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (11f).
Compound 11f was prepared from 8 and dimethylamine hydro-
chloride in a manner similar to that described for 11b. Yield 30%,
pale yellow oil. ESI-MS m/z 434.1 [MH]+. IR: 1669.4, 1516.0,
1257.4 cmꢀ1 1H NMR (CDCl3) d: 1.90–2.30 (3H, m), 2.23 (6H, s),
.
2.82 (1H, br s), 3.03 (1H, br d, J = 17.0 Hz), 3.25–3.40 (1H, m),
3.93 (2H, s), 3.92 (6H, s), 4.97 (2H, dd, J = 49.0 Hz, 14.2 Hz), 5.60–
5.90 (2H, m), 6.85 (1H, d, J = 8.4 Hz), 7.23 (2H, d, J = 8.4 Hz), 7.35
(2H, d, J = 8.0 Hz), 7.45 (1H, d, J = 1.8 Hz). Anal. Calcd for
C26H31N3O3ꢂ0.5H2O: C, 70.56; H, 7.29; N, 9.49. Found: C, 70.89;
H, 7.29; N, 9.49.
4.1.2.7. cis-4-(3,4-Dimethoxyphenyl)-2-{4-[(4-oxopiperidin-1-yl)-
methyl]benzyl}-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (11b).
To a solution of 8 (250 mg, 0.533 mmol) in THF (5 mL) was added
4-piperidone monohydrate (409 mg, 2.665 mmol) and potassium
carbonate (737 mg, 5.33 mmol). The mixture was stirred at room tem-
perature for 12 h. The precipitate that formed was removed by filtra-
tion, the filtrate was extracted with AcOEt, and the organic layer was
washed successively with water and brine, dried over MgSO4, and
concentrated in vacuo. The product was purified by flash column chro-
matography (SiO2, 40 g; CHCl3?CHCl3/MeOH = 100:1) to give 11b as a
colorless powder (175 mg, 67%). ESI-MS m/z 488.3 [MH]+. IR: 1669.9,
4.1.2.12. cis-( )-4-(3,4-Dimethoxyphenyl)-2-[4-(morpholi-
nomethyl)benzyl]-4a,5,8,8a-tetrahydrophthalazin-1(2H)-
one (11g). Compound 11g was prepared from 8 and morpholine
in a manner similar to that described for 11b. Yield 54%, white
powder. ESI-MS m/z 456.3 [MH]+. IR: 3277.7, 1688.7, 1515.0,
1515.2, 1342.9, 1257.4, 1138.9 cmꢀ1
.
1H NMR (CDCl3) d: 1.90–2.35
1257.6 cmꢀ1 1H NMR (CDCl3) d: 1.95–2.30 (3H, m), 2.42 (4H, t,
.
(3H, m), 2.47 (4H, t, J = 5.8 Hz), 2.72 (4H, t, J = 5.8 Hz), 2.83 (1H, t,
J = 5.8 Hz), 3.02 (1H, d, J = 16.8 Hz), 3.34 (1H, dt, J = 11.2 Hz, 5.8 Hz),
3.58 (2H, s), 3.92 (3H, s), 3.93 (3H, s), 5.05 (2H, dd, J = 43.2 Hz,
14.2 Hz), 5.60–5.85 (2H, m), 6.86 (1H, d, J = 8.6 Hz), 7.22–7.39 (5H,
m), 7.46 (1H, d, J = 2.2 Hz). Anal. Calcd for C29H33N3O4ꢂ0.1H2O: C,
71.17; H, 6.84; N, 8.59. Found: C, 70.90; H, 6.68; N, 8.58.
J = 4.8 Hz), 2.82 (1H, t, J = 6.2 Hz), 3.04 (1H, br d, J = 18.8 Hz), 3.35
(1H, dt, J = 11.4 Hz, 6.0 Hz), 3.46 (2H, s), 3.69 (4H, t, J = 4.8 Hz),
3.92 (3H, s), 3.93 (3H, s), 5.04 (2H, dd, J = 43.6 Hz, 14.2 Hz), 5.60–
5.90 (2H, m), 6.85 (1H, d, J = 8.4 Hz), 7.20–7.37 (5H, m), 7.45 (1H,
d, J = 1.8 Hz). Anal. Calcd for C28H33N3O4ꢂ0.2H2O: C, 70.18; H,
7.03; N, 8.77. Found: C, 70.12; H, 7.10; N, 8.75.

K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
6967
4.1.2.13. cis-(+)-4-(3,4-Dimethoxyphenyl)-2-[4-(morpholino-
methyl)benzyl]-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one
((+)-11g). Compound (+)-11g was prepared from (+)-8 in a
1.8 Hz), 7.50 (1H, d, J = 1.8 Hz). Anal. Calcd for C14H16O5ꢂ0.1H2O:
C, 63.20; H, 6.14. Found: C, 63.29; H, 5.94.
manner similar to that described for 11b. ½a D25
ꢁ
+417.39 (c 1.0,
4.1.3.4. cis-2-(3,4-Dimethoxybenzoyl)cyclohexanecarboxylic
acid (15d). A solution of 4-bromoveratrole (24.1 g, 111 mmol) in
THF (77 mL) was added dropwise to a suspension of magnesium
turnings (2.7 g, 111 mmol) in THF (200 mL) at room temperature
and the mixture was stirred for 1.5 h. at 85 °C. After cooling at
0 °C, the solution was added dropwise to a solution of cis-1,2-
cyclohexanedicarboxylic anhydride (17.1 g, 111 mmol) in THF
(277 mL) at 0 °C. Stirring was continued for 1 h at 0 °C and the mix-
ture was then allowed to warm to room temperature and was kept
at room temperature for 15 h. The reaction was quenched by the
addition of a saturated NH4Cl solution and the reaction mixture
was concentrated in vacuo and extracted with AcOEt. The organic
layer was washed successively with water and brine, dried over
MgSO4, and concentrated in vacuo. The product was crystallized
from isopropyl alcohol to give 15d as white crystals (6.0 g, 19%).
Mp 130.5–132.3 °C. ESI-MS m/z 293.2 [MH]+. IR: 1699.1, 1669.7,
CHCl3).
4.1.2.14. cis-(ꢀ)-4-(3,4-Dimethoxyphenyl)-2-[4-(morpholino-
methyl)benzyl]-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one
((ꢀ)-11g). Compound (ꢀ)-11g was prepared from (ꢀ)-8 in a man-
ner similar to that described for 11b. ½a D25
ꢀ419.19 (c 1.0, CHCl3).
ꢁ
4.1.2.15. cis-4-(3,4-Dimethoxyphenyl)-2-[3-(morpholinometh-
yl)benzyl]-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (12). Com-
pound 12 was prepared from 9 and morpholine in a manner
similar to that described for 11b. Yield 59%, white powder. ESI-
MS m/z 476.3 [MH]+. IR: 1669.4, 1514.9, 1257.5 cmꢀ1 1H NMR
.
(CDCl3) d: 1.80–2.30 (3H, m), 2.39 (4H, t, J = 4.8 Hz), 2.83 (1H, t,
J = 5.8 Hz), 3.04 (1H, d, J = 18.2 Hz), 3.37 (1H, dt, J = 11.8 Hz,
5.8 Hz), 3.46 (2H, s), 3.64 (4H, t, J = 4.8 Hz), 3.92 (6H, s), 5.03 (2H,
dd, J = 71.6 Hz, 14.2 Hz), 5.72 (2H, m), 6.85 (1H, d, J = 8.4 Hz),
7.21–7.44 (5H, m), 7.44 (1H, d, J = 2.2 Hz). Anal. Calcd for
C28H33N3O4ꢂ0.2H2O: C, 70.18; H, 7.02; N, 8.77. Found: C, 70.15;
H, 7.07; N, 8.72.
1513.3, 1417.8, 1260.1, 1147.1, 1020.9 cmꢀ1 1H NMR (CDCl3) d:
.
1.30–1.60 (3H, m), 1.70–2.35 (6H, m), 2.65–2.75 (1H, m), 3.92
(1H, s), 3.94 (1H, s), 6.87 (1H, d, J = 8.2 Hz), 7.47 (1H, dd,
J = 8.2 Hz, 2.2 Hz), 7.53 (1H, d, J = 2.2 Hz). Anal. Calcd for
C16H20O5: C, 65.74; H, 6.90. Found: C, 65.39; H, 6.76.
4.1.2.16. cis-4-(3,4-Dimethoxyphenyl)-2-[2-(morpholinometh-
yl)benzyl]-4a,5,8,8a-tetrahydrophthalazin-1(2H)-one (13). Com-
pound 13 was prepared from 10 and morpholine in a manner
similar to that described for 11b. Yield 42%, white powder. ESI-
4.1.3.5. 6-(3,4-Dimethoxyphenyl)-4,5-dihydropyridazin-3(2H)-
one (16a). A suspension of 15a (1.65 g, 6.93 mmol) in EtOH
(10 mL) was added to hydrazine monohydrate (0.67 mL,
13.9 mmol) at room temperature and the mixture was refluxed
for 4 h. After cooling to room temperature, the crystallized product
was filtered off and washed successively with isopropyl alcohol,
aqueous 1 N HCl, and H2O, again with isopropyl alcohol, and finally
with Et2O to give 16a as white crystals (1.46 g, 90%). Mp 169.7–
172.5 °C. ESI-MS m/z 235.0 [MH]+. IR: 3251.9, 1671.8, 1517.6,
1419.9, 1338.0, 1271.1, 1230.3, 1201.7, 1170.6, 1120.45, 1022.3,
MS m/z 476.3 [MH]+. IR: 1670.5 cmꢀ1 1H NMR (CDCl3) d: 1.90–
.
2.53 (3H, m), 2.46 (4H, t, J = 4.2 Hz), 2.82 (1H, t, J = 2.6 Hz), 3.03
(1H, br d, J = 18.8 Hz), 3.35 (1H, dt, J = 11.8 Hz, 6.0 Hz), 3.65 (2H,
s), 3.67 (4H, t, J = 4.8 Hz), 3.90 (3H, s), 3.92 (3H, s), 5.25 (2H, dd,
J = 31.6 Hz, 15.0 Hz), 5.60–5.85 (2H, m), 6.85 (1H, d, J = 8.6 Hz),
7.20–7.27 (4H, m), 7.38–7.41 (2H, m). Anal. Calcd for
C28H33N3O4ꢂ0.7H2O: C, 68.89; H, 7.10; N, 8.61. Found: C, 68.95;
H, 6.84; N, 8.62.
765.5, 576.7 cmꢀ1 1H NMR (CDCl3) d: 2.61 (2H, dd, J = 8.4 Hz,
.
6.6 Hz), 2.98 (2H, dd, J = 8.6 Hz, 6.8 Hz), 3.93 (3H, s), 3.94 (3H, s),
6.87 (1H, d, J = 8.6 Hz), 7.19 (1H, dd, J = 8.0 Hz, 1.8 Hz), 7.42 (1H,
d, J = 1.8 Hz), 8.73 (1H, br s). Anal. Calcd for C12H14N2O3: C,
61.53; H, 6.02; N, 11.96. Found: C, 61.47; H, 5.83; N, 12.06.
4.1.3. Preparation of 17a–d
4.1.3.1. 4-(3,4-Dimethoxyphenyl)-4-oxobutanoic acid (15a). Com-
pound 15a was prepared from veratrole and succinic anhydride
in a manner similar to that described for 3. Yield 12%, white crys-
tals. Mp 126.3–130.5 °C. ESI-MS m/z 261.2 [M+Na]+. IR: 1709.5,
4.1.3.6. cis-5-(3,4-Dimethoxyphenyl)-3,4-diazabicyclo[4.1.0]-
hept-4-en-2-one (16b). Compound 16b was prepared from 15b
in a manner similar to that described for 16a. Yield 71%, white
crystals. Mp 147.0–149.1 °C. ESI-MS m/z 247.2 [MH]+. IR:
1664.5, 1516.5, 1424.7, 1372.6, 1268.6, 1216.8, 1172.7, 1137.6,
1671.1, 1591.2, 1516.0, 1427.6, 1276.7, 1162.0 cmꢀ1 1H NMR
.
(CDCl3) d: 2.81 (2H, t, J = 6.6 Hz), 3.29 (2H, t, J = 6.6 Hz), 3.94 (3H,
s), 3.96 (3H, s), 6.90 (1H, d, J = 8.4 Hz), 7.60 (1H, d, J = 2.2 Hz),
7.62 (1H, dd, J = 8.6 Hz, 2.0 Hz).
1023.4, 579.3, 458.0 cmꢀ1 1H NMR (CDCl3) d: 1.50 (1H, dt,
.
4.1.3.2. cis-2-(3,4-Dimethoxybenzoyl)cyclopropanecarboxylic
acid (15b). Compound 15b was prepared from veratrole and 3-
oxabicyclo[3.1.0]hexane-2,4-dione in a manner similar to that de-
scribed for 3. Yield 28%, white crystals. Mp 133.1–134.9 °C. ESI-MS
m/z 251.1 [MH]+. IR: 1702.9, 1670.7, 1513.9, 1419.3, 1262.0,
J = 8.8 Hz, 5.2 Hz), 1.91 (1H, q, J = 6.6 Hz), 2.33 (1H, q,
J = 8.4 Hz), 2.88 (1H, q, J = 8.8 Hz), 3.93 (3H, s), 3.96 (3H, s),
6.86 (1H, d, J = 8.4 Hz), 7.54 (1H, d, J = 2.2 Hz), 7.73 (1H, dd,
J = 8.4 Hz, 2.2 Hz). Anal. Calcd for C13H14N2O3: C, 63.40; H,
5.73; N, 11.38. Found: C, 63.11; H, 5.74; N, 11.25.
1018.4 cmꢀ1 1H NMR (CDCl3) d: 1.55 (1H, dt, J = 8.4 Hz, 4.6 Hz),
.
1.90 (1H, dd, J = 12.0 Hz, 6.6 Hz), 2.33 (1H, dd, J = 15.8 Hz, 8.8 Hz),
2.92 (1H, dd, J = 15.4 Hz, 8.4 Hz), 3.94 (3H, s), 3.97 (3H, s), 6.92
(1H, d, J = 8.6 Hz), 7.56 (1H, s), 7.72 (1H, d, J = 8.4 Hz). Anal. Calcd
for C13H14O5ꢂ0.1H2O: C, 61.95; H, 5.68. Found: C, 61.93; H, 5.76.
4.1.3.7. cis-5-(3,4-Dimethoxyphenyl)-3,4-diazabicyclo[4.2.0]-
oct-4-en-2-one (16c). Compound 16c was prepared from 15c in
a manner similar to that described for 16a. Yield 62%, pale yel-
low crystals. Mp 184.6–187.1 °C. ESI-MS m/z 261.2 [MH]+. IR:
3233.7, 1665.4, 1515.4, 1462.4, 1420.6, 1338.8, 1265.9, 1171.5,
4.1.3.3. cis-2-(3,4-Dimethoxybenzoyl)cyclobutanecarboxylic
acid (15c). Compound 15c was prepared from veratrole and per-
hydrocyclobuta[c]furan-1,3-dione in a manner similar to that de-
scribed for 3. Yield 46%, pale yellow crystals. Mp 102.4–105.2 °C.
ESI-MS m/z 265.1 [MH]+. IR: 2978.5, 2350.6, 2005.1, 1704.2,
1136.6, 1022.9, 567.4 cmꢀ1 1H NMR (CDCl3) d: 2.35–2.70 (4H,
.
m), 3.30–3.50 (1H, m), 3.80–3.95 (1H, m), 3.91 (3H, s), 3.93
(3H, s), 6.83 (1H, d, J = 8.4 Hz), 7.00 (1H, dd, J = 8.4 Hz, 2.0 Hz),
7.38 (1H, d, J = 2.2 Hz). Anal. Calcd for C14H16N2O3: C, 64.60; H,
6.20; N, 10.76. Found: C, 64.68; H, 6.20; N, 10.99.
1595.0, 1515.2, 1263.9, 1021.9 cmꢀ1 1H NMR (CDCl3) d: 2.20–
.
2.50 (4H, m), 3.40–3.55 (1H, m), 3.92 (3H, s), 3.94 (3H, s), 4.20–
4.35 (1H, m), 6.85 (1H, d, J = 8.4 Hz), 7.41 (1H, dd, J = 8.2 Hz,
4.1.3.8. cis-4-(3,4-Dimethoxyphenyl)-4a,5,6,7,8,8a-hexahydro-
phthalazin-1(2H)-one (16d). Compound 16d was prepared from

6968
K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
15d in a manner similar to that described for 16a. Yield 78%, white
crystals. Mp 166.7–169.0 °C. ESI-MS m/z 289.2 [MH]+. IR: 2934.1,
6.26. Found: C, 59.10; H, 5.19; N, 6.24. Compound 17c was
prepared from cis-3-[4-(bromomethyl)benzyl]-5-(3,4-dimethoxy-
phenyl)-3,4-diazabicyclo[4.2.0]oct-4-en-2-one in a manner similar
to that described for 11b and converted to its HCl salt. Yield 79%,
white powder. FAB-MS m/z 450 [MH]+. IR: 3397.9, 2312.7,
1670.2, 1515.9, 1336.8, 1251.9, 1132.8, 1024.5 cmꢀ1 1H NMR
.
(CDCl3) d: 1.35–1.90 (7H, m), 2.53–2.65 (1H, m), 2.75–2.80 (1H,
m), 3.06–3.20 (1H, m), 3.92 (3H, s), 3.94 (3H, s), 6.87 (1H, d,
J = 8.4 Hz), 7.20 (1H, dd, J = 8.4 Hz, 2.2 Hz), 7.45 (1H, d, J = 1.8 Hz),
8.50 (1H, br s). Anal. Calcd for C16H20N2O3: C, 66.65; H, 6.99; N,
9.72. Found: C, 66.25; H, 7.08; N, 9.75.
1647.1, 1514.3, 1348.2, 1265.2, 1033.1, 772.9 cmꢀ1 1H NMR
.
(CDCl3) d: 2.30–2.95 (6H, m), 3.25–3.50 (3H, m), 3.90 (3H, s),
3.91 (3H, s), 3.80–4.00 (3H, m), 4.12 (2H, d, J = 4.4 Hz), 4.37 (2H,
t, J = 11.8 Hz), 5.05 (2H, s), 6.83 (1H, d, J = 8.4 Hz), 7.03 (1H, dd,
J = 8.4 Hz, 2.0 Hz), 7.33 (1H, d, J = 1.8 Hz), 7.51 (2H, d, J = 8.2 Hz),
7.62 (2H, d, J = 8.4 Hz), 13.24 (1H, br s). Anal. Calcd for
C26H31N3O4ꢂHClꢂ2H2O: C, 59.82; H, 6.95; N, 8.05. Found: C, 59.73;
H, 6.80; N, 8.02.
4.1.3.9. 6-(3,4-Dimethoxyphenyl)-2-[4-(morpholinomethyl)be-
nzyl]-4,5-dihydropyridazin-3(2H)-one (17a). 2-[4-(Bromo-
methyl)benzyl]-6-(3,4-dimethoxyphenyl)-4,5-dihydropyridazin-
3(2H)-one was prepared from 16a and
a,
a0-p-dibromoxylene
in a manner similar to that described for 6a. Yield quant.,
white crystals. Mp 130.2–141.6 °C. ESI-MS m/z 417.0 [MH]+.
4.1.3.12. cis-4-(3,4-Dimethoxyphenyl)-2-[4-(morpholinomethyl)
benzyl]-4a,5,6,7,8,8a-hexahydrophthalazin-1(2H)-one (17d). cis-
2-[4-(Bromomethyl)benzyl]-4-(3,4-dimethoxyphenyl)-4a,5,6,7,8,8a-
hexahydrophthalazin-1(2H)-one was prepared from 16d in a manner
similar to that described for 6a. Yield 43%, colorless oil. ESI-MS m/z
471.1 [MH]+. IR: 1661.4, 1513.2, 1337.6, 1253.7, 1127.9,
IR: 3404.2, 1669.5, 1517.9, 1344.9, 1271.0, 1024.4 cmꢀ1 1H
.
NMR (CDCl3) d: 2.62 (2H, dd, J = 8.8 Hz, 6.6 Hz), 2.94 (2H, dd,
J = 8.4 Hz, 6.6 Hz), 3.92 (6H, s), 4.48 (2H, s), 5.02 (2H, s), 6.86
(1H, d, J = 8.4 Hz), 7.18 (1H, dd, J = 8.4 Hz, 2.2 Hz), 7.31–7.45
(6H, m). Anal. Calcd for C20H21BrN2O3: C, 57.56; H, 5.07; N,
6.71. Found: C, 57.34; H, 4.90; N, 6.65. Compound 17a was
1022.6 cmꢀ1 1H NMR (CDCl3) d: 1.20–1.50 (3H, m), 1.60–1.90 (3H,
.
prepared
from
2-[4-(bromomethyl)benzyl]-6-(3,4-dime-
manner
m), 2.50–2.65 (1H, m), 2.70–2.75 (1H, m), 3.05–3.20 (1H, m), 3.91
(6H, s), 4.47 (2H, s), 5.03 (2H, dd, J = 34.8 Hz, 14.8 Hz), 6.85 (1H, d,
J = 8.4 Hz), 7.20 (1H, dd, J = 8.6 Hz, 2.0 Hz), 7.30–7.45 (6H, m). Anal.
Calcd for C24H27BrN2O3ꢂ0.5H2O: C, 60.00; H, 5.87; N, 5.83. Found: C,
59.76; H, 5.52; N, 5.68. Compound 17d was prepared from cis-2-
[4-(bromomethyl)benzyl]-4-(3,4-dimethoxyphenyl)-4a,5,6,7,8,8a-
hexahydrophthalazin-1(2H)-one in a manner similar to that de-
scribed for 11b. Yield 59%, white powder. FAB-MS m/z 478 [MH]+.
thoxyphenyl)-4,5-dihydropyridazin-3(2H)-one in
a
similar to that described for 11b. Yield quant., white powder.
FAB-MS m/z 424 [MH]+. IR: 3343.9, 1663.8, 1513.2,
1269.9 cmꢀ1 1H NMR (CDCl3) d: 2.42 (4H, t, J = 4.8 Hz), 2.61
.
(2H, dd, J = 8.8 Hz, 6.6 Hz), 2.92 (2H, dd, J = 8.8 Hz, 6.6 Hz),
3.47 (2H, s), 3.69 (4H, t, J = 4.8 Hz), 3.92 (6H, s), 5.01 (2H, s),
6.85 (1H, d, J = 8.4 Hz), 7.19 (1H, dd, J = 8.4 Hz, 2.2 Hz), 7.23–
7.45 (6H, m). Anal. Calcd for C24H29N3O4ꢂ0.75H2O: C, 65.96;
H, 7.03; N, 9.62. Found: C, 65.86; H, 6.74; N, 9.51.
IR: 3277.0, 1663.6, 1514.8, 1255.5 cmꢀ1 1H NMR (CDCl3) d: 1.20–
.
1.50 (4H, m), 1.50–1.70 (2H, m), 1.70–1.90 (1H, m), 2.42 (4H, t,
J = 4.8 Hz), 2.50–2.65 (1H, m), 2.70–2.75 (1H, m), 3.05–3.20 (1H, m),
3.46 (2H, s), 3.69 (4H, t, J = 4.8 Hz), 3.92 (6H, s), 5.02 (2H, dd,
J = 36.2 Hz, 14.8 Hz), 6.85 (1H, d, J = 8.4 Hz), 7.15–7.40 (5H, m), 7.44
(1H, d, J = 1.8 Hz). Anal. Calcd for C26H31N3O2ꢂ0.2H2O: C, 69.89; H,
7.41; N, 8.73. Found: C, 69.75; H, 7.20; N, 8.57.
4.1.3.10. cis-5-(3,4-Dimethoxyphenyl)-3-[4-(morpholinometh-
yl)benzyl]-3,4-diazabicyclo[4.1.0]hept-4-en-2-one
(17b). cis-
3-[4-(Bromomethyl)benzyl]-5-(3,4-dimethoxyphenyl)-3,4-diazabi-
cyclo[4.1.0]hept-4-en-2-one was prepared from 16b in a manner
similar to that described for 6a. Yield 64%, white powder. ESI-MS
m/z 429.1 [MH]+. IR: 1650.2, 1608.0, 1514.9, 1421.2, 1255.2,
4.2. Biological assays
4.2.1. Animals
1023.4 cmꢀ1 1H NMR (CDCl3) d: 0.82 (1H, dd, J = 10.0 Hz, 5.2 Hz),
.
1.80 (1H, dt, J = 9.0 Hz, 4.4 Hz), 2.21–2.26 (1H, m), 2.50–2.62 (1H,
m), 3.92 (3H, s), 3.93 (3H, s), 4.48 (2H, s), 4.98 (2H, dd,
J = 33.0 Hz, 14.4 Hz), 6.89 (1H, d, J = 8.2 Hz), 7.25–7.42 (6H, m).
Anal. Calcd for C21H21BrN2O3ꢂ0.1H2O: C, 58.51; H, 4.96; N, 6.50.
Found: C, 58.36; H, 4.93; N, 6.24. Compound 17b was prepared
For the TNF-a-suppression assay, male Sprague-Dawley rats
were used at 10 weeks of age. For the dermatitis model, male
BALB/c mice were used at eight weeks of age. All animals were
supplied by Japan SLC and were used after quarantine and accli-
mation for a week after delivery to the laboratory. The animals
were housed in rooms at 20–26 °C with a relative humidity of
35–75% under a 12-h light–dark cycle (lights on, 8:00–20:00)
and a ventilation frequency of at least 15 times/h. The animals
were fed standard laboratory chow (F-2; Funabashi Farm) and
were allowed free access to tap water. All experiments were
conducted in compliance with the Law for the Humane Treat-
ment and Management of Animals (Law No. 105, 1 October
1973, as revised on 1 June 2006).
from
cis-3-[4-(bromomethyl)benzyl]-5-(3,4-dimethoxyphenyl)-
3,4-diazabicyclo[4.1.0]hept-4-en-2-one in a manner similar to that
described for 11b. Yield 95%, white powder. ESI-MS m/z 436.3
[MH]+. IR: 1658.5, 1515.6, 1373.1, 1269.3, 1116.9 cmꢀ1 1H NMR
.
(CDCl3) d: 0.81 (1H, dd, J = 10.0 Hz, 5.2 Hz), 1.80 (1H, dt,
J = 30.0 Hz, 16.4 Hz), 2.27 (1H, ddd), 2.42 (4H, t, J = 4.6 Hz), 2.53
(1H, ddd), 3.46 (2H, s), 3.69 (4H, t, J = 4.4 Hz), 3.93 (6H, s), 5.00
(2H, dd, J = 29.2 Hz, 14.2 Hz), 6.89 (1H, d, J = 8.4 Hz), 7.25–7.35
(5H, m), 7.38 (1H, d, J = 1.4 Hz). Anal. Calcd for C25H29N3O4ꢂ0.7H2O:
C, 67.01; H, 6.84; N, 9.38. Found: C, 66.94; H, 6.68; N, 9.55.
4.2.2. Cloning and production of the human PDE4D catalytic
domain
4.1.3.11. cis-5-(3,4-Dimethoxyphenyl)-3-[4-(morpholinometh-
yl)benzyl]-3,4-diazabicyclo[4.2.0]oct-4-en-2-one hydrochloride
(17c). cis-3-[4-(Bromomethyl)benzyl]-5-(3,4-dimethoxyphenyl)-
3,4-diazabicyclo[4.2.0]oct-4-en-2-one was prepared from 16c in a
manner similar to that described for 6a. Yield 71%, white crystals.
Mp 113.3–115.8 °C. ESI-MS m/z 443.2 [MH]+. IR: 2938.5, 1655.1,
1514.5, 1387.0, 1343.3, 1265.32, 1174.15, 1141.2, 1023.7,
The catalytic domain of human PDE4D was cloned from HL-60
cells to include bp 703–1737 of locus NM_006203 (NCBI Reference
Sequence).24 Amplification was performed by polymerase chain
reaction (PCR) with the primers 50-ATGCACAGCTCTAGTCTGAC-30
(forward) and 50-TTTACTAGTTTAGCTCTGAGGGATTGTGCTC-30 (re-
verse). The PCR product was ligated into the EcoRV/SpeI-digested
expression vector pEU3-NII (Toyobo). The vector containing the in-
sert was amplified in Escherichia coli JM109 cells and transcribed
with T7 RNA polymerase. The resulting mRNA was translated
in vitro with the PROTEIOSTM wheat germ cell-free protein synthesis
602.1 cmꢀ1 1H NMR (CDCl3) d: 2.25–2.70 (4H, m), 3.35–3.50 (1H,
.
m), 3.80–3.95 (1H, m), 3.90 (6H, s), 4.49 (2H, s), 5.02 (2H, s), 6.81
(1H, d, J = 8.4 Hz), 7.00 (1H, dd, J = 8.4 Hz, 2.2 Hz), 7.30–7.50 (5H,
m). Anal. Calcd for C22H23BrN2O3ꢂ0.25H2O: C, 59.00; H, 5.29; N,

K. Kagayama et al. / Bioorg. Med. Chem. 17 (2009) 6959–6970
6969
core kit Ver. 2 (Toyobo) according to the manufacturer’s instruc-
tions to give the PDE4D catalytic domain.
10 mM MgCl2) in 100 mM potassium phosphate, pH 7.4. After prein-
cubationat 37 °C for 5 min, the reaction was initiated by the addition
of 5
M. After incubation at 37 °C for 0, 2 or 10 min, the reaction was
stopped by the addition of acetonitrile (500 L). The reaction mix-
ture was then centrifuged at 13,000g for 5 min and a sample of the
supernatant (25 L) was analyzed by HPLC. The HPLC system was
lL of 11g maleate in methanol to give a final concentration of
4.2.3. Assay and determination of IC50 of test compounds
The hydrolysis of cAMP catalyzed by the PDE4D catalytic do-
main was measured in a total volume of 30 lL containing enzyme
2 l
l
and cAMP (10 pmol) in Tris–HCl (pH 7.6), 100 mM NaCl, 150 mM
MgCl2, and 0.5% polyethylene glycol 6000 in the presence or ab-
sence of test compound. After incubation at 30 °C for 90 min, the
reaction was quenched by incubation at 75 °C for 1 min and cAMP
was determined with the HitHunterTM cAMP II Assay Kit (Discov-
eRx). The IC50 was estimated from dose–response curves as the
concentration of test compound required to inhibit cAMP hydroly-
sis by 50% under these conditions.
l
a Waters 2695 separations module fitted with a Waters 2487 dual-
wavelength absorbance detector. The column was an Inertsil ODS-
3 (5
l
m, 4.6 ꢃ 150 mm; GL Science) operated at a flow rate of
1.0 mL/min. The mobile phase was 0.1% ammonium acetate/aceto-
nitrile (50:50) and detection was at 314 nm. The metabolic stability
of 11g was evaluated by its elimination half-life (t1/2). The peak area
of 11g on the HPLC chromatogram was converted to percentage
remaining by taking the peak area at zero-time of incubation as
100%. The elimination rate constant (k) was estimated by linear
regression of the logarithm of the percentage remaining on incuba-
tion time, and t1/2 was calculated as ꢀ0.693/k.
4.2.4. Inhibition of TNF-a production by test compounds in
LPS-treated rat whole blood
The ability of test compounds to suppress TNF-a production by
rat whole blood treated with LPS was evaluated as previously de-
scribed.37 Under ether anesthesia, blood was withdrawn from a
10-week-old male Sprague-Dawley rat through the abdominal vein
into a tube containing 1.6 mg/mL EDTA. A 10 mM stock solution of
test compound in dimethyl sulfoxide was diluted as necessary be-
4.3. Molecular modeling
Molecular modeling was performed with the MOE software
package Version 2003.02 (Chemical Computing Group, Inc.). Active
sites were identified with the Site Finder module, and surfaces
were generated with the Surfaces and Maps module. Inhibitors
were manually docked into the active-site pocket of PDE4 by using
the coordinates of the PDE4–zardaverine complex (PDB entry
1MKD) as a reference. Figures were prepared with PyMOL Version
1.1 (DeLano Scientific LLC).
fore use. Rat blood (80
well plate, test compound (10
ing, and the plate was incubated at 37 °C in 5% CO2 for 30 min.
LPS (10 L of a 100 g/mL solution) was then added with thorough
mixing and incubation was continued for 5 h. Phosphate-buffered
saline (100 L) was then added to each well, the plate was centri-
lL) was dispensed into each well of a 96-
lL) was added with thorough mix-
l
l
l
fuged at 4000 rpm for 2 min, and the resulting supernatants were
Acknowledgments
collected and stored frozen at ꢀ80 °C to await determination of
TNF-a by ELISA. The IC50 value of the test compound was calcu-
We thank Mr. Takahiro Sasagawa for performing the pharmaco-
kinetic evaluation, Dr. Akira Matsuura, Mr. Yoshiaki Shirouchi and
Dr. Masato Matsuoka for practical guidance, and Dr. Gerald E. Smyth
and Dr. Tetsuo Asaki for helpful suggestions during the preparation
of the manuscript. We also thank Prof. Chisato Mukai for his help
with the preparation of the chemical part of the manuscript.
lated as the concentration required for 50% inhibition of TNF-
production under these conditions.
a
4.2.5. Effect of test compounds in a dermatitis model
The ability of test compounds to reduce dermatitis was investi-
gated in a mouse model of dermatitis induced by TNCB.31,32 Eight-
week-old male BALB/C mice were sensitized by applying TNCB/
Supplementary data
acetone solution (1% w/v, 20 lL) dropwise to each side of the right
pinna. From seven days after sensitization, TNCB/acetone solution
was applied to both sides of the right pinna every other day (three
times per week) for a further three weeks. TNCB/acetone solution
alone was applied for the first two of these three weeks so as to
fully induce dermatitis. For the treated group (n = 4), 20 mg of 1%
test compound in a vaseline ointment was applied to each side
of the right pinna five times over the last week in addition to the
TNCB/acetone solution. The diseased control group (n = 4) was
treated in the same way except that vaseline containing no test
compound was applied during the last week. In both groups, the
left pinna was not treated in any way and served as a normal,
non-inflamed pinna for comparison. Six hours after the last appli-
cation, the thickness of the right and left pinnae were measured
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.014.
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