Dual inhibitors of phosphodiesterase-4 and serotonin reuptake

Article abstract of DOI:10.1021/jm8010993

A new class of multitarget compounds was synthesized by linking a novel selective serotonin reuptake inhibitor (SSRI) to a PDE4 inhibitor. The new dual PDE4 inhibitor/SSRI showed antidepressant-like activity in the forced swim test in mice The SSRIs 2-{5-

Full text of DOI:10.1021/jm8010993

1530
J. Med. Chem. 2009, 52, 1530–1539
Dual Inhibitors of Phosphodiesterase-4 and Serotonin Reuptake
John R. Cashman,*,† Troy Voelker,† Han-Ting Zhang,‡ and James M. O’Donnell‡
Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, California 92121, West Virginia UniVersity Health Sciences Center,
Morgantown, West Virginia 26505
ReceiVed September 3, 2008
A new class of multitarget compounds was synthesized by linking a novel selective serotonin reuptake
inhibitor (SSRI) to a PDE4 inhibitor. The new dual PDE4 inhibitor/SSRI showed antidepressant-like activity
in the forced swim test in mice The SSRIs 2-{5-[3-(5-fluoro-2-methoxy-phenyl)-ethyl]-tetrahydro-furan-2-
yl}-ethylamine (14) and 2-{5-[3-(5-fluoro-2-methoxy-phenyl)-propyl]-tetrahydro-furan-2-yl}-ethylamine (15)
were both individually linked to the PDE4 inhibitor 4-(3,4-dimethoxy-phenyl)-4a,5,8,8a-tetrahydro-2H-
phthalazin-1-one (19), via a five-carbon chain. The dual PDE4 inhibitor/SSRI 2-{5-[3-(5-fluoro-2-methoxy-
phenyl)-ethyl]-tetrahydro-furan-2-yl}-ethylamine)-pentyl]-4,5,8,8a-tetrahydro-2H-phthalazin-1-one (21) showed
potent and selective serotonin reuptake inhibition (IC50 value of 127 nM). The dual PDE4 inhibitor/SSRI 21
also inhibited PDE4D3 with a Ki value of 2.0 nM. The dual PDE4 inhibitor/SSRI was significantly more
effective than the individual SSRI alone or fluoxetine in the forced swim test at standard doses. On a molar
basis, the antidepressant-like effect of the dual PDE4 inhibitor/SSRI 21 showed a 129-fold increase in in
vivo efficacy compared to fluoxetine.
Introduction
metabolized, and metabolites are found in the liver, lung, and
brain. Metabolism of antidepressants may play a significant role
in overall efficacy because generally, the secondary amines (i.e.,
desmethyl metabolites) are more potent inhibitors of the uptake
of NE than of 5-HT.4 However, this is not the case with
fluoxetine because the main metabolite (i.e., nor-fluoxetine,
C16H15F3NO) was equally as selective a 5-HT reuptake inhibitor
as its parent.6 With acute treatment, SSRIs will increase 5-HT
receptor-mediated cyclic adenosine monophosphate (cAMP)
signaling.8 Upon repeated treatment with SSRIs, this effect will
be blunted (i.e., some tolerance development) because phos-
phodiesterases (PDEs) are up-regulated and cAMP hydrolysis
is increased.8
The neurodegeneration hypothesis of depression proposes that
depression may be caused by maladaption of immune system
and monoaminergic function.1 Chronic antidepressant treatment
up-regulates hippocampal neurogenesis and could thereby block
or reverse cellular atrophy. Antidepressants imipramine
(C19H24N2) and amitriptyline (C20H23N) inhibit the uptake of
serotonin (5-HT) and norepinephrine (NE) in vitro and in vivo.
On the basis of clinical observations, inhibition of the reuptake
of either of these monoamines is responsible in part for the
mood-elevating action of these compounds.2 Overall, the
literature suggests that 5-HT may be involved in depression and
elevation of 5-HT by reuptake inhibition could be clinically
useful. Reuptake agents such as fluoxetine (C17H18F3NO),
sertraline (C17H17Cl2N), citalopram (C20H21FN2O), and fluvox-
amine (C15H21F3N2O2) are relatively selective serotonin reuptake
inhibitors (SSRIsa) and selectively elevate 5-HT in vitro and in
the brain of animals by inhibiting the human serotonin trans-
porter (hSERT).3,4
SSRIs do not have marked efficacy in animal models of
depression such as the forced swim test (FST) and SSRI
antidepressants may require 1-4 weeks of administration to
observe behavioral improvement involving longer-term adaptive
changes in receptor sensitivity.5 Animals treated repeatedly with
SSRIs such as paroxetine (C19H20FNO3) down-regulate the
SERT.6 The extent of 5-HT reuptake inhibition after repeated
treatment is greater than that observed after acute drug treat-
ment.4 This results in progressive increase in extracellular 5-HT
and stimulation of postsynaptic receptors.7 SSRIs are extensively
Cellular cAMP concentrations are determined by synthesis
(adenylyl cyclases) and hydrolysis (cAMP PDEs). PDEs
comprise a diverse group of enzymes important as regulators
of signal transduction9,10 coded by distinct genes, (e.g., PDE4A,
PDE4B, PDE4C, and PDE4D, referred to as “subtypes”) and
their selective inhibitors as “subtype-selective” inhibitors. PDE4
enzymes are particularly important in neuropsychopharmacol-
ogy9 because PDE4 hydrolyzes cAMP formed by stimulation
of ꢀ adrenergic receptor-linked adenylyl cyclase in rat cerebral
cortical slices.11 Rolipram (C16H21NO3), a selective PDE4
inhibitor, has antidepressant activity and produces memory-
enhancing effects.12 Increased cAMP increases the expression
of a number of PDE4 variants in neurons. Thus, in the case of
SSRIs, the adaptation of PDE4 that occurs in response to
repeated treatment with SSRIs is homeostatic and in opposition
to the acute effects of the drugs.
Selective inhibitors of PDE4 (i.e., rolipram) produce antide-
pressant-like effects in a number of preclinical tests sensitive
to antidepressants. They antagonize the behavioral and physi-
ological effects of reserpine (C33H40N2O9),12 decrease the time
of immobility in the FST in rats and mice,13 and decrease
response rate and increase reinforcement rate of rats under a
differential-reinforcement-of-low-rate (DRL) schedule14 and
support the conclusion that PDE4 inhibitors have antidepressant
activity.9
* To whom correspondence should be addressed. Phone: 858-458-9305.
Fax: 858-458-9311. E-mail: jcashman@hbri.org.
†
Human BioMolecular Research Institute.
West Virginia University Health Sciences Center.
‡
a Abbreviations: SSRI, selective serotonin reuptake inhibitor; FST, forced-
swim test; PDE, phosphodiesterase; cAMP, cyclic adenosine monophos-
phate; ICR mice, inbred Charles River mice; 5-HT, 5-hydroxytryptamine;
5-HIAA, 5-hydroxy indole acetic acid; DA, dopamine; NE, norepinephrine;
hSERT, human serotonin transporter; hNET, human norepinephrine trans-
porter; hDAT, human dopamine transporter; DRL, differential-reinforce-
ment-of-low-rate; ANOVA, analysis of variance; Veh, vehicle.
10.1021/jm8010993 CCC: $40.75
2009 American Chemical Society
Published on Web 03/03/2009

Dual Inhibitors of PDE4 and Serotonin Reuptake
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1531
Scheme 1a
a (a) EtOC(O)CH-PPH3, DCM; (b) H2, Pd/C; (c) DiBAL, toluene; (d) [2-(1,3-dioxolan-2-yl)ethyl]triphenylphosphonium bromide; (e) acetone, H2SO4.
Scheme 2a
If SSRIs produce their effects in part via activation of cAMP
signaling, then loss of activity of the PDE4 subtype involved
would enhance their ability to increase cAMP and alter
subsequent pharmacology. Thus, one adaptation of PDE4 that
occurs in response to repeated treatment with SSRIs increases
5-HT receptor-mediated cAMP signaling. With repeated treat-
ment, this effect will be blunted as tolerance develops because
PDE4 is up-regulated and cAMP hydrolysis is increased. When
animals are repeatedly treated with SSRIs, PDE4 is up-
regulated.7 This may be a consequence of 5-HT receptor-
mediated cAMP signaling. Increased cAMP augments the
expression of a number of PDE4 variants in neurons.8
Compared to imipramine-treated rats, a combination of
rolipram and imipramine showed that chronic coadministration
of a PDE4 inhibitor with an antidepressant may be more
a (a) 1-Butenylmagnesium bromide; (b) NBS, DCM; (c) KCN, NaI,
DMSO; (d) Raney Ni, H2, 1 M NH3 in MeOH.
effective for depression therapy and suggest that elevation of
the cAMP signal transduction pathway is involved in the
Scheme 3a
antidepressive effects.15 Likewise, acute treatment with fluox-
etine to elevate synaptic 5-HT but not dopamine (DA) signifi-
cantly enhanced rolipram binding and accumulation.16 Dual
PDE4 inhibitor/SSRIs offer advantages beyond simple additive
effects of individual agents. One hypothesis is that dual PDE4
inhibitors/SSRIs block the effect of the up-regulation of PDE4
so that the overall increase in 5-HT-mediated cAMP signaling
is preserved with repeated treatment. Herein, we describe an
SSRI that was chemically linked to a PDE4 inhibitor. The dual
PDE4 inhibitor/SSRI was examined in vitro and in vivo for
pharmacological activity.
Results
a (a) Mg turlings, THF, reflux; (b) cis-1,2,3,6-tetrahydrophthalic anhy-
dride, THF; (c) H2NNH2, EtOH, reflux; (d) 1,5-dibromopentane, NaH, DMF.
Chemistry. The chemical synthesis of the dual inhibitors
consisted of coupling an SSRI (i.e., a 2,5-disubstituted tetrahy-
drofuran) with a known PDE4 inhibitor (i.e., a phthalazinone)
via a five-carbon linker to afford the target compound. The SSRI
portion of the dual inhibitor was obtained as follows. The
requisite aldehyde (i.e., 4 (3-(5′-fluoro-2′-methoxyphenyl)pro-
panal) and 7 (4-(5′-fluoro-2′-methoxyphenyl)butyraldehyde),
synthesized by standard chemistry (as described in Scheme 1),
was combined with butenyl magnesium bromide to afford the
alcohols 7-(5′-fluoro-2′-methoxyphenyl)hept-1-en-5-ol (8) and
8-(5′-fluoro-2′-methoxyphenyl)oct-1-en-5-ol (9), respectively
(Scheme 2). Cyclization of 8 and 9 in the presence of NBS
afforded the 2,5-disubstituted tetrahydrofurans 2-(bromomethyl)-
5-(2′-methoxy-5′-fluorophenethyl)tetrahydrafuran (10) and 2-(bro-
momethyl)-5-(3′-(2′′-methoxy-5′′-fluorophenyl)-1′-propyl)tet-
rahydrofuran (11), respectively (Scheme 2). Treatment of 10
or 11 with KCN resulted in the cyano compounds 2-cyanom-
ethyl-5-(5′-fluoro-2-methoxyphenethyl)tetrahydrofuran (12) and
2-cyanomethyl-5-(3′-(2′′-methoxy-5′′-fluorophenyl)-1′-propy-
l)tetrahydrafuran (13), respectively, that were hydrogenated with
H2 in the presence of Raney nickel and ammonia to afford 2-{5-
[3-(5-fluoro-2-methoxy-phenyl)-ethyl]-tetrahydro-furan-2-yl}-
ethylamine (14) and 2-{5-[3-(5-fluoro-2-methoxy-phenyl)-
propyl]-tetrahydro-furan-2-yl}-ethylamine (15), respectively.
The SSRIs 14 and 15 were used to couple with the PDE4
inhibitor portion of the molecule to afford the dual PDE4/SSRI.
The PDE4 inhibitor portion of the dual inhibitor was
synthesized by a previously described route17,18 (Scheme 3).
Because it was known that the PDE4 inhibitor 4-(3,4-dimethoxy-
phenyl)-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one (19) possessed
increased inhibitory potency upon N-alkylation, we hypothesized
that a more potent PDE4 inhibitor could arise from linking the
SSRI and PDE4 compounds via N-alkylation. The final target
compounds 2-{5-[3-(5-fluoro-2-methoxy-phenyl)-ethyl]-tetrahy-
dro-furan-2-yl}-ethylamine)-pentyl]-4,5,8,8a-tetrahydro-2H-ph-
thalazin-1-one (21) and 2-{5-[3-(5-fluoro-2-methoxy-phenyl)-
propyl]-tetrahydro-furan-2-yl}-ethylamine)-pentyl-4a,5,8,8a-
tetrahydro-2H-phthalazin-1-one (22) were prepared in a two step
sequence involving: (1) attachment of a five-carbon linker by
treating the phthalazinone 19 with NaH and 1,5-dibromopentane

1532 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Cashman et al.
Scheme 4a
with a molecule possessing potent SSRI function to make a
compound with a dual PDE4 inhibition/SSRI reuptake inhibition
profile.
The pattern of effects of PDE4 inhibitors on behavior is
distinct from that of selective inhibitors of other PDE families.9,14
Generalized increases in cAMP do not adequately explain the
behavioral effects of PDE4 inhibitors. Thus, changes in cAMP
signaling in particular pathways (e.g., ꢀ adrenergic or NMDA
receptor-mediated signaling) result in the pattern of behavioral
changes observed.11 There are four PDE4 genes (i.e., PDE4A,
PDE4B, PDE4C, and PDE4D). The enzyme PDE4C is mainly
in the periphery and was not studied herein9,10 but PDE4A,
PDE4B, and PDE4D are present in the brain and their
distribution suggest distinct roles in neuropsychopharmacology.
Repeated treatment of animals with the antidepressant de-
sipramine (C18H21N2), which indirectly stimulates ꢀ receptors
alters PDE4A and PDE4B expression.8 The PDE4 inhibitor
rolipram does not produce antidepressant-like effects in PDE4D
knockout mice and PDE4D may play a role in pharmacotherapy
of depression.9,10 Consequently, the inhibition of these PDE4
forms was studied (Table 1).
a (a) CsOH ·H2O, 4 Å sieves, 14 or 15, DMF.
Scheme 5a
a (a) Ethylchloroformate, K2CO3, THF; (b) LAH, THF.
Table 1. Inhibition of Recombinant PDE4 Isoforms with PDE4
Inhibitors and Dual PDE4 Inhibitor/SSRIs
Selective Serotonin Reuptake Inhibitors. Compounds 14
and 15 were derived from molecular dissection of aryltropanes21
and were potent inhibitors of serotonin reuptake (Table 2).
Compounds 14 and 15 were potent inhibitors of radiolabeled
25 binding to the hSERT (i.e., Ki values of 2.1 and 1.1 nM,
respectively) and reuptake (i.e., Ki values of 2.5 and 2.3 nM,
respectively) (Table 2). Compounds 14 and 15 were equal to
or more potent and selective than fluoxetine in reuptake
inhibition of the hSERT. For 14 and 15, the selectivity of either
binding potency or reuptake efficacy for hSERT was very great.
Thus, the binding selectivity ratios Ki(hDAT)/Ki(hSERT) and
Ki(hNET)/Ki(hSERT) for 14 and 15 were 3095 and 1162 and
6642 and 9636, respectively. The reuptake inhibition selectivity
ratios IC50(hDAT)/IC50(hSERT) and IC50(hNET)/IC50(hSERT)
for 14 and 15 were 11140 and 192 and 7386 and 839,
respectively. Compounds related to the PDE4 inhibitor (i.e., 19
and 20) were ineffective at inhibiting 25 binding to the hDAT,
hSERT, or hNET (i.e., Ki values 39-100 µM).
Dual PDE4 Inhibitor/SSRIs. Compounds 19, 21, and 22
were tested for inhibition of cAMP hydrolysis by recombinant
forms of human PDE4D3, human PDE4B3, and human PDE4A1,
in vitro. The Ki value for inhibition of human PDE4D3 for
rolipram, 19, 21, and 22 was 58.9, 6.3, 2.0, and 1.2 nM,
respectively (Table 1). The increase in potency for 21 and 22
compared with 19 suggested that N-substitution increased
inhibition of human PDE4D.17 For human PDE4B3, the Ki
values for inhibition of cAMP hydrolysis of 19, 21, and 22 were
250, 199, and 500 nM, respectively. In the case of human
PDE4A1, compounds 19, 21, and 22 all showed Ki values for
inhibition of cAMP hydrolysis with values greater than 1 µM.
Thus, 19, 21, and 22 possessed considerable inhibitory selec-
tivity for PDE4D.
enzyme
human PDE4D3
human PDE4B3
human PDE4A1
compd
19
20
21
22
Ki (nM)
Ki (nM)
Ki (nM)
6.3
2.3
2.0
1.2
58.9
250
NDa
199
500
ND
>1000
ND
>1000
>1000
ND
Rolipram
a ND, not done.
to afford 2-(5-bromo-pentyl)-4-(3,4-dimethoxy-phenyl-4a,5,8,8a-
tetrahydro-2H-phthalazin-1-one (20) and (2) linking 20 to 14
and 15 with the aid of CsOH (to diminish formation of the
tertiary amine) to afford 21 and 22, respectively (Scheme 4).
The N-methyl derivative of 14 was synthesized in a two-step
sequence starting with protection with ethylchloroformate in the
presence of base to from 23 and then reduction of 23 to 24
with lithium aluminum hydride in the presence of THF
(Scheme 5).
Biology. Inhibition of PDE4 enzymes was conducted with
the synthetic agents (Table 1). In addition, binding affinity of
each compound (i.e., Ki value) was calculated from the IC50
value and measured for each compound by assessing the potency
of inhibition of binding radiolabeled 25 (RTI-55)19 to the hDAT,
hSERT, and hNET20,21 (Table 2). In vitro functional potency
was measured by determining the reuptake inhibition (i.e., IC50
value) of [3H]-DA, [3H]-5-HT, or [3H]-NE at the recombinant
hDAT, hSERT, or hNET in the presence of HEK-293 cells
transfected with the respective transporter cDNA.
PDE4 Inhibitors. Compound 19 and rolipram are known
PDE4 inhibitors, and we confirmed this observation (Table 1).
It was known that N-substitution was beneficial for PDE4
inhibition potency,17 and in the phthalazinone series, the cis-
(()-4a,5,8,8a-tetra- and cis-(()-4a,5,6,7,8,8a-hexahydro-2H-
phthalazin-1-ones showed potent inhibition of PDE4 by occu-
pying a region of space different from that of other fused ring
systems to afford selective inhibition.17,18 N-Substituted deriva-
tives distal to the fused ring system were synthesized to examine
whether additional pharmacological properties could be intro-
duced into 19 while still preserving PDE4 inhibitory activity.
In fact, the N-substituted phthalazinone 20 was a more potent
inhibitor of PDE4D3 than was 19 (Table 1). Thus, a template
with potent PDE4 inhibitory activity was chemically combined
The dual PDE4 inhibitor/SSRIs (i.e., compounds 21 and 22)
were moderately potent inhibitors of binding of radiolabeled
25 to the hSERT having Ki values of 156 and 194 nM,
respectively. For compounds 21 and 22, inhibition of neu-
rotransmitter reuptake was highly selective for the hSERT (i.e.,
IC50 values of 127 and 194 nM, respectively) (Table 2). For 21
and 22, the selectivity of either binding potency or reuptake
efficacy was greatest for hSERT. Finally, the data showed that
dual PDE4 inhibitor/SSRIs 21 and 22 were relatively selective
for the hSERT and did not potently interact with hDAT or hNET
(Table 2).

Dual Inhibitors of PDE4 and Serotonin Reuptake
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1533
Table 2. Inhibition of Radioligand Binding and [3H] Neurotransmitter Uptake in HEK-hDAT, HEK-hSERT, and HEK-hNET Cellsa
binding (Ki, nM)
reuptake (IC50, nM)
hSERT
compd
description
hDAT
hSERT
hNET
hDAT
hNET
cocaine
371 ( 81
276 ( 87
1.1 ( 0.5
2.1 ( 0.2
1.1 ( 0.1
>100000
>100000
156 ( 30
194 ( 87
21.2 ( 4.3
1115 ( 198
1560 ( 300
2440 ( 215
10600 ( 1880
>100000
39119 ( 4915
1748 ( 257
7441 ( 1269
19332 ( 3577
303 ( 74
416 ( 135
7.3 ( 2.9
2.5 ( 0.6
2.3 ( 0.6
>100000
>100000
127 ( 15
194 ( 87
18.4 ( 6.1
835 ( 229
1020 ( 180
481 ( 158
1930.0 ( 463.0
>100000
>100000
957 ( 146
7441 ( 1269
20370 ( 3751
Fluoxetineb
6670 ( 850
6500 ( 828
7307 ( 1331
>100000
19500 ( 7600
27850 ( 5129
16987 ( 1352
>100000
14
15
19
20
21
22
24
SSRI
SSRI
PDE4 inhibitor
N-modified PDE4 inhibitor
dual inhibitor
dual inhibitor
SSRI
>100000
>100000
2478 ( 451
5340 ( 1083
66988 ( 14450
3556 ( 377
5340 ( 1083
>100000
a Values are the mean ( SEM of three to four independent experiments conducted with duplicate (binding) or with triplicate (uptake) determinations
unless the mean of three experiments exceeded 100 µM. Drug inhibition of [125I]-25 binding in HEK-hDAT, HEK-hSERT, or HEK-hNET cell membranes.
Inhibition of [3H]-DA, [3H]-5-HT, [3H]-NE in the presence of HEK-hDAT, HEK-hSERT, or HEK-hNET cells, respectively; b Data taken from ref 20.
In Vivo Studies. The PDE4 inhibitor (i.e., 19), the SSRI (i.e.,
14), and the dual PDE4 inhibitor/SSRI (i.e., 21) were separately
examined for acute and subchronic antidepressant-like effects
in vivo. The two compounds were not administered in combina-
tion. Acute treatment of inbred Charles River (ICR) mice with
either 19 or 21, at doses of 0.3 and 3 mg/kg, decreased duration
of immobility in a dose-dependent manner in the forced-swim
test (FST)22,23 (Figure 1). Compared to vehicle control, the
higher dose of 19 or 21 (3 mg/kg) significantly decreased
immobility in the FST (P < 0.05). In contrast, acute administra-
tion of fluoxetine (40 mg/kg) or repeated treatment with rolipram
(0.5 mg/kg, ip, once a day for 8 days) (data not shown), was
required to produce similar antidepressant-like effects in the
FST. On a molar basis, compound 21 (0.5 µmol/kg) was 129-
fold more efficacious than fluoxetine (65 µmol/kg) in the FST
to give the same effect. Acute treatment with rolipram (0.5 mg/
kg, data not shown) did not alter FST behavior in agreement
with the literature.8 The SSRI, compound 14, at the doses used
(i.e., 10 and 30 mg/kg) tended to decrease immobility, but this
effect was not statistically significant (Figure 1).
Because Balb/c mice have been reported to exhibit a relatively
high immobility baseline and are sensitive to fluoxetine chal-
lenge in the FST,23 the in vivo studies described above were
repeated. Acute administration of fluoxetine (20 mg/kg), 14 (10
mg/kg), or 15 (10 mg/kg) did not markedly alter the duration
of immobility in the FST in Balb/c mice (Figure 2). In contrast,
subchronic administration of a lower dose of fluoxetine (10 mg/
kg) significantly decreased immobility (P < 0.05). However,
subchronic treatment with 14 (10 mg/kg) or 21 (1 mg/kg) did
not change immobility compared to vehicle control (Figure 3).
It is possible that the lack of in vivo activity for 14 or 15 was
a consequence of metabolic instability. To test this possibility,
the in vitro metabolic stability of 14, 15, 19, 21, 22, and 24
was examined.
In Vitro Metabolic Studies. The in vitro metabolism of
compounds 14 and 2-{5-[3-(5-fluoro-2-methoxy-phenyl)-ethyl]-
tetrahydro-furan-2-yl}-ethylamine)-methyl-amine (24) were ex-
Figure 1. Effect of acute treatment of test compounds on immobility
in the forced-swim test in ICR mice: (A) compound 14 (10 mg/kg, 37
µmol/kg and 30 mg/kg, 112 µmol/kg), (B) compound 19 (0.3 mg/kg,
1.0 µmol/kg and 3 mg/kg, 10 µmol/kg), and (C) compound 21 (0.3
mg/kg, 0.5 µmol/kg and 3 mg/kg, 5 µmol/kg). Vehicle (Veh) or test
agent was injected ip 30 min prior to the test. n ) 9-11; * P < 0.05
vs Veh.
amined in the presence of mouse and human liver microsomes
supplemented with NADPH to examine the metabolic stability
of the compounds and because metabolism plays a large role
in the pharmacological activity of SSRIs. We hypothesized that
by blocking a prominent route of metabolism of SSRIs (i.e.,
aliphatic amine metabolism), a more efficacious agent could
be obtained. Compounds 19, 21, and 22 were examined in the
presence of mouse and human liver microsomes supplemented
with NADPH. An efficient HPLC method was established to
afford separation of the starting material from any putative
metabolites (e.g., aldehyde, hydroxylamine, nitrone). Aerobic
incubations of 14 and 24 in the presence of mouse and rat liver
microsomes supplemented with NADPH showed a time-
dependent loss of substrate. For 14 and 24, the calculated half-
life was 56 or 106 and 58 or 129 min, respectively, in the
presence of mouse and rat liver microsomes (Table 3). In the
presence of mouse liver microsomes, compounds 19 and 21
showed considerable metabolic stability, having calculated half-
lives of 211 and 154 min, respectively. Compound 21 was not

1534 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Cashman et al.
possessed more potency at inhibiting PDE4. The dual PDE4
inhibitor/SSRIs 21 and 22 possessed considerable potency for
inhibition of binding of 25 and inhibition of 5-HT reuptake in
the presence of the hSERT. Compared to 14 and 15, however,
some functional activity was lost. Because studies have shown
that the trans diastereomer of 14 or 15 possess about 7-fold
more hSERT reuptake inhibition activity than the cis diastere-
omer (unpublished data) preparation of a dual inhibitor with
only the trans diastereomer should increase potency. While some
loss of in vitro binding potency and reuptake inhibition of the
hSERT was observed in chemically linking SSRIs 14 and 15
to compound 20, nevertheless, significant in vitro activity was
retained.
Figure 2. Effect of acute treatment with fluoxetine (20 mg/kg, 65 µmol/
kg), compound 14 (10 mg/kg, 37.4 µmol/kg), and compound 15 (10
mg/kg, 35.5 µmol/kg) on immobility in the forced-swim test in Balb/c
mice. Vehicle or test agent was injected ip 30 min prior to the test. n
) 10-11.
A dual PDE4 inhibitor/SSRI offers advantages beyond simple
additive effects of coadministration of the individual agents
including providing greater symptomatic efficacy and better
utility. The “message-address” concept of a dual agent could
afford proximal inhibition of PDE4, thus keeping ample cAMP
concentrations present near the activated transporter for greater
functional selectivity. Decrease in the catabolism of cAMP might
be expected to increase the sensitivity to and effectiveness of
SSRI antidepressants. Evidence points to the PDE4D subtype
as an important component of the signaling pathway involved
in mediation of antidepressant effects on behavior. Dual PDE4
inhibitor/SSRIs can affect local control of 5-HT and cAMP in
a stimulus-selective manner because of the compartmentalization
of the PDE enzymes. In fact, acute treatment of animals with
fluoxetine that elevated synaptic 5-HT and NE but not DA levels
significantly enhanced rolipram binding and retention.16 By
chemically combining a selective PDE4 inhibitor with an SSRI,
synergistic antidepressant effects might be observed.
Figure 3. Effect of subchronic administration of fluoxetine (10 mg/
kg, 32.3 µmol/kg), compound 14 (10 mg/kg, 37.4 µmol/kg), and
compound 21 (1.0 mg/kg, 1.6 µmol/kg) on immobility in the forced-
swim test in Balb/c mice. Vehicle or the test agent was injected ip 23,
5, and 1 h prior to the test. n ) 10-11; * P < 0.05 vs vehicle.
The greater PDE4 inhibitor potency of the dual agent will
allow lower doses to be used and decrease side effects. Because
CNS disorders are recognized as polyetiological in nature, drugs
that modulate multiple targets will contribute to the multifac-
torial processes in disease treatment. PDE4D inhibition results
in a pattern of behavior that is indicative of an antidepressant-
like effects.13,14 This suggests that PDE4D is critical in
mediating the antidepressant-like effects of PDE4 inhibitors. The
PDE4 inhibitor 19 and the dual PDE4 inhibitor/SSRI 21 at doses
of 0.3 and 3 mg/kg, respectively, decreased duration of
immobility in the FST in a dose-dependent manner in ICR mice.
Compared to vehicle control, 3 mg/kg significantly decreased
immobility in the FST (P < 0.05). Acute administration of 14
decreased immobility in the FST, but this did not reach statistical
significance. In contrast, 40 mg/kg of fluoxetine or repeated
treatment with rolipram (0.5 mg/kg, ip, once a day for 8 days)
was required to produce similar antidepressant-like effects in
the FST; acute treatment with 0.5 mg/kg rolipram did not alter
FST behavior.
Table 3. Effect of Hepatic Microsomes on the Metabolic Stability of
SSRIs, PDE4 Inhibitors, and Dual PDE4 inhibitor/SSRIs
compd
RLMa t1/2 (min)
MLMb t1/2 (min)
HLMc t1/2 (min)
14
19
21
22
24
106.3
NA
NA
NA
129.0
56.0
211.3
153.9
ND
NAd
NDe
ND
ND
NA
58.5
a RLM, rat liver microsomes. b MLM, mouse liver microsomes. c HLM,
human liver microsomes. d NA, not available. e ND, no detectable degrada-
tion observed in the presence of animal liver microsomes supplemented
with NADPH for 60 min.
detectably metabolized in the presence of human liver mi-
crosomes. No detectable metabolic instability of compound 22
was observed in the presence of either mouse or human liver
microsomes up to 60 min (Table 3), and we judged these
compounds were metabolically stable enough to undertake in
vivo studies. Lack of efficacy of 14 and 15 was likely not due
to metabolism.
Acute administration of fluoxetine (20 mg/kg) or 14 (10 mg/
kg) did not alter the duration of immobility in the FST in Balb/c
mice, but SSRIs are known to not significantly affect changes
in FST after acute administration. In contrast, subchronic
administration of a lower dose of fluoxetine (10 mg/kg)
significantly decreased immobility (P < 0.05). However, sub-
chronic treatment with 14 or 15 (10 or 1 mg/kg, respectively)
did not change immobility compared to the vehicle control.
Discussion
A dual PDE4 inhibitor/SSRI was synthesized by linking an
SSRI with a PDE4 inhibitor via a five-carbon linker. Previously,
it was shown that the potency of PDE4 inhibition of 19 was
not diminished by addition of lipophilic bulk to the phthalazin-
1-one nitrogen atom and 20 was a more potent PDE4D3
inhibitor than 19. Thus, elaboration of 21 and 22 afforded PDE4
inhibitors linked to an SSRI that retained considerable phar-
macological activity. Compared to 19, compounds 21 and 22
In summary, we hypothesize that dual PDE4 inhibitor/SSRIs
offer an advantage over simple additive effects of coadminis-
tered agents. Dual PDE4 inhibitor/SSRIs offer the advantage
of blocking the effect of the up-regulation of PDE4 in a
compartmentalized manner. While PDE4 expression will still
increase, its hydrolytic activity will be blocked. Thus, the overall

Dual Inhibitors of PDE4 and Serotonin Reuptake
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1535
Transporter Binding Assays. HEK-hDAT, -hSERT, or -hNET
cells were grown until confluent as described previously.20,24 Cells
were scraped from plates and centrifuged for 20 min at 30000g
and the pellet was resuspended in 0.32 M sucrose with a Polytron
at a setting of 1 for 5 s. Assays contained 50 µL of membrane
preparation, 25 µL of the test compound and 25 µL of [125I]-25
(40-80 pM final concentration) in a final volume of 250 µL Krebs
HEPES buffer (25 mM HEPES, 122 mM NaCl, 5 mM KCl, 1.2
mM MgSO4, 2.5 mM CaCl2, 1 µM pargyline, 100 µM tropolone,
2 mg glucose/mL, and 0.2 mg ascorbic acid/mL at pH 7.4.).
Membranes were preincubated with test compounds for 10 min
before addition of [125I]-25. Specific binding was determined as
the difference in binding observed in the presence and absence of
5 µM mazindol (HEK-hDAT and hNET) or 5 µM imipramine
(HEK-hSERT). The incubations were done in the dark and
terminated by filtration onto a Whatman GF/C filters using a 96-
well Tomtech cell harvester (Tomtech, Orange, CT). Scintillation
fluid was added to each filter spot and radioactivity remaining on
the filter was determined using a Wallace ꢀ-plate reader (Wallace
Laboratories, Cranbury, NJ). Specific binding was defined as the
difference in binding in the presence and absence of 5 µM mazindol
(hDAT and hNET) or 5 µM imipramine (hSERT).
Inhibition of Substrate Uptake. HEK-hDAT, -hSERT, or
-hNET cells were grown as described above. Cells were scraped
from the plates and suspended cells were added to a 96-well plate
containing test compounds and Krebs-HEPES buffer in a final assay
volume of 0.5 mL. After a 10 min preincubation in a 25 °C water
bath, [3H]-labeled neurotransmitter (50 µL, 20 nM final concentra-
tion) was added and the assay was initiated. After 10 min, the
incubation was terminated by filtration onto GF/C filters presoaked
with 0.05% polyethylenimine using a Tomtech call harvester and
radioactivity remaining on the filters was determined as described
above. Specific uptake was defined as the difference in uptake in
the presence and absence of 5 µM mazindol (hDAT and hNET) or
5 µM imipramine (hSERT).
increase in serotonin receptor-mediated cAMP signaling will
be preserved with repeated treatment. Moreover, we suggest
that compounds such as 21 show the utility of addressing
multiple targets to alleviate complex disease such as depression.
Experimental Section
General. Chemicals used in this study were of the highest purity
available. Commercially available reagents including 2-methoxy-
5-fluoro benzaldehyde were purchased from Aldrich Chemical Co.
(Milwaukee, WI) or VWR (San Diego, CA) and were used as
received. All moisture sensitive reactions were carried out in flame-
dried glassware under an argon atmosphere. Tetrahydrofuran (THF)
and toluene were freshly distilled from calcium hydride under an
argon atmosphere. Methanol (CH3OH) was passed through a
column of neutral alumina and stored over 3 Å molecular sieves
prior to use. Rolipram, fluoxetine, diethyleneaminetetracetic acid
(DETAPAC), and all the compounds of the NADPH-generating
system were obtained from Sigma Chemical Company (St. Louis,
MO). Human liver, pooled female liver, and pooled male rat liver
microsomes were purchased from BD Gentest Corp. (Woburn, MA).
The human liver microsomes had the following functional activities
(nmol/min/mg of protein): phenacetin O-deethylase (0.18), coumarin
7-hydroxylase (2.0), (S)-mephenytoin N-demethylase (0.05), di-
clofenac 4′-hydroxylase (1.9), (S)-mephenytoin 4′-hydroxylase
(0.03), bufuralol 1′-hydroxylase (0.12), chlorzoxazone 6-hydroxy-
lase (1.5), testosterone 6ꢀ-hydroxylase (4.6), and methyl p-tolyl
sulfide oxidase (4.3). The pooled male rat liver microsomes had
the following functional activities (nmol/min/mg of protein):
testosterone 6ꢀ-hydroxylase (5.8) and nicotine oxidase (2.2). The
pooled female mouse liver microsomes had the following functional
activities (nmol/min/mg of protein): 7-ethoxyresorufin O-deethylase
(0.39), p-nitrophenol hydroxylase (1.9), lauric acid-hydroxylase
(0.86), and testosterone 6ꢀ-hydroxylase (5.0). Cocaine was provided
by the National Institute on Drug Abuse, NIH (Bethesda, MD).
Compound 25 was a kind gift of Dr. Ivy Carroll (RTI, Research
Triangle Park, NC). [3H]-DA, [3H]-5-HT, [3H]-NE, and [125I]-25
were purchased from Perkin-Elmer Life Sciences (Boston, MA).
The preparation of the hDAT used was described previously.20 The
hSERT cDNA and HEK cells transfected with hNET cDNA was
generously supplied by Dr. Randy Blakely (Vanderbilt University,
Nashville, TN). Analytical thin-layer chromatography (TLC) was
done on K6F silica gel 60 Å glass-backed plates from Whatman
(Clifton, NJ). Compounds were detected using UV absorption at
254 nm and/or stained with I2 (iodine). Flash chromatography was
done on (60 Å) pore silica gel from E. Merck (Darmstadt,
Germany).
PDE4 Assay. PDE4 enzyme assays were carried out as described
previously.25 Studies with recombinant PDE4 enzymes were carried
out in the laboratory of Professor Marco Conti (Stanford University,
Palo Alto, CA).
Data Analysis. GraphPad Prism (GraphPad Software, San Diego,
CA) was used to determine the saturation and binding kinetic data.
IC50 values were converted to Ki values using the Cheng-Prusoff
equation.
Mouse and Rat Liver Microsome Stability Assay. Microsomal
incubations (final volume 0.25 mL) with the SSRIs, dual PDE4
inhibitor/SSRIs or PDE4 inhibitor was done with an HPLC assay
as described above. Diluted stocks of either mouse, human, or rat
liver microsomes (0.5 mg of protein), 100 µM potassium phosphate
buffer (pH 7.4), 0.5 mM NADP+, 0.5 mM glucose-6-phosphate, 5
IU/mL glucose-6-phosphate dehydrogenase, 0.6 mM DETAPAC,
and 3 mM MgCl2 were combined and placed on ice. For a metabolic
stability assay, either vehicle or 40 µM test compound was added
and the incubation was initiated at 37 °C with constant shaking.
After 0, 10, 25, 40, and 60 min, the incubations were stopped by
the addition of 1 mL CH2Cl2/2-propanol (3:1, v:v). After thorough
mixing, the organic layer was separated from the aqueous portion
by centrifugation at 12000g. The organic material was evaporated
with a stream of argon. The residue was dissolved in methanol
(200 µL), and the sample was analyzed by HPLC.
In Vivo Evaluation of Selected Compounds. Separate nonha-
bituated male mice were used in the conduct of this work. Fully
approved animal protocols were used and the studies were done in
keeping with the NIH standards for use of experimental animals.
Experiment 1. Forty male ICR mice, weighing 26.2 ( 0.3 g,
were housed in a temperature-controlled room (22-23 °C) and
maintained on a 12 h on/12 h off light cycle (lights on at 6:00
a.m.). Water and food were freely available in the home cages.
The mice were randomly divided into four groups: (A) vehicle,
(B) compound 14, (C) compound 19, and (D) compound 21 and
administered compounds were dissolved in 0.9% saline containing
5% dimethyl sulfoxide (DMSO). Saline containing the same
Instrument Analysis. NMR spectra were recorded with a Bruker
spectrometer operating at 500 MHz (NuMega Resonance Labora-
tories, Inc., San Diego, CA) or at 300 MHz by an in-house Varian
spectrometer (Palo Alto, CA) using the solvent specified. Chemical
shifts were reported in parts per million (ppm, δ) using residual
solvent signals as internal standards. Low resolution mass spec-
troscopy (LRMS) was done with an HP 1100 mass spectrometer
(HT Laboratories, San Diego, CA) using electrospray ionization
(ESI) or with an in-house Hitachi M-8000 3DQMS (ion trap) mass
spectrometer using ESI. High resolution mass spectroscopy (HRMS)
was done with a Micromass LCT time-of-flight mass spectrometer
at the University of Montana Mass Spectral Facility (Missoula, MT)
using ESI.
The 2,5-disubstituted tetrahydrofurans and the dual PDE4
inhibitor/SSRIs were characterized by 1H NMR, LRMS, and HRMS
and their purities (>95%) were quantified by HPLC in two distinct
solvent systems. Analytical HPLC measurements were run on a
Hitachi L-6200 system equipped with a Hitachi L-7400 UV
detector. Separations were done with a 5 µm, 4.6 mm × 250 mm,
Axxi-chrom silica column (Richard Scientific, Novato, CA) or with
a 5 µm, 4.6 mm × 250 mm Supelco HS F5 pentafluorophenyl
column (Supelco Inc., Bellefonte, PA). HPLC analysis is described
1
in the Supporting Information. H NMR and mass spectra were
consistent with the assigned structures.

1536 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Cashman et al.
percentage of DMSO was used as vehicle. The lower doses (i.e.,
0.3 mg/kg for 19 and 21 or 10 mg/kg for 14) were tested followed
by the larger doses (3 mg/kg for 19 and 21 or 30 mg/kg for 14) 1
week later, when the mice were again randomly divided into four
groups as described above (Figure 1). The forced-swim test (FST)
was carried out as described previously.23 Mice were given a
swimming pretest session once a day for two successive days. Then
24 hours after the last session, mice were injected with vehicle or
the lower dose of each test compound by the ip route 30 min prior
to the FST. One week later, administration of the larger dose of
each of the test compounds was repeated. During the pretest and
test sessions, each mouse was placed for 6 min in a plastic cylinder
(45 cm high × 20 cm diameter), which was filled to a depth of 28
cm with water (23 ( 1 °C). The duration of immobility, which
was defined as floating in an upright position without additional
activity other than that necessary for the animal to keep its head
above water, was recorded.22,23 In a control experiment, acute
administration of fluoxetine (40 mg/kg) or repeated treatment with
rolipram (0.5 mg/kg, ip, once a day for 8 days, was required to
produce similar antidepressant-like effects in the FST (data not
shown).
Experiment 2. Forty Balb/c male mice weighing 22.4 ( 0.1 g
were used in this experiment, which was carried out in two sessions.
In each session, the mice were divided into four groups: (A) vehicle
(saline containing 5% DMSO), (B) fluoxetine, 20 mg/kg, (C)
compound 21, 10 mg/kg, and (D) compound 14, 10 mg/kg for the
first session and (A) vehicle, (B) Fluoxetine, 10 mg/kg, (C)
compound 14, 10 mg/kg, and (D) compound 21, 10 mg/kg for the
second session, which was carried out 4 weeks later. Mice in the
first session (Figure 2) were treated with vehicle or the test agents
acutely, whereas mice in the second sessions (Figure 3) were treated
subchronically, i.e., vehicle or drugs were given ip 23, 5, and 1 h
before the FST. The experiment was done similar to Experiment 1
except for the pretest training, which was not carried out in the
second session.
atmosphere of Ar was added a DIBAL solution (1 M in toluene,
30 mL, 30 mmol, 1.2 equiv) at -78 °C. The reaction was stirred
at -78 °C for 2 h. Methanol (2 mL) was then added to the mixture,
and the reaction was allowed to warm to 0 °C. The reaction mixture
was then poured into a separatory funnel containing HCl solution
(1 N, 100 mL). The organic fraction was extracted with EtOAc (3
× 60 mL), and the combined organic layers were washed with brine
(80 mL) and dried over sodium sulfate. The crude product obtained
by removal of the solvent in vacuo was purified by flash column
chromatography (EtOAc/hexane, 10:90, v:v, Rf ) 0.1) to give the
1
product (3.1 g, 66%). H NMR (CDCl3, 500 MHz): δ 9.80 (s, 1
H), 6.88-6.86 (m, 2 H), 6.75 (dd, J ) 4.6, 8.6 Hz, 1 H), 3.79 (s,
3 H), 2.91 (t, J ) 7.4 Hz, 2 H), 2.72 (t, J ) 7.4 Hz, 2 H).
1-(5′-Fluoro-2′-methoxyphenyl)-4-ethylenedioxy-1-butene (5).
To a suspension of 2-(1,3-dioxolan-2-yl)ethyltriphenylphosphonium
bromide (5.0 g, 11.3 mmol, 1 equiv) in THF (30 mL) under an
atmosphere of Ar was added NaH (60% in mineral oil, 0.48 g,
11.3 mmol, 1 equiv), and the mixture obtained was heated to reflux
for 1 h. The orange suspension obtained was cooled to 0 °C and
5-fluoro-2-methoxybenzaldehyde (1.54 g, 10 mmol, 0.9 equiv) was
added and the reaction was warmed to room temperature and
continued with stirring for 12 h. The reaction mixture was poured
into a separatory funnel containing ammonium chloride aqueous
solution (sat. NH4Cl/H2O 50:50, v:v, 50 mL). The organic material
was extracted with EtOAc (3 × 80 mL), and the combined organic
layers were washed with brine (60 mL) and dried over sodium
sulfate. The solvent was then removed in vacuo and the crude
product obtained was purified by flash column chromatography
(EtOAc/hexane, 7:93, v:v, Rf ) 0.15) to give the product (2.63 g,
1
66%) as a 4:1 mixture of cis and trans diastereomers. H NMR
(major isomer) (CDCl3, 500 MHz): δ 7.08 (dd, J ) 3.1, 8.7 Hz, 1
H), 6.93 (dt, J ) 3.1, 8.7 Hz, 1 H), 6.78 (dd, J ) 4.3, 8.7 Hz, 1 H),
6.63 (d, J ) 11.9 Hz, 1 H), 5.83 (td, J ) 7.3 Hz, 11.9 Hz, 1 H),
4.99 (t, J ) 4.6 Hz, 1 H), 4.02-3.99 (m, 2 H), 3.9-3.87 (m, 2 H),
3.8 (s, 3 H), 2.64-2.62 (m, 2 H).
In Vivo Statistics. All in vivo data were analyzed by one-way
analyses of variance (ANOVA) followed by Dunnett’s tests for post
hoc comparisons of individual means.
4-(5′-Fluoro-2′-methoxyphenyl)-1-ethylenedioxybutane (6). To
a solution of 5 (1.75 g, 7.3 mmol, 1 equiv) in ethanol (200 proof,
80 mL) under an atmosphere of Ar was added Pd/C (10%, 300
mg, 0.1 equiv), and the flask containing the mixture was evacuated
and purged with H2 three times. H2 gas was attached to the flask
and the reaction was allowed to proceed for 72 h at room
temperature. The reaction mixture was then filtered through a pad
of celite and eluted with EtOH. The crude product was obtained
Synthesis. Ethyl-3-(2′- Methoxy-5′-fluorophenyl)-2-propenoate
(2). To a solution of 2-methoxy-5-fluoro benzaldehyde (4.3 g, 28.0
mmol, 1 equiv) in dry CH2Cl2 (30 mL) at 0 °C was added
carbethoxymethylenetriphenylphosphorane (10.7 g, 30.1 mmol, 1.1
equiv) portionwise and the reaction was warmed to room temper-
ature and stirred for 12 h. The solvent was then removed in vacuo,
and the residue was purified by flash column chromatography
(EtOAc/hexane, 10:90, v:v, Rf ) 0.2) to yield the product (6.0 g,
95%) as a 4:1 mixture of trans/cis diastereomers. 1H NMR (CDCl3,
500 MHz): δ 7.93 (d, J ) 16.2 Hz, 0.8 H), 7.33 (dd, J ) 3.1, 9.2
Hz, 0.2 H), 7.21 (dd, J ) 3.1, 9.2, Hz, 0.8 H), 7.08 (d, J ) 12.5
Hz, 0.2 H), 7.03 (dt, J ) 3.0, 8.8 Hz, 0.8 H), 6.99 (dt, J ) 3.0,
8.8 Hz, 0.2 H), 6.84 (dd, J ) 3.1, 8.6 Hz, 0.8 H), 6.79 (dd, J )
3.3, 9.1 Hz, 0.2 H), 6.47 (d, J ) 16.2 Hz, 0.8 H), 5.99 (d, J ) 12.5
hz, 0.2 H), 4.25 (q, J ) 7.1 Hz, 1.6 H), 4.15 (q, J ) 7.0 Hz, 0.4
H), 3.86 (s, 2.4 H), 3.81 (s, 0.6 H), 1.34 (t, J ) 7.1 Hz, 2.4 H),
1.22 (t, J ) 7.0 Hz, 0.6 H).
Ethyl-4-(5′-fluoro-2′-methoxyphenyl)propanoate (3). To a
solution of 2 (6.0 g, 27.0 mmol, 1 equiv) in ethanol (200 proof, 40
mL) under Ar was added Pd/C (10%, 250 mg, 0.1 equiv), and the
flask containing the mixture was evacuated and purged with H2
three times. H2 was attached to the flask and the reaction was
allowed to stir for 15 h at room temperature. The reaction mixture
was filtered through a pad of silica and eluted with EtOAc/hexane,
50:50, v:v. The crude product obtained by removal of the solvent
in vacuo was purified by a flash column chromatography (EtOAc/
hexane, 10:90, v:v, Rf ) 0.27) to give the product (5.8 g, 95%). 1H
NMR (CDCl3, 500 MHz): δ 6.89-6.84 (m, 2 H), 6.74 (dd, J )
4.7, 9.8 Hz, 1 H), 4.12 (q, J ) 7.1 Hz, 2 H), 3.79 (s, 3 H), 2.91 (t,
J ) 7.7 Hz, 2 H), 2.59 (t, J ) 7.7 Hz, 2 H), 1.24 (t, J ) 7.2 Hz,
3 H).
1
by removal of the solvent in vacuo. H NMR (CDCl3, 500 MHz):
δ 6.87-6.72 (m, 3 H), 4.87 (t, J ) 3.8 Hz, 1 H), 3.97-3.95 (m, 2
H), 3.86-3.83 (m, 2 H), 3.79 (s, 3 H), 2.64-2.62 (m, 2 H), 2.44
(td, J ) 1.7, 7.2 Hz, 2 H), 1.92 (quintet, J ) 7.5 Hz, 2 H).
4-(5′-Fluoro-2′-methoxyphenyl)butyraldehyde (7). To a solu-
tion of 6 (1.7 g, 7.0 mmol, 1 equiv) in THF (60 mL) was added
HCl (1 N, 3 mL), and the reaction was stirred at room temperature
for 48 h. The reaction mixture was then poured into a separatory
funnel containing water (60 mL). The organic fraction was extracted
with EtOAc (3 × 60 mL), and the combined organic layers were
washed with brine (80 mL) and dried over sodium sulfate. The
crude product obtained by removal of the solvent in vacuo was
purified by flash column chromatography (EtOAc/hexane, 10:90,
v:v, Rf ) 0.1) to give the product (1.03 g, 53% from 3a). 1H NMR
(CDCl3, 500 MHz): δ 9.76 (s, 1 H), 6.88-6.83 (m, 2 H), 6.75 (dd,
J ) 4.6, 8.6 Hz, 1 H), 3.79 (s, 3 H), 2.63 (t, J ) 7.4 Hz, 2 H), 2.44
(t, J ) 7.4 Hz, 2 H), 1.92 (quintet, J ) 7.4 Hz, 2 H).
7-(5′-Fluoro-2′-methoxyphenyl)hept-1-en-5-ol (8). To a solu-
tion of 4 (3.0 g, 19.5 mmol, 1 equiv) in THF (20 mL) at 0 °C was
added a solution 1-butenylmagnesium bromide (0.5 M in THF, 45
mL, 22.5 mmol, 1.1 equiv) dropwise for 15 min. The reaction
mixture was poured into a solution of saturated ammonium chloride
(80 mL) in a separatory funnel. The organic fraction was extracted
with EtOAc (3 × 60 mL) and the combined organic layers were
washed with brine (50 mL) and dried over sodium sulfate. The
product was purified by flash column chromatography (EtOAc/
hexane, 15:85, v:v, Rf ) 0.2) to give the product as a colorless oil
(3.7 g, 94%). 1H NMR (CDCl3, 500 MHz): δ 6.86-6.82 (m, 2 H),
3-(5′-Fluoro-2′-methoxyphenyl)propanal (4). To a solution of
3 (5.8 g, 25.7 mmol, 1 equiv) in dry toluene (40 mL) under an

Dual Inhibitors of PDE4 and Serotonin Reuptake
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1537
6.76 (dd, J ) 4.6, 8.6 Hz, 1 H), 5.82 (m, 1 H), 5.04 (d, J ) 17.3
Hz, 1 H), 4.95 (d, J ) 10.2 Hz, 1 H), 3.81 (s, 3 H), 3.55 (m, 1
H),2.75-2.69 (m, 2 H), 2.2 (m, 1 H), 2.1 (m, 1 H), 1.96 (bs, 1 H),
1.73-1.68 (m, 2 H), 1.6-1.53 (m, 3 H).
removal of the solvent was purified by preparative TLC and eluted
with (MeOH/CH2Cl2, 10:90, v:v, Rf ) 0.1) to give the product (55
1
mg, 70%) as a 2:1 mixture of trans/cis diastereomers. H NMR:
(CDCl3, 500 MHz): δ 6.87 (m, 1 H), 6.82 (dd, J ) 3.0, 8.5 Hz, 1
H), 6.72 (m, 1 H), 4.03 (m, 0.65 H), 3.96 (m, 0.65 H), 3.89 (m,
0.35 H), 3.82 (m, 0.35 H), 3.78 (s, 3 H), 2.68 (m, 1 H), 2.58 (m,
1 H), 2.03 (m, 2 H), 1.83 (bs, 2 H), 1.86-1.6 (m, 4 H), 1.54
(m, 2 H). HRMS (ESI) [M + H]+ calcd for C15H23FNO2 268.1713,
found 268.1701.
The following compound was prepared in a similar manner.
8-(5′-Fluoro-2′-methoxyphenyl)oct-1-en-5-ol (9). The product
was purified by flash column chromatography (EtOAc/hexane, 12.5:
1
87.5, v:v, Rf ) 0.15) as a colorless oil (1.06 g, 61%). H NMR
(CDCl3, 500 MHz): δ 6.86-6.82 (m, 2 H), 6.74 (dd, J ) 4.6, 8.6
Hz, 1 H), 5.85 (m, 1 H), 5.04 (d, J ) 17.3 Hz, 1 H), 4.97 (d, J )
10.2 Hz, 1 H), 3.79 (s, 3 H), 3.66 (m, 1 H), 2.6 (m, 2 H), 2.22-2.1
(m, 2 H), 1.74-1.47 (m, 7 H).
The following compound was prepared in a similar manner.
2-{5-[3-(5-Fluoro-2-methoxy-phenyl)-propyl]-tetrahydro-fu-
ran-2-yl}-ethylamine (15). The product (24 mg, 47%), was isolated
as 3:1 mixture of trans/cis diastereomers (MeOH/CH2Cl2, 10:90,
2-(Bromomethyl)-5-(2′-methoxy-5′-fluorophenethyl)tetrahy-
drafuran (10). To a solution of 8 (3.24 g, 18.0 mmol, 1 equiv) in
dry CH2Cl2 (50 mL) at 0 °C was added N-bromosuccinimide (3.56
g, 20 mmol, 1.1 equiv) portionwise, and the reaction was warmed
to room temperature and stirred for 12 h. Solvent was then removed
in vacuo and the product was purified by flash column chroma-
tography (EtOAc/hexane, 5:95, v:v, Rf ) 0.1) and resulted in a
colorless oil as a 2:1 mixture of trans/cis diastereomers. 1H NMR
(CDCl3, 500 MHz): δ 6.86-6.81 (m, 2 H), 6.72 (dd, J ) 4.6, 8.6
Hz, 1 H), 4.22 (m, 1 H), 4.03 (m, 1 H), 3.78 (s, 3 H), 3.44 (dd, J
) 4.3, 9.7 Hz, 1 H), 3.34 (dd, J ) 6.4, 9.7 Hz, 1 H), 2.6 (t, J ) 7.3
Hz, 2 H), 2.13 (m, 1 H), 2.05 (m, 1 H), 1.75 (m, 1 H), 1.67-1.46
(m, 5 H).
The following compound was prepared in a similar manner.
2-(Bromomethyl)-5-(3′-(2′′-methoxy-5′′-fluorophenyl)-1′-pro-
pyl)tetrahydrofuran (11). The product was purified by flash
column chromatography (EtOAc/hexane, 5:95, v:v, Rf ) 0.1) to
provide a product (530 mg, 40%) as a colorless oil. 1H NMR
(CDCl3, 500 MHz): δ 6.86-6.81 (m, 2 H), 6.72 (dd, J ) 4.6, 8.6
Hz, 1 H), 4.22 (m, 1 H), 4.03 (m, 1 H), 3.78 (s, 3 H), 3.44 (dd, J
) 4.3, 9.7 Hz, 1 H), 3.34 (dd, J ) 6.4, 9.7 Hz, 1 H), 2.6 (t, J ) 7.3
Hz, 2 H), 2.13 (m, 1 H), 2.05 (m, 1 H), 1.75 (m, 1 H), 1.67-1.46
(m, 5 H).
2-Cyanomethyl-5-(5′-fluoro-2-methoxyphenethyl)tetrahydro-
furan (12). To a vial under an atmosphere of Ar(g) was added 10
(1.23 g, 4.3 mmol, 1 equiv), NaI (100 mg, 1.3 mmol, 0.3 equiv),
potassium cyanide (0.7 g, 10.6 mmol, 2.4 equiv), and dry DMSO
(15 mL). The mixture obtained was heated to 70 °C under an
atmosphere of Ar for 12 h. After cooling to room temperature, the
reaction mixture was poured into a separatory funnel containing
sodium bicarcarbonate aqueous solution (sat. NaHCO3/H2O, 50:
50, v:v, 80 mL). The organic fraction was extracted with EtOAc
(3 × 60 mL), and the combined organic layers were washed with
brine (50 mL) and dried over sodium sulfate. The solvent was then
removed in vacuo and the crude product obtained (0.45 g, 86%)
was purified by flash column chromatography (EtOAc/hexane, 10:
90, v:v, Rf ) 0.11) as a 2:1 mixture of trans/cis diastereomers. 1H
NMR (500 MHz, CDCl3) δ 6.88-6.82 (m, 2 H), 6.74 (dd, J )
4.3, 8.5 Hz, 1 H), 4.26 (m, 1 H), 4.09 (m, 0.65 H), 3.90 (m, 0.35
H)3.79 (s, 3 H), 2.72-2.53 (m, 4 H), 2.22-2.04 (m, 2 H), 1.84-1.6
(m, 4 H).
1
v:v, Rf ) 0.1). H NMR: (CDCl3, 500 MHz): δ 6.86-6.8 (m, 2
H), 6.73 (m, 1 H), 4.02 (m, 0.65 H), 3.96 (m, 0.65 H), 3.92 (m,
0.35 H), 3.82 (m, 0.35 H), 3.78 (s, 3 H), 3.09 (m, 1 H), 2.99 (m,
1 H), 2.6 (bs, 2 H), 1.96 (m, 2 H), 1.83-1.77 (m, 2 H), 1.65-1.47
(m, 8 H). HRMS (ESI) [M + H]+ calcd for C16H25FNO2 282.1875,
found 282.1869.
3,4-Dimethoxyphenylmagnesium bromide (17). Into a flame-
dried round-bottom flask with a magnetic stir bar was placed
anhydrous THF (150 mL) and Mg(s) turnings (1.43 g, 58.8 mmol,
1.0 equiv). The flask was then fitted with a pressure equalizing
addition funnel containing 4-bromo-1,2-dimethoxybenzene (8.2 g,
58.8 mmol, 1 equiv) (16) solution that was added slowly over a
period of 45 min while stirring at rt. Once the addition was
complete, I2(s) (500 mg) was added and the reaction was brought
to reflux and stirred for 12 h. The Grignard reagent was cooled
and used immediately in the following reaction.
6-(3,4-Dimethoxy-benzoyl)-cyclohex-3-enecarboxylic acid (18).
A solution of 17 (9.6 g, 59 mmol, 0.29 M, 1 equiv) in THF was
added dropwise to an ice-cooled solution of cis-1,2,3,6-tetrahydro-
phthalic anhydride (8.9 g, 59 mmol, 1 equiv) in THF (120 mL)
over a 1 h period. After the addition was complete, the resulting
mixture was stirred for another 30 min at 0 °C. The reaction mixture
was allowed to warm to rt and stirred overnight. The reaction was
then stopped with sat. NH4Cl and the pH adjusted to 2 with
concentrated HCl(aq) and extracted with diethyl ether. The organic
layer was washed with water and subsequently extracted with 1 M
NaOH. The combined aqueous extract was acidified with concen-
trated HCl and extracted with EtOAc (3 × 100 mL). The organic
layers were combined and dried over MgSO4, filtered, and
concentrated under reduced pressure to afford oil. The oil was
dissolved in CH2Cl2 and filtered through silica gel to remove the
dicarboxylic acid formed during workup. The product was recrys-
tallized from diethyl ether to afford pure product (1.62 g, 10%) as
a white solid; mp ) 109 °C. LRMS ESI [M - H]- calcd for
1
C16H18O5 289, found m/z 289. H NMR (CDCl3, 500 MHz): δ
7.60-7.53 (m, 2 H), 6.92 (d, J ) 8.4 Hz, 1 H), 5.84-5.82 (m, 1
H), 5.71-5.68 (m, 1 H), 4.03-4.00 (m, 1 H), 3.98 (s, 3 H), 3.95
(s, 1 H), 3.10-3.07 (m, 1 H), 2.90-2.84 (m, 1 H), 2.54-2.44 (m,
3 H).
The following compound was prepared in a similar manner.
2-Cyanomethyl-5-(3′-(2′′-methoxy-5′′-fluorophenyl)-1′-propyl)-
tetrahydrafuran (13). The product (0.2 g, 80%) was purified by
chromatography (EtOAc/hexane, 10:90, v:v, Rf ) 0.11) to give a
2:1 mixture of trans/cis diastereomers. 1H NMR (CDCl3, 500 MHz):
δ 6.86-6.81 (m, 2 H), 6.73 (dd, J ) 4.2, 8.5 Hz, 1 H), 4.22 (p, J
) 6.8 Hz, 0.65 H), 4.11 (m, 0.35 H) 4.09 (m, 0.65 H), 3.9 (m,
0.35 H), 3.79 (s, 3 H), 2.62-2.52 (m, 4 H), 2.18 (m, 1 H), 2.12
(m, 1 H), 1.78 (m, 1 H), 1.69-1.46 (m, 3 H).
2-{5-[3-(5-Fluoro-2-methoxy-phenyl)-ethyl]-tetrahydro-furan-
2-yl}-ethylamine (14). To a round-bottom flask under an atmo-
sphere of Ar was added Raney Ni (5 mg, 0.03 mmol, 0.1 equiv)
that was washed with methanol. Compound 12 (80 mg, 0.3 mmol,
1 equiv), dissolved in 2 M NH3 in methanol, was added to the
flask. The flask was evacuated and purged with H2 once. H2 gas
was then attached to the flask, and the reaction was allowed to
stirr for 4 h at room temperature. The reaction mixture was filtered
through a plug of celite and the crude product obtained after the
4-(3,4-Dimethoxy-phenyl)-4a,5,8,8a-tetrahydro-2H-phthalazin-
1-one (19). A mixture of 18 (610 mg, 2.1 mmol, 1.0 equiv) and
hydrazine hydrate (168 mg, 5.25 mmol, 2.5 equiv) in EtOH (10
mL) was refluxed for 4 h. The reaction was then cooled to rt and
concentrated under reduced pressure to afford a white precipitate.
The precipitate was dissolved in EtOAc and washed with Na2SO4(aq)
,
1 N HCl(aq), and water. The organic layer was then dried over
MgSO4, filtered, and concentrated to give a white precipitate. The
precipitate was recrystalized in EtOH to afford the product as white
crystals (376 mg, 63%). HRMS ESI [M + H]+ calcd for
C16H18N2O3 287.3385, found m/z 287.3379. 1H NMR (CDCl3, 500
MHz): δ 8.53 (bs, 1 H), 7.46 (d, J ) 2.0 Hz, 1 H), 7.23 (dd, J )
2.0, 8.4 Hz, 1 H), 6.87 (d, J ) 8.4 Hz, 1 H), 5.8-5.77 (m, 1 H),
5.72-5.7 (m, 1 H), 3.94 (s, 3 H), 3.93 (s, 3 H), 3.4 (dt, J ) 5.5,
8.7 Hz, 1 H), 3.01-2.97 (m, 1 H), 2.85 (t, J ) 6.0 Hz, 1 H),
2.26-2.19 (m, 3 H). HRMS (ESI) [M + H]+ calcd for C16H19N2O3
287.3385, found 287.3376. HPLC > 98% pure (tR ) 4.93, 70(A):
30(E); tR ) 5.33, 50(A):50(E)).

1538 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Cashman et al.
2-(5-Bromo-pentyl)-4-(3,4-dimethoxy-phenyl-4a,5,8,8a-tet-
rahydro-2H-phthalazin-1-one (20). Sodium hydride (60% disper-
sion in oil, 44 mg, 1.1 mmol, 1.1 equiv) was added to a solution
19 (287 mg, 1.0 mmol, 1.0 equiv). The mixture was stirred at rt
for 30 min, whereupon it took on a slight yellow color. 1,5-
Dibromopentane (600 mg, 2.6 mmol, 2.6 eq) was added via syringe,
and the reaction was stirred for 30 min at rt while the yellow color
dissipated to an almost clear solution. The reaction was stopped
with the addition of water and then transferred to a separatory funnel
and extracted with Et2O (3 × 30 mL). The organic fractions were
combined and dried over MgSO4, filtered, and concentrated to afford
an oil. The product was purified on silica and eluted with CH2Cl2
H), 6.74-6.71 (m, 1 H), 4.12 (q, J ) 7.1 Hz, 2 H), 4.05-3.77 (m,
2 H), 3.78 (s, 3 H), 2.71-2.56 (m, 2 H), 2.08-1.51 (m, 10 H) 1.3
(t, J ) 7.2 Hz, 3 H). LRMS (ESI) [M + H]+ calcd for C18H27FNO4
340, found 340.
2-{5-[3-(5-Fluoro-2-methoxy-phenyl)-ethyl]-tetrahydro-furan-
2-yl}-ethylamine)-methyl-amine (24). Into a flame-dried round-
bottom flask, purged with Ar(g), was placed 23 (112 mg, 0.33 mmol,
1.0 equiv) and anhydrous THF (1.6 mL). The flask was cooled
with an ice bath and stirred for 15 min. LAH (1.0 M, 1.32 mL,
1.32 mmol, 4.0 equiv) was then slowly added via a syringe over a
period of 5 min. The reaction was allowed to warm to rt and stirred
an additional 4 h. The reaction was then stopped with ice-cold
MeOH and then stirred for an additional 15 min at rt. The resulting
solution was acidified with 1 N HCl(aq) and then transferred to a
beaker and then made basic with 10 M NaOH(aq). The basic solution
was extracted with diethyl ether (3 × 20 mL), and then the organic
layer was washed with brine. The diethyl ether extract was dried
over Mg2SO4 for 15 min and then filtered and concentrated to oil
under reduced pressure to afford the crude product. The oil was
purified with preparative TLC (developed with MeOH/CH2Cl2, 5:95,
1
to afford a clear oil. H NMR (CDCl3, 500 MHz): δ 7.47 (d, J )
2.0 Hz, 1 H), 7.25 (dd, J ) 2.0, 8.5 Hz, 1 H), 6.87 (d, J ) 8.5 Hz,
1 H), 5.8-5.77 (m, 1 H), 5.7-5.67 (m, 1 H), 4.01-3.97 (m, 1 H),
3.95 (s, 3 H), 3.92 (s, 3 H), 3.79-3.74 (m, 1 H), 2.22-2.18 (m, 2
H), 2.1-2.04 (m, 1 H), 1.94-1.86 (m, 4 H), 1.74-1.69 (m, 4 H),
1.63-1.56 (m, 1 H), 1.52-1.46 (m, 2 H). LRMS ESI [M + H]+
calcd for C21H28BrN2O3 436, found m/z 435 (Br79), 437 (Br81).
HRMS (ESI) [M + H]+ calcd for C21H28BrN2O3 436.8007, found
436.8025.
1
v:v, Rf 0.05). H NMR (CDCl3, 500 MHz) 6.87-6.82 (m, 2 H),
6.74-6.71 (m, 1 H), 4.05-3.77 (m, 2 H), 3.78 (s, 3 H), 2.71-2.56
(m, 5 H), 2.08-1.51 (m, 10 H). HRMS (ESI) [M + H]+ calcd for
C16H25FNO2 282.3784, found 282.3762.
2-{5-[3-(5-Fluoro-2-methoxy-phenyl)-ethyl]-tetrahydro-furan-
2-yl}-ethylamine)-pentyl]-4,5,8,8a-tetrahydro-2H-phthalazin-1-
one (21). Into a flame-dried round-bottom flask under an atmosphere
of Ar(g), cesium hydroxide monohydrate (23 mg, 0.14 mmol, 1.0
equiv) and DMF (0.5 mL) was stirred for 30 min at rt. Compound
14 (37 mg, 0.14 mmol, 1.0 equiv) in DMF (0.3 mL) was then added
via syringe and the resulting mixture was stirred at rt. After stirring
an additional 30 min, compound 20 (72 mg, 0.16 mmol, 1.2 eq)
dissolved in DMF (0.3 mL) was added and stirred for an additional
18 h at rt. The mixture was washed with EtOAc (30 mL) and then
filtered, and the filtrate was washed with water in a separatory
funnel. The organic layer was dried with Na2SO4, filtered, and
concentrated to oil. The product was purified by preparative TLC
(developed with MeOH/CH2Cl2, 5:95, v:v, Rf ) 0.2) to afford an
oil (27 mg, 0.04 mmol, 31%). 1H NMR (CDCl3, 500 MHz) δ 7.47
(d, J ) 2.0 Hz, 1 H), 7.25 (dd, J ) 2.0, 8.5 Hz, 1 H), 6.87 (d, J )
8.5 Hz, 1 H), 6.86-6.81 (m, 2 H), 6.76-6.72 (m, 1 H), 5.81-5.79
(m, 1 H), 5.71 (m, 1 H), 4.01-3.97 (m, 1 H), 3.94 (s, 3 H), 3.91
(s, 3 H), 3.8 (s, 3 H), 3.79-3.74 (m, 1 H), 3.36-3.31 (m, 1 H),
3.03-3.0 (m, 1 H), 2.81-2.78 (m, 2 H), 2.73-2.37 (m, 5 H),
2.30-2.19 (m, 3 H), 2.09-1.97 (m, 3 H), 1.87-1.26 (m, 14 H).
HRMS (ESI) [M + H]+ calcd for C36H49FN3O5 622.8007, found
622.8025.
Acknowledgment. We are indebted to Professor Marco
Conti (Stanford University) for providing the results shown in
Table 1. We also thank Dr. Aaron Janowsky and Robert Johnson
(Portland VA Hospital, Portland, OR) for doing the binding and
reuptake inhibition assays shown in Table 2. The work was
supported in part by research grants and an Independent Scientist
Award from the National Institute of Mental Health (J.O.D.).
Supporting Information Available: Analytical data of the target
compounds is provided in the Supporting Information. This
information is available free of charge via the Internet at http://
pubs.acs.org.
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The following compound was prepared in a similar manner.
2-{5-[3-(5-Fluoro-2-methoxy-phenyl)-propyl]-tetrahydro-fu-
ran-2-yl}-ethylamine)-pentyl-4a,5,8,8a-tetrahydro-2H-phthalazin-
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1
(developed with MeOH/CH2Cl2, 5:95, v:v, Rf ) 0.2). H NMR
(CDCl3, 500 MHz) δ 7.47 (d, J ) 2.0 Hz, 1 H), 7.25 (dd, J ) 2.0,
8.5 Hz, 1 H), 6.87 (d, J ) 8.5 Hz, 1 H), 6.86-6.81 (m, 2 H),
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1
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