Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.04.027

Abstract A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compounds showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compound 3b, chosen among the synthesized compounds as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analogue. Spontaneous locomotor activity, assessed in an open field apparatus, showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compounds 3b, 5b and 7b, did not affect locomotion, whereas compound 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.

Full text of DOI:10.1016/j.bmc.2015.04.027

Bioorganic & Medicinal Chemistry 23 (2015) 3426–3435
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological activities and pharmacokinetic properties
of new fluorinated derivatives of selective PDE4D inhibitors
a
a
a
b
b
Chiara Brullo , Matteo Massa , Carla Villa , Roberta Ricciarelli , Daniela Rivera ,
b
c
d
d
Maria Adelaide Pronzato , Ernesto Fedele , Elisabetta Barocelli , Simona Bertoni ,
Lisa Flammini , Olga Bruno
Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, 3, 16132 Genoa, Italy
Department of Experimental Medicine, Section of General Pathology, School of Medical and Pharmaceutical Sciences, University of Genoa, Via LB Alberti, 2, 16132 Genoa, Italy
Department of Pharmacy, Section of Pharmacology and Toxicology, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Cembrano, 4, 16147 Genoa, Italy
Department of Pharmacy, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy
d
a,
⇑
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 February 2015
Revised 9 April 2015
Accepted 11 April 2015
Available online 16 April 2015
A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy
group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All com-
pounds showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 iso-
forms (PDE4A4, PDE4B2 and PDE4C2). Compound 3b, chosen among the synthesized compounds as the
most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacoki-
netic profile compared to its non fluorinated analogue. Spontaneous locomotor activity, assessed in an
open field apparatus, showed that, differently from rolipram and diazepam, selective PDE4D inhibitors,
such as compounds 3b, 5b and 7b, did not affect locomotion, whereas compound 1b showed a tendency
to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Phosphodiesterases type 4D inhibitors
PDE4D
cAMP enhancers
Pharmacokinetic properties
Microwave-assisted reaction
1
. Introduction
Phosphodiesterase enzymes (PDEs) regulate the intracellular
successful agents for the treatment of neurological disorders char-
acterized by cognitive deficits. Particularly, in the hippocampus,
cAMP increases cAMP response element-binding protein (CREB)
levels of the second messengers cAMP and cGMP, thus exerting
control over a number of cellular functions.1,2 The large PDEs fam-
ily consists of eleven enzymes ubiquitously expressed in the body.
Four different PDE type 4 isoforms (PDE4A, PDE4B, PDE4C and
PDE4D) and twenty five splice variants (e.g., PDE4A2, PDE4B2. . .)
constitute a subfamily that specifically hydrolizes cAMP in several
phosphorylation and activates transcription of proteins related to
6–11
synaptic plasticity and memory formation.
Different lines of
evidence have recently indicated PDE4D as the isoform mainly
1
2–14
involved in cognitive mechanisms.
However, most of the first generation of pan class PDE4-Is, such
as rolipram, showed some undesirable side effects, in particular
3
–5
15
16
districts.
Some PDE4 inhibitors (PDE4Is), enhancing cAMP
sedation and emesis.
dependent signal transduction in lung and endothelial cells, exert
beneficial effects in several inflammatory pathologies (chronic
obstructive pulmonary disease, psoriatic arthritis) and have been
recently approved for therapeutic use (roflumilast, ibudilast, apre-
milast). On the other hand, PDE4Is have been proposed as
Our research on PDE4Is led to several series of small molecules
structurally related to rolipram and characterized by good and
1
7–19
selective PDE4D inhibition.
Among them, compound GEBR-
7b (7b, Fig. 1) increased memory performances in the object loca-
tion test (OLT) and the object recognition test (ORT) in rodents,13
and improved spatial memory in the APPswe/PS1dE9 mouse model
2
0
of Alzheimer’s disease (AD). Interestingly, 7b did not induce any
emetic-like effect at doses 33–100 times higher than those effec-
tive on memory, as assessed with the taste reactivity test on rats
Abbreviations: AD, Alzheimer disease; cAMP, cyclic adenosine monophosphate;
CREB, cAMP response element-binding protein; cGMP, cyclic guanosine mono-
phosphate; OLT, object location test; ORT, object recognition test; PDE, phospho-
diesterase; PDE4Is, PDE4 inhibitors; PTC, phase transfer catalyzes; SAR, structure–
activity-relationship.
1
3
and the xylazine/ketamine test on mice.
In 2010, Burgin et al. reported a PDE4D allosteric modulator
able to revert the scopolamine-induced cognitive impairment in
⇑
Corresponding author. Tel.: +39 010 353 8367; fax: +39 010 353 8358.
E-mail address: obruno@unige.it (O. Bruno).
http://dx.doi.org/10.1016/j.bmc.2015.04.027
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0

C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
3427
NR
2
O
A
B
N
H
O
OH
OH
O
H
N
a
b
c
N
N
N
O
O
R
NR
2
R1
NR
2
1
2
3
4
5
6
7
8
a-c R = CHF
R = CHF
a-c R = CHF
2
2
2
2
R
R
R
R
1
= A NR
2
= a-c
= b
NR
2
b
1
1
1
= B NR
2
C
D
= C NR = a-c
2
2
2
O
OH
b
R = CHF
= D NR
= A NR
= b
= a-c
NR2
a-c R = CH
R = CH
a-c R = CH
R = CH
3
3
3
3
R
R
R
R
1
1
1
1
b
= B NR = b
2
O
= C NR
= D NR
2
2
= a-c
= b
b
Figure 1. Molecular structure of new compounds 1a–c, 2b, 3a–c, and 4b and of former 5a–c, 6b, 7a–c, and 8b.
the Y maze test and to improve memory performance in the ORT.
That compound was 100–3000 less emetic than rolipram in three
different species that are able to vomit (Suncus murinus, beagle
dog and Cynomolgus monkey).12
Finally, preliminary in vivo pharmacokinetic studies were per-
formed on our lead 7b and its fluorinated analogue 3b, to verify
the effects of isosteric substitution on BBB crossing ability.
In line with this study, our results, obtained with 7b, supported
the idea that PDE4D selective modulators or inhibitors could exert
pro-cognitive effects with reduced emetic potential.21
2. Results and discussion
2.1. Chemistry
In addition, two different studies reported no changes in loco-
motor activity between PDE4D knock out and wild type mice, indi-
cating that PDE4D inhibition does not necessarily induce
The limitative step to obtain all title compounds is the already
described starting product 4-(difluoromethoxy)-3-hydroxybenzal-
dehyde 9. Most methods reported in the literature use
3,4-dihydroxybenzaldehyde as a substrate and methyl 2-chlorodiflu-
2
2,14
sedation.
The halogen or halogenated group introduction in
different active molecules enhances their lipophilicity and
improves the blood–brain barrier (BBB) crossing, with a little
change in their steric hindrance. Moreover, the fluorine inductive
effects could increase the binding force to specific molecular sites
and therefore could increase drug potency.23–26
On the basis of these well-established lead optimization para-
digms, we planned a further chemical development of our PDE4D
inhibitors to improve their druggability. To investigate the role of
fluorine substitution on enzyme inhibition activity and to improve
the pharmacokinetic properties, a new series of derivatives (com-
pounds 1a–c, 2b, 3a–c, and 4b, Fig. 1) were designed by replacing
the 3-methoxy group with a 3-difluoromethoxy isoster moiety in
our previously synthesized compounds. In particular, we selected,
among the numerous derivatives, some that were active and selec-
tive (5a–c and 7a–c, Fig. 1) and some that were inactive (6b and 8b,
Fig. 1).
oroacetate as a reagent in the presence of different basic salts (as
potassium or caesium carbonate) in a solvent medium.2
7a–j
The
yields are ranging from 12% to 44%, but the crude requires an
expensive and time-consuming phase of work up and
chromatographic purification due to the large amount of by-
products. A recent alternative strategy starting from vanillin
gives, in two steps, the desired compound 9 in high yields.28
However, this method involves the use of chlorodifluoromethane,
a gas reagent difficult to manipulate and responsible for ozone
depletion. Thus, we developed an alternative green method to opti-
mize the reaction conditions and to improve the product yields. A
microwave irradiation system has been used and an experimental
design was applied by changing the type and amount of basic salts,
the heating method and the reaction time (Supporting informa-
tion). The new microwave assisted procedure achieved an
improved yield (57%) and cleaner crude, which was purified by
rapid work up and simple Silicagel filtration.
Preliminary tests on PDE4D3 activity were performed using the
recombinant human enzymes expressed in a baculoviral system.
All compounds were tested in duplicate at the concentration of
1
0
l
M, as previously reported.18 We subsequently assessed their
The second intermediate 3-(cyclopentyloxy)-4-(difluorome-
thoxy)benzaldehyde (10), was obtained by alkylation with cyclo-
pentylbromide, as reported in the literature.
inhibitory potency on PDE4D3 (IC50 values) and their selectivity
compared with PDE4A4, PDE4B2, and PDE4C2 isoforms.
2
9
Compound 10
All compounds were analyzed to preliminarily evaluate their
cytotoxicity on human neuroblastoma cells (HTLA); subsequently,
genotoxicity was assessed only on selected, not cytotoxic, com-
pounds. On the basis of its PDE4D3 inhibitory activity and selectiv-
ity, and given the absence of cytotoxic and genotoxic effects, 3b has
been selected among the synthesized compounds and tested
in vitro to verify its capability of enhancing cAMP accumulation,
using 7b as reference compound.
Moreover, on the basis of their PDE4D3 inhibitory activity and
selectivity, we selected some compounds to assess their capability
of affecting spontaneous locomotor activity on mouse exploratory
behavior using the open-field test. In detail, we tested the previ-
ously reported compound 5b (PDE4D3 active and selective), its
fluorinated analogue 1b (PDE4D3 very active, but not selective),
our lead 7b, and its fluorinated analogue 3b (PDE4D3 selective,
but less active than 7b), in comparison with rolipram and diaze-
pam used as reference compounds.
reacted then with hydroxylamine in ethanol 95% and water to give
2
7g
the corresponding oxime (11).
The latter, by reacting with
epichlorohydrin in anhydrous dimethylformamide at 40–50 °C
for 24 h, in the presence of sodium ethoxide, gave the 3-
(cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde O-(oxiran-2-
ylmethyl)oxime (12), which finally reacted with the suitable
amines or with 2,6-dimethylmorpholin-4-amine to give the
desired products 1a–c and 2b, respectively, with good yields
(Scheme 1).
A convergent reaction between the oxime intermediate (11)
and the suitable chloroacetylamines or 4-(3-chloropropanoyl)-
2,6-dimethylmorpholine gave compounds 3a–c and 4b, as
reported in the Scheme 1.
The syntheses of the building blocks 2,6-dimethylmorpholine-
4-amine,
4-(chloroacetyl)morpholine,
4-(chloroacetyl)-2,6-
dimethylmorpholine and 4-(3-chloropropanoyl)morpholine have
3
0–33
been already reported in the literature,
but we prepared them

3
428
C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
O
O
N
O
O
O
N
O
NR
2
N O
O
O
O
2
F HC
2
F HC
3
a-c
4b
vii
viii
F2HC
F2HC
F2HC
OH
O
O
O
OH
OH
O
i or ii
iii
iv
N
OH
O
H
O
H
O
H
10
11
9
NR
2
O
N
O
OH
1a-c
F2HC
O
F2HC
O
O
v
vi
O
N
O
N
NH
1
2
2
b
O
N
O
OH
O
O
F2HC
Scheme 1. Synthetic route for the preparation of 1a–c, 2b, 3a–c, and 4b. Reagents and conditions: (i) Methyl 2-chlorodifluoroacetate, Cs
0 °C, 6 h, 39%. (ii) Methyl 2-chlorodifluoroacetate, Cs CO
CO /KI, an. DMF, 65 °C, 22 h, 87%. (iv) NH OHÁHCl, NaHCO
morpholine or dimethylmorpholine, 40–50 °C, overnight, 36% and 60%, respectively. For 1c and 2b: 4-hydroxypiperidine or 2,6-dimethylmorpholine-4-amine, an. DMF, 40–
2
CO
3 2
, an. DMF, under N atm, 60–
7
K
2
3
, an. DMF, 300 W, under pressure, 90 °C, compressed air cooling, 5 cycles of 3 min, 57%. (iii) Cyclopentylbromide,
, EtOH/H O, rt, 4 h, 95%. (v) (1) Na, abs EtOH, rt; (2) epichlorohydrin, an. DMF, 45 °C, 24 h, 67%. (vi) For 1a and 1b:
2
3
2
3
2
5
K
0 °C, overnight, 45% and 21%, respectively. (vii) Chloroacetylamines, an. K
CO , an. DMF, 50–60 °C, 24 h, 18%.
2
CO
3
, an. DMF, 50–60 °C, 24 h, 47–85%. (viii) 4-(3-Chloropropanoyl)-2,6-dimethylmorpholine, an.
2
3
using the suitable amine and chloroacetyl- or chloropropanoyl
chloride in toluene in the presence of potassium carbonate, a
new profitable method that we have recently described. We used
the same method also for the synthesis of the 4-(3-chloropropa-
noyl)-2,6-dimethyl-morpholine, while, 1-(chloroacetyl)piperidin-
analogues (5b, 7a, 7c and 8b, respectively). In particular, the result
of compounds 3c and 4b (IC50 values of 2.63 and 0.47 lM, respec-
tively) has to be underlined, since the corresponding non fluori-
nated analogues (7c and 8b) were almost inactive. Moreover,
compounds 1a and 2b showed the same potency against PDE4D3
as the analogues 5a and 6b, while compounds 1c and 3b were less
potent than 5c and 7b, respectively.
19
4
-ol was prepared from 4-hydroxypiperidine and chloroacetyl
chloride in the presence of a sodium carbonate saturated solu-
tion/ethyl acetate mixture (1:2), as previously reported in the
3
4
literature.
Table 1
Compounds 1–8 inhibitory activity and IC50 values toward the PDE4D3 isoforma
2
.2. Enzymatic assays
Compd PDE4D3 % inhib
10 M)
IC50
M)
Compd PDE4D3 % inhib
IC50
M)
Preliminary tests on PDE4D3 activity were performed using the
(
l
(
l
(10
l
M)
(
l
recombinant human enzymes expressed in a baculoviral system.
All compounds were tested in duplicate at the concentration of
b
c
1
1
1c
a
b
83
84
51
87
63
74
60
63
1.49
0.19
2.54
0.19
1.12
4.84
2.63
0.47
5a
5b
5c
81
68
65
80
73
67
30
24
1.45
3.46
1.79
0.21
3.48
1.91
d
1
0
l
M, as previously reported and briefly described in the
1
9
d
Section 4. Subsequently, all compounds showing percent of inhi-
bition higher than 50% were tested on PDE4D3 at five concentra-
tions (5 Â 10 –10 M) and IC50 values were determined by non
2b
6b
7a
b
c
3a
À8
À4
3b
7b
d
e
3
4
c
b
7c
nd
nd
linear regression analysis of their inhibition curves.
8b
All synthesized compounds showed a good inhibitory activity
on PDE4D3 enzyme, ranging from 60% to 87% and IC50 values in
the micromolar or sub-micromolar range, being 1b, 2b and 4b
the most active compounds with IC50 values of 0.19, 0.19 and
a
Results are expressed as % inhibition in respect to the control. Compounds have
been tested in duplicate at 10 M concentration.
l
b
18
Results already reported for generic PDE4.
c
d
e
18
Results already reported.
Results already reported.
19
0
.47
l
M, respectively (Table 1). Compounds 1b, 3a, 3c and 4b
Not determined.
showed a good increase in potency with respect to the isosteric

C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
Table 3
3429
We, therefore, suggest that introducing fluorine in the cathecol-
Relative cytotoxic potential of compounds 1a–c, 2b, 3a–c, and 4b and 7b as reference
ic system generally improves the interaction with the catalytic
domain and causes a better PDE4D3 inhibition.
Subsequent screening on different PDE4 isoforms (particularly
PDE4A4, PDE4B2 and PDE4C2) were performed on all compounds
in order to determine their selectivity (Table 2).
The majority of the tested compounds, except 1b and 2b,
showed a good PDE4D3 selectivity, having inhibitory activity, at
M concentration, lower than 50% toward PDE4A4, PDE4B2
and PDE4C2 (Table 2). The MOLCAD surface studies performed by
Srivani et al. showed a higher lipophilicity in the PDE4B M-loop
compared to that of PDE4D. Such observations could explain the
decreased selectivity of compounds 1b and 2b in comparison with
their isoster 5b and 6b.19
On the other hand, the in silico calculations on PDE4B and
PDE4D interaction with our previous compounds were not partic-
ularly explicatory and only gave an approximate picture of the
ligand–enzyme interaction phenomenon, which is actually very
complex and influenced by many different factors.
a
compound
Compd
Cytotoxicity%
Positive control
DMSO
100.00 ± 1.98
0.67 ± 0.24
0.21 ± 1.40
8.83 ± 6.36
0.01 ± 0.02
31.37 ± 4.13
3.42 ± 2.49
1.54 ± 0.91
2.06 ± 1.61
0.01 ± 0.42
4.62 ± 4.62
0.01 ± 0.17
0.01 ± 0.40
0.14 ± 0.18
1
1
1
1
a (100
b (100
l
l
M)
M)
M)
M)
M)
M)
M)
b (10
l
1
0
l
c (100
l
1c (10
l
3
5
2
3
3
3
b (100 l
a (100
b (100
c (100
l
l
l
M)
M)
3c (10
4b (100
b (100
l
M)
l
l
M)
M)
7
a
Data represent the mean ± SEM for three independent experiments.
which had been selected among all the new and previous synthe-
sized compounds on the basis of its PDE4D3 inhibitory activity and
selectivity, and the absence of cytotoxic and genotoxic effects. In
the same assay, the non fluorinated analogue 7b was used as refer-
ence compound. In order to slightly stimulate the activity of aden-
ylyl cyclase, the cells received forskolin (FSK) during the last
2
.3. Cytotoxicity and genotoxicity assays
To evaluate the cytotoxic potential of the new molecules, we
analyzed the lactate-dehydrogenase release in human HTLA cells
that were exposed for 24 h to very high concentration
100 M) of the test compounds and 7b (as reference compound).
Compounds exhibiting a cytotoxic potential higher than 3% (i.e.,
b, 1c and 3c) have also been tested at a lower concentration
10 M), finally showing negligible cytotoxicity (Table 3).
To evaluate the genotoxicity, we analyzed the phosphorylation
of the chromatin-bound histone H2AX ( -H2AX), which is a quan-
titative marker for the DNA damage response at the site of double-
a
(
l
2
0 min of incubation. Both 7b and 3b have been able to signifi-
cantly increase the FSK-induced cAMP accumulation, without
affecting the basal levels of the cyclic nucleotide (Fig. 3).
Moreover, compound 3b showed higher efficacy in increasing
cAMP intracellular levels than 7b, despite having a lower PDE4D3
inhibitory activity. As the introduction of fluorine is known to
increase the lipophilicity of drugs, we feel that the greater effect
of compound 3b on cAMP accumulation is likely to depend on its
increased cell penetration and intracellular availability (see also
below). However, given that, in addition to PDE4A4, B2, C2 and
D3 (see Table 2), there are other different PDE4D variants, the pos-
sibility that 3b inhibits more enzymes than 7b, leading higher lev-
els of intracellular cAMP, cannot be completely ruled out.
1
(
l
c
3
6
strand breaks. To this purpose, we performed immunoblot anal-
ysis on protein extracts from HTLA cells that had been exposed for
2
4 h to the compounds 1a, 3b and 4b, which showed a cytotoxic
potential below 1% at the concentration of 100 M. As a positive
l
control, we used etoposide, a topoisomerase II inhibitor that
induces DNA double-stranded breaks.37
While the etoposide treatment of HTLA cells led to a robust DNA
damage, compounds 1a, 3b and 4b, as well as 7b, did not produce
2
3
.5. Spontaneous motor activity in an open field task
the
c-H2AX immunoreactive band, typical marker of genotoxicity
(Fig. 2).
In order to verify the potential sedative effects, compounds 1b,
b, 5b, and 7b (10 mg/kg) were tested on mouse exploratory
2
.4. In vitro intracellular cAMP quantification
behavior using the open-field test in comparison with rolipram
(
10 mg/kg) and diazepam (10 mg/kg), used as reference drugs.
Compounds 3b, 5b and 7b did not affect mice locomotor activ-
To verify the capability of enhancing cAMP intracellular accu-
mulation, we used a cAMP-specific enzymatic immunoassay (EIA)
in HTLA cells that had been exposed for 30 min to compound 3b,
ity at the same dose of rolipram and diazepam, which, on the
Table 2
Inhibition activity % toward different PDE4 isoforms of compounds 1a–c, 2, 3a–c, and 4 in comparison with previously reported compounds 5–8, and rolipram as reference
compounda
Compd
PDE4A4 %inhib
PDE4B2 %inhib
PDE4C2 %inhib
Compd
PDE4A4 %inhib
PDE4B2 %inhib
PDE4C2 %inhib
1
1
1
2
3
3
3
4
a
b
c
b
a
b
c
41
68
15
58
36
32
28
34
79
43
68
15
51
31
29
26
37
65
ntb
62
nt
nt
nt
9
nt
nt
57
5a
nt
34
10
41
nt
34
nt
nt
—
nt
39
5
51
nt
23
nt
nt
—
nt
19
9
25
nt
25
nt
nt
—
c
5b
d
5c
d
6b
c
7a
c
7b
7c
8b
—
b
Rolipram
a
b
c
Results expressed as % inhibition in respect to the control at 10
Not tested.
lM concentration, in duplicate.
18
Results already reported.
d
19
Results already reported.

3
430
C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
Under the same experimental conditions, absorption and elim-
ination of compound 3b were also rapid (Fig. 5B), since peak
plasma concentration was reached 10 min after the sc administra-
tion of the same dose and the half-life being 0.82 h. In this case, the
brain to plasma ratio was 0.429 ± 0.037 (mean ± SD; n = 3), which
is significantly higher (P <0.04; unpaired, two-tailed Student’s t-
test) than that of 7b and indicates that the introduction of a fluo-
rine in compound 3b improved BBB penetration.
For both compounds, a marked rebound in the plasma levels
was observed 5 h after their administration, which could be due
to drug redistribution or enterohepatic circulation. Finally, both
Figure 2. Genotoxic potential of compounds 1a, 3b, 4b, and 7b. Western blot
analysis of -H2AX in HTLA cells treated for 24 h with 100 M etoposide, 7b and the
test compounds, or an equal volume of solvent (DMSO). The H2AX signals represent
the internal loading control. The figure is representative of three independent
experiments all showing essentially similar results.
c
l
drugs were quickly eliminated with a plasma t
. Conclusion
A new series of 3-cyclopentyloxy-4-difluoromethoxy deriva-
tives has been synthesized to prove the influence of fluorine intro-
duction both in PDE4D inhibitory activity and selectivity. All
synthesized compounds showed a good PDE4D3 inhibition and
most of them were also selective in relation to the other PDE4
isoforms.
½
of 0.7–0.8 h.
contrary, significantly, reduced the distance traveled by mice (P
3
<0.01) and prolonged their immobility time (P <0.01 and P
<0.001, respectively) measured over a period of 60 min (Fig. 4).
Compound 1b showed a slight, even if not significant, tendency
to reduce the distance traveled and to prolong the immobility time.
2
.6. In vivo pharmacokinetic studies
Our lead 7b and its fluorinated analogue 3b were subjected to
3
b, selected as the best compound in terms of selectivity and
preliminary in vivo pharmacokinetic studies to verify the isosteric
substitution effects on BBB crossing. The main pharmacokinetic
parameters of 7b and 3b in mice, following sub-cutaneous (sc)
administration of a 10 mg/kg dose, are summarized in Table 4.
safety, enhanced the intracellular cAMP levels more than its non
fluorinated analogue (7b), despite having a lower PDE4D3 inhibi-
tory activity. This result suggests that 3b, due to the fluorine sub-
stitution, can better cross the cell membrane. For this reason 3b
has been chosen besides 7b to verify its ability to cross the BBB.
In our preliminary tests, 3b showed a better BBB penetration, its
brain/plasma ratio being 20% higher than that of its non fluorinated
analogue 7b.
7
b was rapidly absorbed as indicated by peak plasma concentra-
tion occurring at the first sampling time-point (10 min post-injec-
tion); also the peak brain concentration was achieved 10 min after
administration (Fig. 5A). The elimination of 7b from plasma was
fast with a half-life of 0.7 h and a similar pattern occurred in the
brain, where tissue concentrations fell below the detection limit
Finally, our selective PDE4D3 inhibitors (3b, 5b and 7b) did not
affect any locomotor activity in vivo, whereas the new non selec-
tive fluorinated derivative 1b slightly, though not significantly,
reduced the distance traveled and prolonged the immobility per-
iod. Therefore, our data indicate that selective PDE4D inhibition
is not associated with sedative effects, a result that fits with those
5
h after administration. The brain to plasma ratio (AUC0–t ratio)
for 7b was 0.358 ± 0.021 (mean ± SD; n = 3), indicating that the
compound was able to enter the brain. This result is in line with
our previous in vivo data showing that subcutaneous administra-
tion of this PDE4D inhibitor evoked significant central effects as
demonstrated by its capability to improve memory in rodents in
14,22
previously reported for PDE4D KO mice.
In conclusion, the introduction of fluorine in our previous selec-
tive PDE4D inhibitors generally provided more active compounds
and also slightly improved their CNS penetration, an essential fea-
ture to develop drugs for neurodegenerative disorders such as
Alzheimer’s disease (AD). On the other hand, in some molecules,
the lipophilicity increase could be responsible of selectivity loss
that might relate to undesirable side-effects, such as sedation.
Thus, our study provides important information for the future
development of new molecules that need to possess a balanced
lipophilicity to have both potent and selective PDE4D inhibition
together with a favorable CNS penetration.
1
3
specific behavioral tasks.
4
4
. Experimental protocols
.1. Synthesis
All chemicals were purchased from Chiminord and Aldrich
Chemical, Milan, Italy. Solvents were reagent grade. Unless other-
wise stated, all commercial reagents were used without further
purification.
Aluminum backed silica gel plates (Kieselgel 60F254 DC-
Alufolien, E. Merck, Darmstadt, Germany) were used in thin-layer
chromatography (TLC) for routine monitoring the course of reac-
tions and testing all compounds for purity. Detection of spots
was made by UV light. Merck silica gel, 100–200 mesh, was used
for chromatography.
Figure 3. Quantification of intracellular cAMP by specific enzyme immunoassay
(
EIA). HTLA cells were pre-treated for 10 min with 7b (100
lM), compound 3b
(
100 M) or an equal volume of DMSO. Then, 1 M forskolin (FSK) was added,
l
l
where indicated, for 20 min. At the end of the incubation periods, intracellular
cAMP was measured with a cAMP-specific EIA kit, according to the manufacturer’s
instructions. The histogram shows the mean ± SEM for three independent exper-
⁄
⁄⁄⁄
All amines were purified by distillation before the use. Melting
points are not ‘corrected’ and were measured with a Buchi M-560
iments. P <0.05 vs FSK + 7b-treated cells,
P <0.0001 vs FSK-treated cells (one-
way ANOVA, Bonferroni post test).

C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
3431
Figure 4. Effects of compounds 1b, 3b, 5b and 7b, on the spontaneous motor activity in an open field task. Rolipram and diazepam have been used as reference compounds.
All compounds were orally administered at 10 mg/kg. (A) Total distance traveled (m), (B) immobility time (min) both measured in a period of 60 min. Data are mean ± SEM
⁄⁄
⁄⁄⁄
with n = 6–8. P <0.01 and P <0.001 vs vehicle-treated mice, one-way Anova followed by Dunnett’s post-test.
Table 4
power 300 Watt, temperature control by optical fiber, magnetic
stirring of the sample, air cooling system.
Main pharmacokinetic parameters of compounds 7b and 3b in mice following sc
administration of a 10 mg/kg dose
Parameters
Compd
4.1.1. 4-(Difluoromethoxy)-3-hydroxybenzaldehyde (9)
Method (a):
1.38 g, 10 mmol) in an. DMF (15 mL) was stirred under nitrogen
atmosphere for 10 min. Then, Cs CO (3.26 g, 10 mmol) and methyl
A suspension of 3,4-dihydroxybenzaldehyde
7
b
3b
(
Plasma
Brain
Plasma
Brain
2
3
T
C
t
max (h)
max (ng/mL or g)
1/2 (h)
AUC0–t (ng h/mL or g)
0.17
4406
0.70
1225
0.17
1567
1.36
391
0.17
2157
0.82
1893
0.17
936
0.54
812
chlorodifluoroacetate (1.73 g, 12 mmol) were added. The mixture
was stirred at 60–70 °C for 6 h. After cooling to room temperature,
the solution was poured into water (30 mL) and extracted with
ethylacetate (3 Â 20 mL), washed with brine (3 Â 10 mL), dried
(
4
MgSO ) and concentrated under reduced pressure. The obtained
oil was purified by Silicagel (100–200 mesh) column chromatogra-
phy using a mixture of ethylacetate/exane (15:85) as the eluent.
The pure product was obtained as a white solid. Yield: 39%
instrument. IR spectra were recorded with a Perkin-Elmer 398
spectrophotometer. H NMR spectra were recorded on a Varian
Gemini 200 (200 MHz) instrument; chemical shifts are reported
as d (ppm) relative to tetramethylsilane (TMS) as internal stan-
dard; signals were characterized as s (singlet), d (doublet), t (trip-
let), m (multiplet), br s (broad singlet); J in Hz.
1
2
7
(lit.:15–30%). Mp: 86–88 °C.
Method (b): The 4-(difluoromethoxy)-3-hydroxybenzaldehyde
was prepared with improved yield by the following microwave
assisted procedure.
Elemental analyses were determined with an elemental ana-
lyzer EA 1110 (Fison-Instruments, Milan, Italy). Analyses indicated
by the symbols of the elements or functions were within ±0.4% of
the theoretical values.
To a solution of 3,4-dihydroxybenzaldehyde (0.415 g, 3 mmol)
in an. DMF (5 mL), Cs CO (0.975 g, 3 mmol) and methyl chlorodi-
fluoroacetate (0.520 g, 3.6 mmol) were added; then the mixture
was irradiated with microwaves, under pressure, increasing the
emitted power until 300 W in 2 min and at the same time cooling
by compressed air to avoid the temperature increasing over 90 °C;
2
3
Microwave apparatus: CEM DiscoverÒ (CEM Corporation,
Matthews, NC, USA), single-mode microwave oven, max emitted
Figure 5. Concentration-time profile of 7b (A) and 3b (B) following subcutaneous administration of a single dose to BALB/c mice. A total of 21 mice (3 mice/time point) were
included in the analysis for each compound. 7b and 3b were administered in 0.5% methylcellulose at the dose of 10 mg/kg. Each point represents mean ± SD.

3
432
C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
the irradiation was carried on for further 3 min and then the mix-
ture was cooled to room temperature, with compressed air, in
4.1.5. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde O-
[3-(2,6-dimethylmorpholin-4-yl)-2-hydroxypropyl]oxime hydro-
chloride (1b)
1
min. The cycle was repeated five times for a total reaction time
of 30 min. The mixture was poured into water (100 mL) and
Yield: 60%. Mp: 126 °C (dec). 1H NMR (CDCl
3
): d 1.18–1.23 (m,
cyclopent.), 2.30–2.60 and
3.10–3.20, 3.40–3.60, 4.00–4.18 (4 m, 8H, 3CH -N + 2CH
morph.) 4.22–4.42 (m, 2H, OCH ), 4.57–4.77 (m, 1H, CH-OH),
4.79–4.95 (m, 1H, OCH cyclopent.), 6.55 (t, 1H, J = 72.6 Hz,
OCHF ), 7.00 (dd, 1H, J = 8.2 Hz, 1.6 Hz, H-6 Ar), 7.12 (d, 1H,
extracted with ethyl acetate (3 Â 20 mL); the organic phase was
3 2
6H, 2CH ), 1.45–1.95 (m, 8H, 4CH
washed with water (20 mL), brine (20 mL), dried (MgSO
4
) and con-
2
centrated under reduced pressure, yielding a brown oil which was
purified by Silicagel (100–200 mesh) column chromatography
eluting with an ethylacetate/exane mixture (15:85). The pure
product was obtained as a light gray solid. Yield: 57% (lit. 15–
2
2
J = 8.2 Hz, H-5 Ar), 7.21 (d, 1H, J = 1.6 Hz, H-2 Ar), 8.01 (s, 1H,
2
7
1
À1
3
0%). Mp: 86–88 °C. H NMR (CDCl
3
): d 6.05 (br s, 1H, OH, 1H dis-
), 7.26 (d, 1H,
CH@N). IR (KBr) cm
:
3437 (OH), 1602 (CH@N). Anal.
appears with D O), 6.65 (t, 1H, J = 72.6 Hz, OCHF
2
2
22 32 2 2 5
C H F N O HCl (C, H, N).
J = 8.2 Hz, H-5 Ar), 7.45 (dd, 1H, J = 8.2 Hz, 2.0 Hz, H-6 Ar), 7.12
À1
(
1
d, 1H, J = 2.0 Hz, H-2 Ar), 9.90 (s, 1H, CH@N). IR (KBr) cm
:
4.1.6. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde O-
[2-hydroxy-3-(4-hydroxypiperidin-1-yl)propyl]oxime (1c)
To a solution of 3-(cyclopentyloxy)-4-(difluoromethoxy)benzal-
dehyde O-(oxyran-2-ylmethyl)oxime 12 (0.51 g, 1.56 mmol) in an.
DMF (1 mL), piperidin-4-ol (0.30 g, 3 mmol) solved in an. DMF
(3 mL) was added. The mixture was heated at 40–50 °C for 18 h.
After cooling to room temperature, the mixture was poured into
water (30 mL) and extracted with dichloromethane (3 Â 20 mL);
the organic phases were washed with brine (3 Â 20 mL), dried
687 (C@O), 3305 (OH). Anal. C
8 6 2 3
H F O (C, H, N).
4
(
.1.2. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde O-
oxiran-2-ylmethyl)oxime (12)
To a solution of sodium (0.1 g, 4.35 mmol) in absolute ethanol
(
5 mL),
3-(cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde
oxime 11 (1.18 g, 4.35 mmol) was added and the mixture was stir-
red at room temperature for 15 min; then, the solvent was
removed under reduced pressure, the crude was solved in an.
DMF (10 mL), epichlorohydrin (0.60 g, 6.52 mmol) was added drop
wise and the mixture was heated at 40–50 °C for 23 h. After cooling
to room temperature, water (30 mL) was added and the solution
was extracted with dichloromethane (3 Â 20 mL). The organic
4
(MgSO ) and concentrated under reduced pressure. The crude was
purified by Silicagel (100–200 mesh) column chromatography using
astheeluentfirstlydiethylether,thenamixtureofdichloromethane/
methanol (9:1) yielding the pure product as light yellow oil. Yield:
1
45%. H NMR (CDCl
3
): d 1.45–2.12 (m, 12H, 4CH
2
cyclopent. + 2CH
2
phase was dried (MgSO
4
) and concentrated under reduced pres-
pip.), 2.20–2.40, 2.44–2.64, 2.70–2.88 and 2.89–3.07 (4 m, 8H,
3CH N + 2OH, 2H disappear with D O), 3.71–3.88 (m, 1H, CH-OH
pip.),4.03–4.26(m,3H,CH O + CH-OH),4.81–4.97(m,1H,OCHcyclo-
pent.),6.58(t,1H,J = 76.0 Hz,OCHF ),7.04(dd,1H,J = 8 Hz,1.6 Hz,H-
sure yielding brown oil, which was purified by Silicagel (100–200
mesh) column chromatography using a mixture of diethyl ether/
petroleum ether (boiling point 46–60 °C) (10:90) as the eluent.
2
2
2
2
The final product was obtained as pale oil.
6 Ar), 7.17 (d, 1H, J = 8.0 Hz, H-5 Ar), 7.25 (d, 1H, J = 1.6 Hz, H-2 Ar),
8.09 (s, 1H, CH@N). IR (film) cm : 3377 (OH), 1601 (CH@N). Anal.
C H F N O (C, H, N).
21 30 2 2 5
1
À1
Yield: 67%. H NMR (CDCl
pent.), 2.53–2.78 and 2.79–3.00 (2 m, 2H, CH
3
): d 1.10–2.10 (m, 8H, 4CH
oxiran), 3.18–3.40
m, 1H, CH oxiran), 3.95–4.21 and 4.25–4.55 (2 m, 2H, OCH ),
.71–5.00 (m, 1H, OCH cyclopent.), 6.56 (t, 1H, J = 72.6 Hz,
2
cyclo-
2
(
4
2
4.1.7. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde O-
{3-[(2,6-dimethylmorpholin-4-yl)amino]-2-hydroxypropyl}oxime
(2b)
OCHF
2
), 6.89–7.54 (3 m, 3H, H-5 + H-6 + H-2 Ar), 8.05 (s, 1H,
À1
CH@N). IR (KBr) cm 1602 (CH@N). Anal. C16
H
19
F
2
NO
4
(C, H, N).
To a solution of 3-(cyclopentyloxy)-4-(difluoromethoxy)benzal-
dehyde O-(oxiran-2-ylmethyl)oxime 12 (0.51 g, 1.56 mmol) in an.
DMF (1 mL), a solution of 2,6-dimethylmorpholin-4-amine30 in
an. DMF (4 mL) was added drop wise. The mixture was heated at
40–50 °C for 18 h.
4
.1.3. General procedure for 3-(cyclopentyloxy)-4-(difluorometh-
oxy)benzaldehyde O-(2-hydroxy-3-cycloamine-4 ylpropyl)oximes
1a and 1b
A solution of 3-(cyclopentyloxy)-4-(difluoromethoxy)benzalde-
hyde O-(oxyran-2-ylmethyl)oxime 12 (0.51 g, 1.56 mmol) and
morpholine or 2,6-dimethylmorpholine (1.5 mL) was heated at
After cooling to room temperature, the mixture was diluted
with water (50 mL) and extracted with dichloromethane
(2 Â 20 mL). The organic phase was washed with brine (20 mL),
4
(
0–50 °C for 18 h. After cooling to room temperature, diethyl ether
20 mL) was added and the organic phases were washed with
) and concentrated under reduced
4
dried (MgSO ), and concentrated under reduced pressure yielding
water (2 Â 20 mL), dried (MgSO
4
oil, which was purified by Silicagel (100–200 mesh) chromatogra-
pressure. The crude was purified by Silicagel (100–200 mesh) col-
umn chromatography using as the eluent firstly diethyl ether, then
a mixture of dichloromethane/methanol (9:1) yielding the final
product as yellow oil. In the case of compound 1b the oil was trea-
ted with anhydrous HCl saturated diethyl ether solution to give the
corresponding hydrochloride salt as light yellow solid.
phy column using diethyl ether as the eluent. The pure product
1
was obtained as light yellow oil. Yield: 21%. H NMR (CDCl
3
): d
1.19 (d, 6H, J = 6.4 Hz, 2CH
(2 m, 8H, 4CH cyclopent.), 2.35–3.08 (m, 6H, 3CH
(m, 2H, 2CHO morph.), 4.02–4.35 (m, 3H, CH O + CH-OH), 4.82–
4.98 (m, 1H, OCH cyclopent.), 6.58 (t, 1H, J = 80.0 Hz, OCHF ),
3
morph.), 1.22–1.38 and 1.53–2.07
2
2
N), 3.62–3.92
2
2
Compounds 1a and 1b were characterized as follows:
7.00–7.38 (m, 3H, H-6 + H-5 + H-2 Ar), 8.09 (s, 1H, CH@N). IR
À1
(
KBr): cm
:
3617 (NH) 3441 (OH), 1580 (CH@N). Anal.
4
.1.4. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde O-
C H
22 33
F
2
N
3
O
5
(C, H, N).
(
2-hydroxy-3-morpholin-4 ylpropyl)oxime (1a)
1
Yield: 36%. H NMR (CDCl
3
): d 1.57–2.18 (m, 8H, 4CH
NCHOH), 2.99–3.18 (m, 4H,
N morph.), 3.82–4.12 (m, 4H, 2CH O morph.), 4.12–4.39 (m,
H, OCH ), 4.39–4.59 (m, 1H, CH-OH), 4.79–4.97 (m, 1H, OCH
), 7.04 (dd, 1H,
2
cyclo-
4.1.8. General procedure for 3-(cyclopentyloxy)-4-(difluorometh-
oxy)benzaldehyde O-[2-cycloamino-2-oxoethyl]oximes 3a–c
To a solution of 3-(cyclopentyloxy)-4-(difluoromethoxy)benzal-
pent.), 2.94 (d, 2H, J = 6 Hz, CH
2
2
2
CH
2
2
2
2 3
dehyde oxime 11 (0.68 g, 2.50 mmol) in an. DMF (2 mL), K CO
cyclopent.), 6.59 (t, 1H, J = 76.0 Hz, OCHF
2
(0.69 g, 5 mmol) and the suitable chloroacetylamines (5.58 mmol)
were added, and the mixture was heated at 50–60 °C for 24 h. After
cooling to room temperature, the mixture was poured into water
(50 mL) and extracted with diethyl ether (3 Â 20 mL); the organic
J = 8.0 Hz, 2.0 Hz, H-6 Ar), 7.18 (d, 1H, J = 8.0 Hz, H-5 Ar), 7.24 (d,
À1
1
H, J = 2.0 Hz, H-2 Ar), 8.07 (s, 1H, CH@N). IR (film) cm : 3366
(
28 2 2 5
OH), 1602 (CH@N). Anal. C20H F N O (C, H, N).

C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
3433
phase was washed with water (3 Â 20 mL), dried (MgSO
4
) and con-
IMAP technology (Molecular Devices), which is based on the high
affinity binding of phosphate by immobilized metal coordination
complexes on nanoparticles. The binding reagent complexes with
phosphate groups on nucleotide monophosphate generated from
cyclic nucleotides (cAMP) through phosphodiesterases. With fluo-
rescence polarization detection, binding causes a change in the rate
of the molecular motion of the phosphate bearing molecule and
results in an increase in the fluorescence polarization value
observed for the fluorescent label attached to the substrate.
For PDE4D3 tests, all compounds were preliminary tested at
centrated under reduced pressure. The crude was purified by
Silicagel (100–200 mesh) column chromatography using as the
eluent a mixture of dichloromethane/methanol (9:1) (compounds
3
3
a and 3c) or diethyl ether/dichloromethane (8:2) (compound
b) yielding the final pure product as light yellow oil.
Compounds 3a and 3b were characterized as follows:
4
.1.9. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde O-
(
2-morpholin-4-yl-2-oxoethyl)oxime (3a)
1
Yield: 85%. H NMR (CDCl
pent.), 3.48–3.84 (m, 8H, 2CH
H, OCH cyclopent. + OCH ), 6.58 (t, 1H, J = 78.0 Hz, OCHF
2
3
): d 1.57–2.08 (m, 8H, 4CH
N + 2CH O morph.), 4.78–4.98 (m,
), 7.08
2
cyclo-
10 lM concentration in duplicate; then, IC50 values were deter-
2
2
mined for compounds that showed inhibition of PDE4D3 activity
higher than 50% (the most common cut-off value for further inves-
tigation). The compounds were tested at five concentrations in the
3
2
(
dd, 1H, J = 10, 1.8 Hz, H-6 Ar), 7.18, (d, 1H, J = 10 Hz, H-5 Ar),
À1
À8
À4
7
1
.25 (d, 1H, J = 1.8 Hz, H-2 Ar), 8.15 (s, 1H, CH@N). IR (KBr) cm
655 (C@O), 1602 (CH@N). Anal. C19 (C, H, N).
:
interval 5 Â 10 –10 M. IC50 values were determined by non lin-
ear regression analysis of their inhibition curves (GraphPad Prism
software) (Table 1).
24 2 2 5
H F N O
4
.1.10. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde
The same protocol was applied to determine inhibitory activity
on PDE4A4, PDE4B2 and PDE4C2 for all compounds. The results are
reported in Table 2.
O-[2-(2,6-dimethylmorpholin-4-yl)-2-oxoethyl]oxime (3b)
1
Yield: 47%. H NMR (CDCl
.66–2.00 (m, 8H, 4CH cyclopent.), 2.36–2.87 (m, 2H, CH
.30–3.71 (m, 4H, CH
OCH cyclopent. + OCH
7
3
): d 1.17–1.22 (m, 6H, 2CH
3
morph.),
1
3
2
2
morph.),
4
.2.2. Cytotoxicity and genotoxicity assays
2
morph. + 2CH morph.), 4.72–4.90 (m, 3H,
), 6.54 (t, 1H, J = 78.0 Hz, OCHF ), 6.99–
.31 (m, 3H, H-6 + H-5 + H-2 Ar), 8.11 (s, 1H, CH@N). IR (CHCl
The cells used in this study (human neuroblastoma cell line
2
2
HTLA-230, HTLA) were grown in Roswell Park Memorial Institute
medium (RPMI), with 0.1 mM non-essential aminoacids and 10%
fetal bovine serum.
3
)
À1
28 2 2 5
cm : 1647 (C@O), 1581 (CH@N). Anal. C21H F N O (C, H, N).
For the cytotoxicity assay, cells were treated for 24 h with 100
4
.1.11. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde
or 10
lM of the indicated compounds dissolved in DMSO. Control
O-[2-(4-hydroxypiperidin-1-yl]-2-oxoethyl]oxime (3c)
1
cells received the same volume of solvent (1
l
L/mL medium). At
Yield: 50%. H NMR (CDCl
cyclopent. + 2CH pip.), 3.20–3.39 (m, 2H, CH
m, 1H, CH-OH pip.), 3.90–4.18 (m, 3H, CH N pip. + OH, 1H disap-
O), 4.78–4.95 (m, 3H, OCH cyclopent. + OCH ), 6.58
), 7.05 (dd, 1H, J = 8.0 Hz, 1.8 Hz, H-6
3
): d 1.47–2.15 (m, 12H, 4CH
2
the end of the incubation period, conditioned media were analyzed
2
2
N pip.), 3.68–3.90
for lactate-dehydrogenase release using the Cytotoxicity Detection
(
2
PLUS
Kit
(Roche, Germany) according to manufacturer protocols.
pears with D
2
2
To evaluate genotoxicity, HTLA cells were treated for 24 h with
(
t, 1H, J = 80.0 Hz, OCHF
2
the indicated compounds dissolved in DMSO and then processed
for total protein extraction as previously described.38
Immunoblots were done according to standard methods, using
the following antibodies: mouse monoclonal [2F3] to gamma
H2A.X (phospho S139) and rabbit polyclonal to Histone H2A.X
Ar), 7.17 (d, 1H, J = 8.0 Hz, H-5 Ar), 7.25 (d, 1H, J = 1.8 Hz, H-2
Ar), 8.15 (s, 1H, CH@N). IR (film) cm : 3407 (OH), 1652 (C@O),
À1
1
581 (CH@N). Anal. C20
26 2 2 5
H F N O (C, H, N).
4
.1.12. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzaldehyde
(
Abcam, UK); anti-mouse and anti-rabbit secondary antibodies
O-[3-(2,6-dimethylmorpholin-4-yl)-3-oxopropyl]oxime (4b)
To suspension of 3-(cyclopentyloxy)-4-(difluorome-
thoxy)benzaldehyde oxime 11 (0.68 g, 2.5 mmol) and an. K CO
0.69 g, 5 mmol) in an. DMF (4 mL), a solution of 4-(3-chloroprop-
anoyl)-2,6-dimethylmorpholine (0.52 g, 2.5 mmol) in an. DMF
1 mL) was added drop wise. The mixture was heated at 50–
0 °C overnight. After cooling to room temperature, the mixture
was diluted with water (50 mL) and extracted with diethyl ether
coupled to horseradish peroxidase (GE Healthcare, UK). Proteins
were visualized with an enzyme-linked chemiluminescence detec-
tion kit according to the manufacturer instructions (GE
Healthcare). Chemiluminescence was monitored by exposure to
films and signals were analyzed under non-saturating condition
with an image densitometer (Bio-Rad, CA, USA).
a
2
3
(
(
6
4
.2.3. cAMP enzymatic immunoassay (EIA)
(
(
2 Â 20 mL). The organic phase was washed with brine
Quantification of intracellular cAMP was performed with
2 Â 10 mL), dried (MgSO
4
), and concentrated under reduced pres-
DetectXÒ Direct Cyclic AMP Enzyme Immunoassay Kit (Arbor
sure yielding oil, which was purified by Silicagel (100–200 mesh)
column chromatography using diethyl ether as the eluent. The
Assay, MI, USA), following the manufacturer protocol. cAMP levels
were calculated according to the standard curves prepared on the
same EIA plates.
pure product was obtained as light yellow oil.
1
Yield: 18%. H NMR (CDCl
3
): d 1.08–1.32 (m, 6H, 2CH
cyclopent.), 2.64–2.93 (m, 2H, CH
.17–3.39 (m, 1H, OCH morph.), 3.43–3.92 (m, 4H, CH N morph.),
.93–4.17 (m, 1H, OCH morph.), 4.42–4.60 (m, 2H, CH -O), 4.81–
.95 (m, 1H, OCH cyclopent.), 6.58 (t, 1H, J = 76.0 Hz, OCHF ),
3
morph.),
1
3
3
4
7
.59–2.06 (m, 8H, 4CH
2
2
CO),
4.2.4. Spontaneous motor activity
2
Spontaneous locomotor activity was assessed in the open field
apparatus, consisting in four square chambers (height 40 cm,
width 45 cm, depth 45 cm) with a video camera fixed above them.
The tests were performed from 9 a.m. to 12 a.m. in mice fasted 18 h
before the experiments, but with free access to water. 30 min
before the trial, mice received orally the compounds under study
or the vehicle (0.5% methylcellulose, 10 mL/kg body weight). The
subjects were placed individually in the center of the square cham-
ber and locomotor activity was recorded for 60 min by means of a
video-tracking software (Any-maze, Ugo Basile, Comerio (VA),
Italy). The total traveled distance (meters) and the total time spent
2
2
.02–7.28 (m, 3H, H-6 + H-5 + H-2 Ar), 8.02 (s, 1H, CH@N). IR (film)
À1
cm : 1648 (C@O), 1579 (CH@N). Anal. C22
H
30
F
2
N
2
O
5
(C, H, N).
4
4
.2. Biological evaluation
.2.1. Enzymatic assays
PDE4A4, PDE4B2, PDE4C2, PDE4D3 enzyme assays were per-
formed by Scottish Biomedical (Glasgow, Scotland, UK) using
recombinant human PDE4 enzymes expressed in a baculoviral sys-
tem. The preliminary screening assays were performed by the
15
on the same place (immobility time; minutes) were registered.
Data are given as mean ± SEM of 6–8 observations.

3
434
C. Brullo et al. / Bioorg. Med. Chem. 23 (2015) 3426–3435
4
.2.5. Pharmacokinetic analysis
The pharmacokinetic analysis for 7b and 3b has been carried
Calibration curves were prepared in heparinized blank
mice plasma (1–10000 ng/mL) or blank brain homogenate
(1–10000 ng/g); lapatinib was used as internal standard.
Concentrations were calculated using a weighted least-squares
linear regression (W = 1/x ). The linear regression equations of
the calibration curves are presented in the following table.
out by the Center for Drug Metabolism and Pharmacokinetics
Research, Shanghai Institute of Materia Medica (Zhangjiang Hi-
Tech Park, Pudong, Shanghai, China). Briefly, a total of 21 male
BALB/c mice were used for each drug and three mice were used
2
Matrix
Plasma
Analyte
Regression equations
Correlation coefficient (r2)
À3
À4
À3
7b
y = 1.01 Â 10 Â +5.18 Â 10
0.9950
0.9960
À3
3
b
y = 2.66 Â 10 Â +1.09 Â 10
À4
À6
À6
Brain
7b
y = 1.36 Â 10 Â +3.76 Â 10
0.9937
0.9968
À4
3
b
y = 3.87 Â 10 Â +9.84 Â 10
for each time point. Drugs were dissolved in DMSO, diluted in
.5% methylcellulose to yield a final concentration of 1 mg/mL
and administered subcutaneously at the dose of 10 mg/kg. Dose
volume for each test animal was determined based on the most
Following sc administration of 10 mg/kg 7b and 3b to BABL/c
mice, plasma and brain concentrations were determined by the
LC–MS/MS method described above. The plasma and brain con-
centrations at each sampling time are listed in the following
table.
0
recent body weight. Blood samples (approximately 250
lL) were
Compd
Matrix
Time (h)
0.17
0.33
0.67
1.0
2.0
3.0
5.0
7
b
b
Plasma
Brain
4406 ± 44
1567 ± 175
1309 ± 613
396 ± 221
343 ± 54
84.2 ± 22.5
32.8 ± 13.5
3.29 ± 5.70
20.0 ± 8.0
2.21 ± 2.57
2.18 ± 1.15
1.19 ± 2.06
10.4 ± 9.6
nd
3
Plasma
Brain
2157 ± 333
936 ± 270
1737 ± 186
790 ± 174
1088 ± 105
453 ± 26
657 ± 53
303 ± 47
217 ± 21
96.0 ± 9.7
11.6 ± 3.3
5.45 ± 1.01
67.2 ± 75.8
2.94 ± 3.02
collected via retro-orbital puncture into tubes containing sodium
heparin at 10 min, 20 min, 40 min, 1 h, 2 h, 3 h, and 5 h post-injec-
tion. Plasma was separated by centrifugation (11000 rpm, 5 min,
The pharmacokinetic parameters, calculated by non-compart-
mental analysis using Phoenix 1.3 (Pharsight USA), were the fol-
lowing: AUC0–t, the area under the serum concentration curve
from time 0 to the last measurable concentration, calculated by
using the linear trapezoidal rule; Cmax, the maximum observed
4
°C) and stored at À70 °C before analysis. After blood harvest,
mice were sacrificed by cervical dislocation and brains were
excised, weighed, rinsed by cold saline and then frozen at À70 °C
until analysis.
serum concentration; Tmax, the time corresponding to Cmax; t1/2
,
the elimination half-life, calculated as 0.693/k which was
z
,
For blood analysis, 25
.5 mL Eppendorf tube and were added with 25
and 25 L of internal standard (500 ng/mL lapatinib), followed by
the addition of 100 L methanol.
As for brains, 100 mg of tissue were placed into a plastic tube
and added with 500 L of methanol to facilitate homogenization,
which was carried out using a Fluko F6/10 superfine homogenizer
for approximately 1 min. Homogenized samples were then treated
by ultrasound for 10 min and centrifuged at 11000 rpm for 5 min.
l
L of plasma were transferred into a
obtained by log-linear regression using the terminal points of the
serum concentration–time curve.
1
l
L of methanol
l
l
Acknowledgments
l
Alzheimer Association is gratefully acknowledged for financial
support to this study (IIRG 11-208306). We thank Dr. M. Anzaldi,
R. Raggio, Mr. O. Gagliardo, and F. Tuberoni for spectral recording
and Mr. V. Ruocco for the informatic support.
A 50
l
L aliquot of the homogenized samples was transferred into
L of internal standard
an Eppendorf tube and added with 20
l
Supplementary data
(
500 ng/mL lapatinib).
After centrifugation at 11000 rpm for 5 min, a 10 (plasma) or a
0 (brain tissue) L aliquot of the upper layer was injected onto the
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.027.
2
l
LC/MS/MS system that consisted of a Shimadzu LC-20A HPLC sys-
tem (Shiseido, Tokyo, Japan) coupled with a TSQ Quantum
Vantage triple quadrupole mass spectrometer equipped with a
HESI source (ThermoFisher, San Jose, CA, USA). Chromatographic
conditions were the following: guard column SecurityGuard C18
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