Catecholic amides as potential selective phosphodiesterase 4D inhibitors: Design, synthesis, pharmacological evaluation and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2015.10.033

In this study, a series of catechol-based amides (8a-n) with different amide linkers linking the catecholic moiety to the terminal phenyl ring was designed and synthesized as potent phosphodiesterase (PDE) 4D inhibitors. The inhibitory activities of these compounds were evaluated against the core catalytic domains of human PDE4 (PDE4CAT), full-length PDE4B1 and PDE4D7 enzymes, and other PDE family members. The results indicated the majority of compounds 8a-n displayed moderate to good inhibitory activities against PDE4CAT. Among these compounds, compound 8j with a short amide linker (-CONHCH₂-) displayed comparable PDE4CAT inhibitory activity (IC₅₀ = 410 nM) with rolipram. More interestingly, compound 8g, a potent and selective PDE4D inhibitor (IC₅₀ = 94 nM), exhibited a 10-fold selectivity over the PDE4B subtypes and an over 1000-fold selectivity against other PDE family members. Docking simulations suggested that 8g forms three extra H-bonds with the N-H of residue Asn487 and two water molecules.

Full text of DOI:10.1016/j.bmc.2015.10.033

Bioorganic & Medicinal Chemistry 23 (2015) 7332–7339
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Catecholic amides as potential selective phosphodiesterase 4D
inhibitors: Design, synthesis, pharmacological evaluation and
structure–activity relationships
a
a
a
b,
a,
Zhong-Zhen Zhou a, , Bing-Chen Ge , Yu-Fang Chen , Xiu-Dong Shi , Xue-Mei Yang , Jiang-Ping Xu
⇑
⇑
⇑
a Neuropharmacology Group, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
b Hygiene Detection Center, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of catechol-based amides (8a–n) with different amide linkers linking the catecholic
moiety to the terminal phenyl ring was designed and synthesized as potent phosphodiesterase (PDE) 4D
inhibitors. The inhibitory activities of these compounds were evaluated against the core catalytic
domains of human PDE4 (PDE4CAT), full-length PDE4B1 and PDE4D7 enzymes, and other PDE family
members. The results indicated the majority of compounds 8a–n displayed moderate to good inhibitory
Received 18 September 2015
Revised 22 October 2015
Accepted 23 October 2015
Available online 24 October 2015
activities against PDE4CAT. Among these compounds, compound 8j with
a short amide linker
Keywords:
(ACONHCH2A) displayed comparable PDE4CAT inhibitory activity (IC50 = 410 nM) with rolipram. More
interestingly, compound 8g, a potent and selective PDE4D inhibitor (IC50 = 94 nM), exhibited a 10-fold
selectivity over the PDE4B subtypes and an over 1000-fold selectivity against other PDE family members.
Docking simulations suggested that 8g forms three extra H-bonds with the NAH of residue Asn487 and
two water molecules.
Catechol-based benzamides
Phosphodiesterase 4D inhibitors
Selectivity
Structure–activity relationship
Molecular docking
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
of undesirable effects.12 Many studies have suggested that PDE4D
may play a pivotal role in the antidepressant-like and cognition
Cyclic nucleotide phosphodiesterases (PDEs) comprise a group
of enzymes that control the rates of hydrolysis of cAMP and cGMP.
Hence, these enzymes are important regulators of the signals
mediated by these second messenger molecules. Eleven different
PDE families (PDE1–PDE11) are ubiquitously expressed in the
body, play specific roles in distinct physiological and pathological
processes.1 Among the large family of PDEs, phosphodiesterase IV
(PDE4) which specifically catalyzes the hydrolysis of cAMP is ubiq-
uitous in the body, such as inflammatory and immune cell type’s
relevant, central nervous system (CNS) tissue2–4 (see Fig. 1).
PDE4 is comprised of four genes (PDE4A–D) and each gene has
multiple transcripts that can produce three isoforms of the protein
termed long, short and super short.5 And PDE4 is highly expressed
in brain regions involved with regulation of memory, anxiety and
depression.6,7 Many PDE4 inhibitors, such as rolipram (1), dis-
played antidepressant activities8 followed by memory-enhancing
effects.9–11 However, these PDE4 inhibitors that can block all the
different PDE4 isoforms have not been brought to market because
enhancing effects of PDE4 inhibitors.13–16 Although there are still
outstanding issues regarding the relationship between PDE4D,
emesis, depression, and cognition, there is clear evidence that the
development of selective PDE4D inhibitors without emesis could
prove a winning strategy to produces antidepressant-like and cog-
nition-enhancing effects, such as PDE4D inhibitors DF159687 (2),17
chlorbipram (3)18 and GEBR-7b (4).19,20
The crystal structure of human PDE4D in complex with
rolipram,17,21 indicates that the catechol moiety of rolipram is an
important pharmacophore, in which conformers interact with the
purine-selective glutamine and hydrophobic clamp pocket
(Fig. 2), and the side-chain region of rolipram still has much space
for structural optimization and exploration of subtype selectivity.
On the other hand, many studies have indicated that the linker
length and its spatial direction play critical roes, particularly in
determining the selectivity toward the different PDE4 isoforms.
For example, the replacement of the AC@NOCH2COA linker of
GEBR-7b with a longer linker (AC@NOCH2CH(OH)CH2NHA) give
compound
5 improved the PDE4D selectivity (85-fold) over
PDE4B.22 In addition, the PDE4D inhibitor CPD-B (6)23 with a short
CONHCH2 linker also displayed 142-fold subtype selectivity for
PDE4D over PDE4B (Fig. 2).
⇑
Corresponding authors. Tel./fax: +86 20 61648235.
E-mail addresses: zhouzz@smu.edu.cn (Z.-Z. Zhou), yxmjp@sina.com
(X.-M. Yang), jpx@smu.edu.cn (J.-P. Xu).
http://dx.doi.org/10.1016/j.bmc.2015.10.033
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

Z.-Z. Zhou et al. / Bioorg. Med. Chem. 23 (2015) 7332–7339
7333
been reported to be inactive towards PDE4,32 3-(cyclopentyloxy)-
4-methoxybenzamides with other substituted benzyl groups were
not evaluated in this paper. Thus, in the present study, we report
the design and synthesis of a series of catechol-based amides in
which the acylhydrazone linker of compound 7 is replaced with
a shorter linker (CONHCH2), or a longer linker (CONHCH2CH2O)
(8a–n, Fig. 2).
It is known that PDE4 enzymes contain unique regulatory
domains, called upstream conserved regions 1 and 2 (UCR1 and
UCR2), and the UCR2 or C-terminal regulatory domain can interact
with the catalytic domains of PDE4 enzymes to regulate enzyme
activity.17 Since the access of PDE4 inhibitors to the catalytic
pocket are controlled by these helix-capped conformations, the
inhibitory activity and selectivity of full length enzymes were dif-
ferent from those with truncated enzymes. Many studies have con-
firmed that compounds with truncated enzymes exhibit high
selectivities, while full-length enzymes exhibit lower selectivi-
ties.33 Therefore, in this study, we used full length human PDE4B1
and PDE4D7 enzymes for the in vitro assay to explore PDE4D sub-
type-selective inhibitors, along with other PDE family members for
isoform selectivity. The structure–activity relationships (SARs) of
these derivatives are also discussed.
MeO
MeO
NH
O
NH2
O
O
NH
Cl
1 Rolipram
2 D159687
Ratio B/D (IC50) = 0.5
Ratio B1/D7 (IC50) = 54
O
MeO
O
N
N
N
O
MeO
O
HN
O
N
O
OMe
Cl
Me
Me
3 Chlorbipram
Ratio B2/D4 (IC50) = 13
4 GEBR-7b
O
O
CH3
MeO
N
H
N
O
O
NH
N
N
OMe
OH
Me
2. Results and discussion
2.1. Chemistry
O
Me
F
5
6
The synthetic pathways employed for the synthesis of com-
pounds 8a–n is illustrated in Schemes 1 and 2. 4-Methoxy-3-
alkoxybenzoic acids 11a–b were obtained by the alkali hydrolysis
of methyl 4-methoxy-3-alkoxybenzoates 10a–b, which were syn-
thesized by the alkylation at position 3 of methyl 4-methoxy-3-
hydroxybenzoate 9 with corresponding bromoalkanes (Scheme 1).
Selective difluoromethylation at position 4 of compound 13 using
sodium chlorodifluoroacetate/NaOH led to the formation of
3-hydroxy-4-difluoromethoxybenzaldehyde 14. Further alkylation
at position 3 with bromoalkane gave 3-alkoxy-4-methoxyben-
zaldehydes 15a–b, which were oxidized to corresponding acids
16a–b by 30% H2O2 (Scheme 2). Acids (11a–b and 16a–b) were
converted by thionyl chloride to acid chlorides (12a–b and
17a–b), which were subsequently reacted with the corresponding
amines to prepare benzamides 8a–n with good yields.
CPD-B
Ratio B2/D3 (IC50) = 85
Ratio B/D (IC50) = 142
Figure 1. General structures of PDE4 inhibitors (1–6).
There is no denying the prevalence of the dialkyl catechol group
in PDE4 inhibitors. The catechol motif, which is known to accept a
double hydrogen bond from the carboxamide of a glutamine resi-
due at the back of the PDE4 catalytic site,24–26 is found in a range
of PDE4 inhibitors, from the earliest described compounds such
as rolipram through to marketed roflumilast27 and apremilast.28
On the other hand, many amide derivatives29,30 as drugs and agri-
chemicals display wonderful activities. For example, catechol-
based N-acylhydrazone 7 showed wonderful inhibitory activities
for PDE4B and PDE4D.31 It provided us with an opportunity to
design selective PDE4D inhibitors by the replacement of acylhydra-
zone linker (ACH@NAN(Me)COA) of compound 7 with shorter or
longer amide linkers linking the catecholic moiety to the terminal
substituted phenyl ring. Although N-(2-chlorobenzyl)-3-
(cyclopentyloxy)-4-methoxybenzamide with CONHCH2 linker has
2.2. Pharmacology
Recent studies have indicated that the catalytic domains of all
subtypes of PDE4 (PDE4A–D) exhibit a high degree of sequence
conservation with a single active-site amino acid determining the
important
R1O
MeO
linker
the terminal
important
side-chian
pharmacophore
linker
phenyl ring
pharmacophore
R2
8a-f
O
OMe
Me
MeO
N
NH
N
H
N
R1O
linker
O
R2
O
O
MeO
the catecholic
moiety
H
O
F2HCO
8g-m
side-chian
Gln
rolipram
7
Me
PDE4B (IC50) 53 nM
PDE4D (IC50) 74 nM
the catecholic
moiety
linker
= CONHCH2, CONHCH2CH2O
R1 = cyclopentyl, cyclopropylmethyl
R2 = H, 2-MeO, 4-MeO, 3,4-MeO,
Figure 2. Design strategy.

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Z.-Z. Zhou et al. / Bioorg. Med. Chem. 23 (2015) 7332–7339
Table 1
MeO
R1O
MeO
R1Br
Inhibition of PDE4CAT by target compounds 8a–n
OCH3
OCH3
a
Compound
R1
R2
IC50 (lM)
HO
Cs2CO3, DMF
yield > 95%
O
O
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
Rolipram
Cyclopentyl
Cyclopentyl
4-MeO
3,4-MeO
4-MeO
3,4-MeO
2-MeO
2-MeO
4-MeO
3,4-MeO
H
0.96
>100
0.91
>100
18.30
11.10
1.10
9
10a R1 = cyclopentyl
10b R1 = cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopentyl
Cyclopropylmethyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopentyl
MeO
R1O
MeO
KOH
SOCl2
refluxing
OH
Cl
R1O
MeOH/H2O
yield > 97%
>100
2.20
O
O
12a R1 = cyclopentyl
11a R1 = cyclopentyl
4-MeO
3,4-MeO
H
0.41
>100
2.10
12b R1 = cyclopropylmethyl
11b R1 = cyclopropylmethyl
MeO
2-MeO
2-MeO
12.40
6.90
R2
H
Cyclopropylmethyl
N
R1O
0.55
RCH2NH2
O
a
Data reported are the mean of two experiments.
CH2Cl2, NEt3
8a-d
8e-f
MeO
R1O
MeO
O
H
N
rolipram. It’s mean that 3,4-dimethoxy group on the terminal
phenyl ring of the compounds with the shorter amide linker
(CONHCH2) may be unfavourable for the PDE4 inhibitory activity.
In addition, compounds 8e, 8f, 8m and 8n with the longer amide
linker (CONHCH2CH2O) showed weaker PDE4 inhibitory activities
O
Scheme 1. Synthetic route of compounds 8a–f.
(IC50 = 6.9–18.3 lM). Further analysis of the SARs of 8a–n suggests
that the N-substituents have profound effects on inhibitory activ-
ity; the activities decrease in the following order: 4-MeOPhCH2 >
PhCH2 > 2-MeOPhOCH2CH2 >> 3,4-MeOPhCH2.
nucleotide selectivity.24,34 Preliminary tests on PDE4CAT activity
were performed according to reported protocols,35 using rolipram
as a positive control. All compounds were tested on PDE4CAT at
nine concentrations (10ꢀ8–10ꢀ4 M) and IC50 values were deter-
mined by nonlinear regression analysis of their inhibition curves
(see Table 1).
It can be seen from Table 1 that the inhibitory activities of com-
pounds with the shorter amide linker (CONHCH2) were strongly
modulated by the substituents on the terminal phenyl ring. For
example, all catechol-based amides containing a 3,4-dimethoxy-
benzyl group (i.e., 8b, 8d, 8h, and 8k) exhibit no activity against
Subsequently, compounds 8a, 8c, 8g, and 8j, which exhibited
good activities, were screened for their ability to inhibit full-length
human PDE4B1 and PDE4D7 enzymes in vitro which were pur-
chased form BPS Bioscience Inc., and using rolipram as a positive
control (Table 2). As shown in Table 2, all screened compounds dis-
played both weaker PDE4B1 inhibition, and more potent PDE4D7
inhibitory activity as compared to rolipram. A careful analysis of
the SAR of N-(4-methoxybenzyl)benzamides (8a, 8c, 8g, and 8j)
reveals that compounds 8g and 8j with 4-difluoromethoxy groups
showed higher inhibitory activities than their corresponding ana-
logs (8a and 8c) with 4-methoxy groups. Moreover, compound
8g displayed good selectivity (ꢁ10 fold) for the PDE4D7 subtype
over the PDE4B1 subtype. This suggests that this compound is
exploitable as a potential lead compound for the design of PDE4D
inhibitors.
PDE4 (IC50 > 100 lM). However, catechol-based amides containing
either a 4-methoxybenzyl group (8a, 8c, 8g, and 8j) or a benzyl
group (8i and 8l) displayed moderate to good PDE4CAT inhibitory
activities (IC50 = 0.41–2.22
lM) of rolipram. Moreover, the PDE4
inhibitory activity of compound 8j was comparable with that of
PDEs exist as 11 different isozymes involved in various physio-
logical processes; therefore the selective inhibition of PDE4 is very
important. Thus, we determined the selectivity of compound 8g
toward the other PDEs isoforms using human PDE1A1, PDE1A,
PDE2A, PDE3B, PDE5A, PDE6C, PDE7A, PDE8A1, PDE9A2, PDE10A2
and PDE11A, which were purchased form BPS Bioscience Inc.
HO
HO
F2HCO
CF2ClCOONa
R1Br
H
H
HO
Cs2CO3, DMF
yield > 95%
NaOH, DMF/ H2O
O
O
120 oC, 2 h, 44%
13
14
F2HCO
R1O
F2HCO
R1O
30% H2O2
SOCl2
H
OH
refluxing
Table 2
KOH, MeOH/H2O
yield > 97%
O
Inhibition (IC50, nM)a of PDE4B1 and PDE4D7
O
15a R1 = cyclopentyl
16a R1 = cyclopentyl
15b R1 = cyclopropylmethyl
MeO
R1O
OMe
16b R1 = cyclopropylmethyl
F2HCO
R1O
OMe
H
N
H
N
F2HCO
R2
O
F2HCO
O
H
N
8g
8j
and
R1O
8a and 8c
RCH2NH2
Cl
R1O
O
CH2Cl2, NEt3
R1
PDE4B1
PDE4D7
Selectivity (B/D)
8g-l
Compound
O
F2HCO
R1O
MeO
O
17a
17b
R
R
1 = cyclopentyl
1 = cyclopropylmethyl
8a
8c
8g
8j
Cyclopentyl
1031
1087
976
301
86
284
342
94
108
160
3.6
3.2
10.3
2.8
H
N
Cyclopropylmethyl
Cyclopentyl
Cyclopropylmethyl
/
O
8m-n
Scheme 2. Synthetic route of compounds 8g–n.
Rolipram
0.54
a
Data reported are the means of two experiments.

Z.-Z. Zhou et al. / Bioorg. Med. Chem. 23 (2015) 7332–7339
7335
Table 3
Inhibition of various PDEs by compound 8g
a
PDEs
Inhibition (%)a at 10
lM
IC50 (nM)
PDE1A
PDE2A
PDE3B
PDE5A
PDE6C
PDE7A
PDE8A1
PDE9A2
PDE10A2
PDE11A
11
2
2
16
5
3
10
6
1
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
>1 ꢂ 105
2
a
Data reported are the mean of two experiments.
(Table 3). As shown in Table 3, compound 8g showed the highest
PDE4 selectivity (at least ꢁ1000-fold) over other PDEs.
2.3. Molecular docking
To support the biological data, we performed a docking study
on compounds 8a and 8g with the active site of PDE4D. In this
investigation, the 3D structure of PDE4D (Protein Data Bank
(PDB) entry 3G4K)17 with rolipram was taken from a PDB entry.
The new inhibitors were built and optimized using the Powell
method in sybyl 7.3. Hydrogen and Gasteiger–Hückel charges were
added to every molecule. Water molecules conserved in all PDE4D
structures deposited into the PDB were considered for calculations.
To validate docking reliability, the co-crystalized ligand (roli-
pram) was removed from the active site and docked back into
the known binding pocket. The root mean square deviation (RMSD)
between the predicted conformation and the actual conformation
from the crystal structure of ligand was 1.29 Å, which is smaller
than the resolution of X-ray crystallography 1.95 Å17). Thus, the
parameters set for this Surflex-dock simulation could successfully
reproduce the ligand-binding motif from the X-ray structure, and
could be extended to predict the binding conformations of the
synthesized inhibitors.
The docking studies highlighted the great conformational vari-
ability of the inhibitors inside the catalytic site: all the minimum
energy conformations of compounds 8a and 8g in the active site
assumed a V-shaped binding conformation (Fig. 3). In the active
site of PDE4D, it is clear that the catechol moieties in 8a and 8g
were predicted to occupy the pocket of the catechol moiety in roli-
pram with subtle apparent variances, and to interact in the binding
site by hydrogen bonding with NH2 of the Gln535 residue and
p-stacking with Phe538. Meanwhile, the linker regions (CONHCH2)
are in position to form an extra hydrogen bond with a conserved
water molecule, which extends to Tyr325.
Compound 8g is one of the most potent and selective PDE4D
inhibitors among the newly synthesized compounds. As observed
in the active site of PDE4D (Fig. 3B), this improvement in activity
could be attributed to the extra H-bond of 4-difluoromethyl group
with the NAH of residue Asn487 and the extra H-bond of the
4-methoxy group in the terminal phenyl ring with a conserved
water molecule in contact with residues Ser374 and Ser521. This
finding is consistent with earlier studies24 that the 4-difluro-
methoxy group fits better in the small sub-pocket and generates
more hydrophobic interactions than the 4-methoxy group.
Figure 3. Potential binding poses of 8a (A) and 8g (B) in the active site of PDE4D.
The C atoms in 8a and 8g are colored in white and green–blue, respectively; total
docking scores of 8a and 8g are 8.522 and 9.537, respectively. The C atoms in the
native ligand (rolipram, total docking score = 9.018) are colored in orange. The H-
bonds between each ligand and active-site amino acids are shown in yellow.
3. Conclusion
bearing other substituted benzyl groups (e.g., benzyl and 3,4-
methoxybenzyl groups), and other benzamides with longer linkers
(CONHCH2CH2O) bearing 2-methoxyphenyl groups. The PDE4 inhi-
bitory activity of compound 8j was comparable with that of roli-
pram. More interestingly, compound 8g displayed a preference
for PDE4D with a 10-fold selectivity over PDE4B1, and an at least
In conclusion, a series of catechol-based amide derivatives was
designed and synthesized as potential PDE4 inhibitors. Among
these compounds, N-(4-methoxybenzyl)benzamides 8a, 8c, 8g,
and 8j showed higher PDE4 inhibitory activities than their analogs

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Z.-Z. Zhou et al. / Bioorg. Med. Chem. 23 (2015) 7332–7339
1000-fold selectivity over other PDEs. In the PDE4D catalytic
domain, docking studies revealed that the catechol moiety in 8g
assumes a similar binding profile to PDE4 with rolipram in the
hydrophobic clamp pocket. In addition, the 4-difluoromethyl group
in the catechol moiety forms an extra H-bond with the NAH of
residue Asn487, while the side chain in 8g forms two extra
H-bonds with two water molecules. Further efforts will be aimed
at developing more potent PDE4D inhibitors with increased
selectivities.
(15 mL) and water (0.3 mL) was added sodium hydroxide (0.48 g,
12 mmol). Then, the mixture was heated at 120 °C and stirred at
this temperature for 2 h. The solvent was removed by vacuum
distillation, and to the residue was added aqueous HCl (2 mL).
The mixture was extracted with Et2O and washed with brine.
The solvent was removed under reduced pressure, and the crude
product was purified by chromatography on silica gel (petroleum
ether/EtOAc = 80:20) to afford 12 as
a white solid (1.05 g,
5.28 mmol, 46%). ESI-MS (m/z): 187.4 ([MꢀH]ꢀ).
4.1.4. Synthesis of 4-difluoromethoxy-3-alkoxybenzoic acid 16
General procedures: A mixture of 4-difluoromethoxy-3-hydroxy-
benzaldehyde 14 (1.88 g, 10 mmol), alkylbromide (20 mmol), and
cesium carbonate (6.51 g, 20 mmol) in DMF (15 mL) was stirred
at 65 °C for 1 h and monitored by TLC. Then, the reaction solution
was poured into water and the solid was collected. The obtained
precipitate 15 was used in all the following reactions without fur-
ther purification.
4. Experimental section
4.1. Chemistry
ESI spectra were obtained using a Waters UPLC/Quattro Premier
XE mass spectrometer. 1H and 13C NMR spectra were recorded for
CDCl3 or DMSO-d6 using a Varian Mercury 400 spectrometer and
TMS as an internal reference. Elemental analyses were performed
using a Vario ELIII CHNSO elemental analyzer. Rolipram was pur-
chased from Sigma (St. Louis, MO, USA). All other chemicals were
of analytical grade and used without further purification.
A
mixture of 4-difluoromethoxy-3-alkoxybenzaldehyde 15
(6.25 mmol), 30% H2O2 (1 mL) and KOH (2.8 g, 50 mmol) in metha-
nol (10 ml) was stirred at room temperature for 1 h and monitored
by TLC. Then, the reaction mixture was diluted by water (30 mL)
and adjusted to pH 2 with concentrated hydrochloric acid. The
formed precipitate was collected by filtration to pure 4-difluo-
romethoxy-3-alkoxybenzoic acid.
4.1.1. Synthesis of methyl 4-methoxy-3-alkoxybenzoate 10
General procedures: A mixture of 4-methoxy-3-hydroxybenzoate
9 (1.82 g, 10 mmol), alkylbromide (20 mmol), and cesium carbon-
ate (6.51 g, 20 mmol) in DMF (15 mL) was stirred at 65 °C for 1 h
and monitored by TLC. Then, the reaction solution was poured
into water and the solid was collected. The obtained precipitate
was chromatographed using a mixture of petroleum ether and
acetone (v/v = 20:1) to give corresponding methyl 4-methoxy-3-
alkoxybenzoate.
4.1.4.1. 3-(Cyclopentyloxy)-4-(difluoromethoxy)benzoic acid
16a. Yield: 98%. 1H NMR (400 MHz, CDCl3) d 7.71–7.68 (m, 2H),
7.22 (d, J = 8.8 Hz, 1H), 6.64 (t, J = 74.9 Hz, 1H), 4.92–4.87 (m,
1H), 2.02–1.79 (m, 6H), 1.71–1.62 (m, 2H). 13C NMR (101 MHz,
CDCl3)
d 171.32, 149.37, 145.34, 145.31, 145.28, 127.24,
123.28, 122.15, 118.42 (ACHF2), 116.46, 115.83 (ACHF2),
113.24 (ACHF2), 81.02, 32.75, 23.92. ESI-MS (m/z): 271.5
([MꢀH]ꢀ).
4.1.1.1. Methyl 3-(cyclopentyloxy)-4-methoxybenzoate 10a.
Yield 95%. ESI-MS (m/z): 273.4 ([M+Na]+), 251.5 ([M+H]+).
4.1.4.2. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)benzoic
acid 16b. Yield: 97%. 1H NMR (400 MHz, CDCl3) d 7.75 (dd,
J = 8.2, 1.8 Hz, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H),
6.77 (t, J = 74.8 Hz, 1H), 3.98 (d, J = 6.8 Hz, 2H), 1.39–1.30 (m,
1H), 0.73–0.66 (m, 2H), 0.43–0.39 (m, 2H). 13C NMR (101 MHz,
CDCl3) d 170.98, 150.19, 144.88, 144.85, 144.82, 127.20, 123.72,
121.83, 118.31 (ACHF2), 115.72 (ACHF2), 115.67, 113.12 (ACHF2),
74.15, 10.04, 3.28. ESI-MS (m/z): 257.6 ([MꢀH]ꢀ).
4.1.1.2. Methyl 3-(cyclopropylmethoxy)-4-methoxy benzoate
10b. Yield 95%. ESI-MS (m/z): 259.4 ([M+Na]+), 237.6 ([M+H]+).
4.1.2. Synthesis of 4-methoxy-3-alkoxybenzoic acid 11
General procedures: A mixture of methyl 4-methoxy-3-alkoxy-
benzoate (5 mmol), KOH (1.12 g, 20 mmol) in methanol (9 mL)
and water (1 mL) was stirred at room temperature for 12 h, and
monitored by TLC. Then, the reaction mixture was adjusted to pH
2 with cool hydrochloric acid (2 M). The formed precipitate was col-
lected by filtration to give pure 4-methoxy-3-alkoxybenzoic acid.
4.1.5. Synthesis of compounds 8a–n
General procedures: To the intermediate alkoxybenzoic acid
(0.5 mmol), excess thionyl chloride (5 mL) was added, and the
reaction mixture was stirred at 60–80 °C for 3 h. Excess thionyl
chloride was then evaporated under reduced pressure to give the
corresponding alkoxybenzoyl chloride as a crude yellow oil, which
was used in the below mentioned reaction without further
purification.
To a solution of amine (0.5 mmol) and anhydrous triethylamine
(1 mmol) in dry CH2Cl2 (5 mL), a solution of previously prepared
alkoxybenzoyl chloride (0.5 mmol) in dry CH2Cl2 (2 mL) was
slowly added at 0 °C. The reaction mixture was stirred at room
temperature for 3 h, monitored by TLC, and then concentrated
under reduced pressure; the obtained residue was purified using
flash chromatography (petroleum ether/acetone 20:1) to give the
desired amide products as colorless solids.
4.1.2.1. 3-(Cyclopentyloxy)-4-methoxybenzoic acid 11a22
. Total
yield: 93%. 1H NMR (400 MHz, CDCl3) d 7.77 (dd, J = 8.4, 2.0 Hz,
1H), 7.63 (d, J = 2.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 4.90–4.85 (m,
1H), 3.94 (s, 3H), 2.07–1.83 (m, 6H), 1.71–1.61 (m, 2H). 13C NMR
(101 MHz, CDCl3) d 172.07, 154.73, 147.34, 124.28, 121.58, 115.65,
110.76, 80.59, 56.08, 32.77, 24.10. ESI-MS (m/z): 235.6 ([MꢀH]ꢀ).
4.1.2.2.
11b36
3-(Cyclopropylmethoxy)-4-methoxybenzoic
acid
.
Total yield: 94%. 1H NMR (400 MHz, CDCl3) d 7.77
(dd, J = 8.4, 2.0 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 6.92 (d,
J = 8.4 Hz, 1H), 3.95 (s, 3H), 3.92 (d, J = 7.2 Hz, 2H), 1.41–1.33
(m, 1H), 0.69–0.64 (m, 2H), 0.40–0.37 (m, 2H). 13C NMR
(101 MHz, CDCl3) d 171.99, 154.22, 148.15, 124.61, 121.59,
114.20, 110.61, 74.06, 56.10, 10.17, 3.47. ESI-MS (m/z): 245.9
([M+Na]+), 223.8 ([M+H]+).
4.1.5.1. 3-(Cyclopentyloxy)-4-methoxy-N-(4-methoxybenzyl)
benzamide (8a). Yield: 66%. 1H NMR (400 MHz, CDCl3) d 7.44
(d, J = 2.0 Hz, 1H), 7.30–7.27 (m, 2H), 7.24 (dd, J = 8.4, 2.1 Hz, 1H),
6.91–6.86 (m, 2H), 6.83 (d, J = 8.4 Hz, 1H), 6.26 (s, 1H, NH),
4.1.3. Synthesis of 4-difluoromethoxy-3-hydroxybenzaldehyde 1437
To a solution of 3,4-dihydroxybenzaldehyde (1.66 g, 12 mmol)
and sodium chlorodifluoroacetate (1.83 g, 12 mmol) in DMF

Z.-Z. Zhou et al. / Bioorg. Med. Chem. 23 (2015) 7332–7339
7337
4.87–4.82 (m, 1H), 4.56 (d, J = 5.6 Hz, 2H), 3.87 (s, 3H), 3.80 (s, 3H),
1.99–1.79 (m, 6H), 1.65–1.59 (m, 2H). 13C NMR (100 MHz, CDCl3) d
167.0, 159.1, 152.8, 147.7, 130.5, 129.3, 126.9, 119.2, 114.1, 114.1,
110.8, 80.6, 56.1, 55.3, 43.6, 32.8, 24.0. ESI-MS (m/z): 378.6 ([M
+Na]+), 356.6 ([M+H]+) and Anal. Calcd for C21H25NO4: C, 70.96;
H, 7.09; N, 3.94. Found: C, 70.77; H, 7.19; N, 4.11.
(ACHF2), 115.4, 113.9 (ACHF2), 113.3, 112.0, 74.1, 69.1, 55.8,
39.6, 10.1, 3.2. ESI-MS (m/z): 408.4 ([M+H]+),430.4 ([M+Na]+) and
Anal. Calcd for C21H23F2NO5: C, 61.91; H, 5.69; N, 3.44. Found: C,
61.64; H, 5.43; N, 3.57.
4.1.5.7. 3-(Cyclopentyloxy)-4-(difluoromethoxy)-N-(4-methoxy-
benzyl)benzamide (8g). Yield: 87%. 1H NMR (400 MHz, CDCl3) d
7.53 (s, 1H), 7.28 (s, 1H), 7.18–7.12 (m, 2H), 6.88 (d, J = 8.4 Hz,
2H), 6.57 (t, J = 75.2 Hz, 1H, –CHF2), 6.36 (s, 1H, NH), 4.92–4.84
(m, 1H), 4.56 (d, J = 5.2 Hz, 2H), 3.80 (s, 3H), 1.96–1.76 (m, 6H),
1.70–1.64 (m, 3H). 13C NMR (100 MHz, CDCl3) d 166.3, 159.2,
149.8, 143.3, 132.8, 130.1, 129.3, 122.3, 118.5 (ACHF2), 118.3,
116.0 (ACHF2), 114.7, 114.2, 113.4 (ACHF2), 80.9, 55.34, 43.78,
4.1.5.2. 3-(Cyclopentyloxy)-N-(3,4-dimethoxybenzyl)-4-methoxy-
benzamide (8b). Yield: 55%. 1H NMR (400 MHz, DMSO-d6) d 8.79
(t, J = 6.0 Hz, 1H), 7.51 (dd, J = 8.4, 2.0 Hz, 1H), 7.46 (d, J = 2.0 Hz,
1H), 7.01 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 1.5 Hz, 1H), 6.89 (d,
J = 8.2 Hz, 1H), 6.83 (dd, J = 8.2, 1.6 Hz, 1H), 4.89–4.78 (m, 1H), 4.39
(d, J = 6.0 Hz, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.72 (s, 3H), 1.95–1.81
(m, 2H), 1.77–1.50 (m, 6H). 13C NMR (100 MHz, DMSO-d6) d 165.6,
152.1, 148.6, 147.7, 146.4, 132.4, 126.6, 120.5, 119.4, 113.6, 111.8,
111.4, 111.3, 79.6, 55.6, 55.6, 55.4, 42.3, 32.2, 23.5. ESI-MS (m/z):
384.5 ([MꢀH]ꢀ) and Anal. Calcd for C22H27NO5: C, 68.55; H, 7.06; N,
3.63. Found: C, 68.37; H, 7.14; N, 3.78.
32.8, 23.9. ESI-MS (m/z): 390.5 ([MꢀH]ꢀ) and Anal. Calcd for C21
-
H
23F2NO4: C, 64.44; H, 5.92; N, 3.58. Found: C, 64.39; H, 5.88; N,
3.65.
4.1.5.8. 3-(Cyclopentyloxy)-4-(difluoromethoxy)-N-(3,4-dimeth-
oxybenzyl)benzamide (8h). Yield: 78%. 1H NMR (400 MHz,
CDCl3) d 7.54 (d, J = 1.2 Hz, 1H), 7.19–7.13 (m, 2H), 6.92–6.82 (m,
3H), 6.58 (t, J = 75.2 Hz, 1H, –CHF2), 6.34 (s, 1H, NH), 4.93–4.86
(m, 1H), 4.57 (d, J = 5.6 Hz, 2H), 3.87 (s, 6H), 1.99–1.75 (m, 6H),
1.70–1.63 (m, 2H). 13C NMR (100 MHz, CDCl3) d 166.3, 149.9,
149.3, 148.7, 143.3, 132.8, 130.6, 122.4, 120.3, 118.5 (ACHF2),
118.3, 115.9 (ACHF2), 114.7, 113.4 (ACHF2), 111.4, 111.3, 80.9,
56.0, 56.0, 44.2, 32.8, 23.9. ESI-MS (m/z): 420.5 ([MꢀH]ꢀ) and Anal.
Calcd for C22H25F2NO5: C, 62.70; H, 5.98; N, 3.32. Found: C, 62.82;
H, 6.02; N, 3.41.
4.1.5.3. 3-(Cyclopropylmethoxy)-4-methoxy-N-(4-methoxyben-
zyl)benzamide (8c). Yield: 45%. 1H NMR (400 MHz, CDCl3) d 7.42
(d, J = 2.0 Hz, 1H), 7.30–7.27 (m, 2H), 7.25–7.24 (m, 1H), 6.91–6.86
(m, 2H), 6.84 (d, J = 8.4 Hz, 1H), 6.26 (s, 1H, NH), 4.56 (d, J = 5.6 Hz,
2H), 3.91–3.89 (m, 5H), 3.80 (s, 3H), 1.39–1.30 (m, 1H), 0.67–0.60
(m, 2H), 0.37–0.33 (m, 2H). 13C NMR (100 MHz, CDCl3) d 166.9,
159.1, 152.2, 148.5, 130.5, 129.3, 127.0, 119.4, 114.1, 112.5,
110.6, 74.1, 56.0, 55.3, 43.6, 10.2, 3.4. ESI-MS (m/z): 342.7 ([M
+H]+) and Anal. Calcd for C20H23NO4: C, 70.36; H, 6.79; N, 4.10.
Found: C, 70.07; H, 6.51; N, 4.32.
4.1.5.9. N-Beyl-3-(cyclopentyloxy)-4-(difluoromethoxy)benza-
mide (8i). Yield: 90%. 1H NMR (400 MHz, CDCl3) d 7.54 (d,
J = 1.6 Hz, 1H), 7.40–7.27 (m, 5H), 7.21–7.12 (m, 2H), 6.79–6.35
(m, 2H), 4.92–4.86 (m, 1H), 4.63 (d, J = 5.6 Hz, 2H), 1.99–1.76 (m,
6H), 1.71–1.59 (m, 2H). 13C NMR (100 MHz, CDCl3) d 166.4,
149.8, 143.3, 138.0, 132.7, 128.8, 127.9, 127.7, 122.4, 118.5
(ACHF2), 118.3, 116.0 (ACHF2), 114.7, 113.4 (ACHF2), 80.9, 44.3,
32.8, 23.9. ESI-MS (m/z): 362.5 ([M+H]+), 384.5 ([M+Na]+) and Anal.
Calcd for C20H21F2NO3: C, 66.47; H, 5.86; N, 3.88. Found: C, 66.25;
H, 5.97; N, 3.72.
4.1.5.4. 3-(Cyclopropylmethoxy)-N-(3,4-dimethoxybenzyl)-4-
methoxybenzamide (8d). Yield: 57%. 1H NMR (400 MHz,
DMSO-d6) d 8.78 (t, J = 6.0 Hz, 1H), 7.51 (dd, J = 8.4, 2.0 Hz, 1H),
7.44 (d, J = 2.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 1.6 Hz,
1H), 6.89 (d, J = 8.0 Hz, 1H), 6.82 (dd, J = 8.0, 1.6 Hz, 1H), 4.39 (d,
J = 5.9 Hz, 2H), 3.87–3.79 (m, 5H), 3.73 (s, 3H), 3.72 (s, 3H), 1.30–
1.16 (m, 1H), 0.60–0.54 (m, 2H), 0.34–0.29 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) d 165.6, 151.4, 148.6, 147.7, 147.5, 132.3,
126.6, 120.5, 119.4, 111.8, 111.5, 111.0, 72.8, 55.6, 55.5, 55.4,
42.4, 10.2, 3.2. ESI-MS (m/z): 370.5 ([MꢀH]ꢀ) and Anal. Calcd for
C21H25NO5: C, 67.91; H, 6.78; N, 3.77. Found: C, 67.73; H, 6.59;
N, 3.58.
4.1.5.10.
3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N-(4-
methoxybenzyl)benzamide (8j). Yield: 87%. 1H NMR (400 MHz,
CDCl3) d 7.50 (d, J = 1.2 Hz, 1H), 7.30–7.27 (m, 1H), 7.21 (dd,
J = 8.4, 1.6 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.91–6.86 (m, 2H),
6.58 (t, J = 75.2 Hz, 1H), 6.34 (s, 1H, NH), 4.55 (d, J = 5.2 Hz, 2H),
3.92 (d, J = 6.8 Hz, 2H), 3.80 (s, 3H), 1.34–1.27 (m, 1H), 0.67–0.62
(m, 2H), 0.37–0.33 (m, 2H). 13C NMR (100 MHz, CDCl3) d 166.2,
159.2, 150.7, 142.8 (t, J = 3.6 Hz), 132.8, 130.0, 129.3, 122.1,
118.7, 118.4 (ACHF2), 115.8 (ACHF2), 114.2, 113.9 (ACHF2),
113.3, 74.1, 55.3, 43.8, 10.1, 3.2. ESI-MS (m/z): 376.5 ([MꢀH]ꢀ)
and Anal. Calcd for C20H21F2NO4: C, 63.65; H, 5.61; N, 3.71. Found:
C, 63.65; H, 5.87; N, 4.01.
4.1.5.5. 3-(Cyclopentyloxy)-4-(difluoromethoxy)-N-(2-(2-meth-
oxyphenoxy)ethyl)benzamide (8e).
Yield: 80%. 1H NMR
(400 MHz, CDCl3) d 7.52 (d, J = 2.0 Hz, 1H), 7.26–7.22 (m, 1H),
7.16 (d, J = 8.4 Hz, 1H), 7.06 (s, 1H, NH), 7.01–6.96 (m, 2H), 6.94–
6.90 (m, 2H), 6.58 (t, J = 75.2 Hz, 1H, –CHF2), 4.91–4.87 (m, 1H),
4.20 (t, J = 4.8 Hz, 2H), 3.87–3.83 (m, 5H), 1.99–1.75 (m, 6H),
1.68–1.64 (m, 2H). 13C NMR (100 MHz, CDCl3) d 166.7, 149.8,
149.7, 147.8, 143.3 (t, J = 3.1 Hz), 132.9, 122.4, 122.3, 121.2, 118.6
(ACHF2), 116.0 (ACHF2), 115.4, 114.7, 113.4 (ACHF2), 112.0, 80.9,
69.1, 55.8, 39.6, 32.8, 23.9. ESI-MS (m/z): 444.6. ([M+K]+) and Anal.
Calcd for C22H25F2NO5:C, 62.70; H, 5.98; N, 3.32. Found: C, 62.39;
H, 5.72; N, 3.47.
4.1.5.11. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N-(3,4-
dimethoxybenzyl)benzamide (8k). Yield: 76%. 1H NMR
(400 MHz, CDCl3) d 7.51 (d, J = 2.0 Hz, 1H), 7.22 (dd, J = 8.4,
2.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.91–6.84 (m, 3H), 6.49 (t,
J = 75.2 Hz, 1H), 6.34 (s, 1H, NH), 4.56 (d, J = 5.6 Hz, 2H), 3.93 (d,
J = 7.2 Hz, 2H), 3.87 (s, 6H), 1.35–1.29 (m, 1H), 0.68–0.63 (m, 2H),
0.37–0.34 (m, 2H). 13C NMR (100 MHz, CDCl3) d 166.2, 150.7,
149.3, 148.7, 142.8 (t, J = 3.1 Hz), 132.8, 130.6, 122.1, 120.3,
118.8, 118.4 (ACHF2), 115.8 (ACHF2), 113.9, 113.2 (ACHF2),
111.4, 111.3, 74.1, 56.0, 55.9, 44.2, 10.1, 3.2. ESI-MS (m/z): 406.5
([MꢀH]ꢀ) and Anal. Calcd for C21H23F2NO5: C, 61.91; H, 5.69; N,
3.44. Found: C, 62.10; H, 5.83; N, 3.62.
4.1.5.6. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N-(2-(2-
methoxyphenoxy)ethyl)benzamide (8f). Yield: 84%. 1H NMR
(400 MHz, CDCl3) d 7.51 (d, J = 1.6 Hz, 1H), 7.28 (dd, J = 8.4,
1.6 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.04 (s, 1H, NH), 7.01–6.95
(m, 2H), 6.95–6.89 (m, 2H), 6.69 (t, J = 75.2 Hz, 1H), 4.20 (t,
J = 4.8 Hz, 2H), 3.93 (d, J = 7.2 Hz, 2H), 3.86–3.82 (m, 5H), 1.33–
1.28 (m, 1H), 0.67–0.63 (m, 2H), 0.37–0.33 (m, 2H). 13C NMR
(100 MHz, CDCl3)
d 166.6, 150.5, 149.8, 147.8, 142.8 (t,
J = 3.1 Hz), 132.9, 122.4, 122.0, 121.3, 119.1, 118.5 (ACHF2), 115.9

7338
Z.-Z. Zhou et al. / Bioorg. Med. Chem. 23 (2015) 7332–7339
All compounds were tested at nine concentrations (10ꢀ8–10ꢀ4 M).
Prism GraphPad software was used to calculate IC50 values using
nonlinear regression and a normalized dose–response fit.
4.1.5.12. N-benzyl-3-(cyclopropylmethoxy)-4-(difluoromethoxy)
benzamide (8l). Yield: 87%. 1H NMR (400 MHz, CDCl3) d 7.51 (d,
J = 1.8 Hz, 1H), 7.40–7.28 (m, 5H), 7.22 (dd, J = 8.3, 1.9 Hz, 1H),
7.16 (d, J = 8.2 Hz, 1H), 6.68 (t, J = 75.2 Hz, 1H), 6.37 (s, 1H), 4.63
(d, J = 5.7 Hz, 2H), 3.93 (d, J = 6.9 Hz, 2H), 1.37–1.27 (m, 1H),
0.70–0.60 (m, 2H), 0.39–0.30 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 166.3, 150.6, 142.8, 138.0, 132.7, 128.8, 127.9, 127.7, 122.1,
118.8, 118.4 (ACHF2), 115.8 (ACHF2), 113.8, 113.2 (ACHF2), 74.1,
44.3, 10.0, 3.2. ESI-MS (m/z):370.4 ([M+Na]+) and Anal. Calcd for
4.3. Molecular docking
Molecular docking was performed using the Surflex-Dock pro-
gram interfaced with Sybyl 7.3, which uses an empirical scoring
function and a patented search engine.39,40 Ligands were docked
into the corresponding enzyme-binding site guided by ProtoMol,
which is an idealized representation of a ligand that considers all
potential interactions with its binding site. In this study, the Proto-
Mol was established using the known ligand from the crystal struc-
ture of PDE4D (PDB entry: 3G4K). Prior to docking, all ligands were
removed and random hydrogen atoms were added. The receptor
structure was then minimized in 10,000 cycles using the Powell
method in Sybyl 7.3. All ligands were constructed using the sketch
molecular module. Hydrogen and Gasteiger–Hückel charges were
added to each molecule, the geometries of which were optimized
using the TRIPOS force field and by employing a conjugate gradient
method. The energy convergence criterion was 0.001 kcal/mol.
Default values were selected to finish these jobs except for cases
in which the threshold was 1 when the ProtoMol was generated.
C39H48F4N2O6: C, 65.35; H, 6.75; N, 3.91. Found: C, 65.08; H, 6.57;
N, 3.77.
4.1.5.13. 3-(cyclopentyloxy)-4-methoxy-N-(2-(2-methoxyphenoxy)
ethyl)benzamide (8m). Yield: 65%. 1H NMR (400 MHz, CDCl3) d 7.42
(d, J = 2.0 Hz, 1H), 7.30 (dd, J = 8.4, 2.0 Hz, 1H), 7.01–6.97 (m, 2H),
6.93–6.89 (m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H, NH), 4.87–4.82
(m, 1H), 4.21 (t, J = 5.1 Hz, 2H), 3.88 (s, 3H), 3.86–3.82 (m, 5H),
1.98–1.79 (m, 6H), 1.66–1.56 (m, 2H). 13C NMR (100 MHz, CDCl3) d
167.3, 152.8, 149.9, 147.9, 147.6, 127.0, 122.3, 121.2, 119.5,
115.5, 114.1, 112.0, 110.9, 80.6, 69.2, 56.1, 55.8, 39.5, 32.8, 24.1.
ESI-MS (m/z): 408.6. ([M+Na]+), 386.7 ([M+H]+) and Anal. Calcd
for C22H27NO5: C, 68.55; H, 7.06; N, 3.63. Found: C, 68.75; H, 6.81;
N, 3.94.
Acknowledgments
4.1.5.14. 3-(Cyclopropylmethoxy)-4-methoxy-N-(2-(2-methoxy-
phenoxy)ethyl) benzamide (8n). Yield: 6%. 1H NMR (400 MHz,
CDCl3) d 7.42 (d, J = 2.0 Hz, 1H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H),
7.01–6.96 (m, 2H), 6.93–6.89 (m, 2H), 6.87 (d, J = 8.4 Hz, 1H),
6.84 (s, 1H, NH), 4.20 (t, J = 5.0 Hz, 2H), 3.92–3.89 (m, 5H), 3.86–
3.82 (m, 5H), 1.40–1.30 (m, 1H), 0.67–0.62 (m, 2H), 0.37–0.34
(m, 2H). 13C NMR (100 MHz, CDCl3) d 167.2, 152.3, 150.0, 148.4,
147.9, 127.0, 122.4, 121.2, 119.7, 115.6, 112.6, 112.1, 110.6, 74.1,
69.3, 56.1, 55.9, 39.5, 10.2, 3.4. ESI-MS (m/z): 394.6. ([M+Na]+),
372.6 ([M+H]+) and Anal. Calcd for C21H25NO5: C, 67.91; H, 6.78;
N, 3.77. Found: C, 67.67; H, 6.59; N, 3.69.
This work was financially supported by the Project of Outstand-
ing Young Teachers in Guangdong Province (No. C1032190) to Z.Z.
Z. and the National Science and Technology Major Projects of China
for ‘‘Major New Drugs Innovation and Development” (No.
2012ZX09J1211003C), NSFC-Guangdong Joint Fund (No.
U1032006), and the National Natural Science Foundation of China
(No. 81373384) to J.P.X.
Supplementary data
4.2. Pharmacology
Supplementary data (structural characterization of compounds
8a–n) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2015.10.033.
4.2.1. Materials
Compounds 8a–n were dissolved in DMSO and stored as stock
solutions (100 mM) at ꢀ20 °C. For experimental use, all assays
were prepared using stock solutions, diluted with a growth med-
ium, and used immediately. FAM-Cyclic-30,50-AMP and IMAP bind-
ing reagent was purchased from Molecular Devices Inc. PDE4CAT
and Other PDEs were purchased from Sbdrugdiscovery Inc. and
BPS Bioscience Inc. respectively.
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l
l
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Ã
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