Montmorillonite K-10 catalyzed green synthesis of 2,6-unsubstituted dihydropyridines as potential inhibitors of PDE4

Article abstract of DOI:10.1016/j.ejmech.2012.12.052

Montmorillonite K-10 mediated MCR of anilines, arylaldehydes and ethyl-3,3-diethoxypropionate in water afforded 2,6-unsubstituted dihydropyridines depending on the nature of anilines employed. A variety of dihydropyridines were prepared by using this gree

Full text of DOI:10.1016/j.ejmech.2012.12.052

European Journal of Medicinal Chemistry 62 (2013) 395e404
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Montmorillonite K-10 catalyzed green synthesis of
2,6-unsubstituted dihydropyridines as potential
inhibitors of PDE4
,
T. Ram Reddy a b, G. Rajeshwar Reddya, L. Srinivasula Reddy a,
Chandana Lakshmi T. Meda c, Kishore V.L. Parsa c, K. Shiva Kumar c,
,
Y. Lingappa b, Manojit Pal c
*
a Custom Pharmaceutical Services, Dr. Reddy’s Laboratories Limited, Bollaram Road Miyapur, Hyderabad 500 049, India
b Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India
c Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Montmorillonite K-10 mediated MCR of anilines, arylaldehydes and ethyl-3,3-diethoxypropionate in
water afforded 2,6-unsubstituted dihydropyridines depending on the nature of anilines employed. A
variety of dihydropyridines were prepared by using this green methodology in good yields and mont-
morillonite K-10 was found to be an inexpensive and reusable catalyst. The structure elaboration of
a representative compound was carried out under Heck conditions. Some of the compounds synthesized
showed significant inhibition of PDE4B when tested in vitro. Docking studies indicated that one of the
ester moieties of these compounds played a key role in their interactions with the PDE4B protein.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 10 August 2012
Received in revised form
28 December 2012
Accepted 29 December 2012
Available online 17 January 2013
Keywords:
Dihydropyridine
MCR
Montmorillonite K-10
PDE4
1. Introduction
agonist [13] BAY K 8644 (D, Fig. 1) that affects airway smooth
muscle function by modulating the voltage-dependent calcium
The development of organic reactions in aqueous media [1] has
attracted particular attention because of the possibility that these
reactions may comply with the green chemistry protocols. Multi-
component reactions (MCRs) [2] on the other hand have emerged
as powerful tools in organic synthesis that allow quick access to the
diversity based library of small organic molecules useful for me-
dicinal/pharmaceutical chemistry. Thus MCRs in aqueous media are
expected to have a major impact and value in the area of modern
organic synthesis [3].
The 1,4-dihydropyridine (1,4-DHP) framework is considered as
one of the privileged structures in the area of drug discovery as
compounds containing this scaffold display a wide range of bio-
logical activities [4e8]. This is exemplified by the calcium channel
blocker drugs such as Nifedipine (A, Fig.1), Felodipine (B, Fig.1) and
Nicardipine (C, Fig. 1) that are used for the treatment of hyper-
tension and related cardiovascular diseases [4,9e12]. The calcium
channel also belongs to this class.
Recently, dihydropyridines E (Fig. 2) have been reported as in-
hibitors of human neutrophil elastase (HNE) for the potential
treatment of chronic obstructive pulmonary diseases (COPD) along
with a number of other complications such as acute coronary
syndrome, acute myocardial infarction and development of heart
failure [14]. Inhibitors of PDE4 (phosphodiesterase 4) on the other
hand have been evaluated for the treatment of COPD and asthma
[15,16]. These reports and our continuing interest in the identifi-
cation of novel PDE4 inhibitors [17] prompted us to evaluate a se-
ries of 2,6-unsubstituted 1,4-DHPs represented by F as potential
inhibitors of PDE4B.
Having identified PDE4 as a valuable therapeutic target for in-
flammatory diseases such as COPD and asthma a number of PDE4
inhibitors were discovered and taken forward into clinical trials
[15,16]. However, side effects such as nausea and emesis that were
also associated with the use of first-generation PDE4 inhibitor
rolipram, have restricted the therapeutic index and consequently
delayed the market launch of the second-generation PDE4 in-
hibitors cilomilast and roflumilast [18]. Encouragingly, roflumilast
* Corresponding author. Tel.: þ91 40 6657 1500; fax: þ91 40 6657 1581.
E-mail addresses: palmanojit@yahoo.co.uk, manojitpal@rediffmail.com (M. Pal).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.052

396
T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
Cl
R
N
CO2Et
EtO2C
NO2
Cl
Cl
NH2
EtO2C
+ RCHO +
H3CO2C
CO2CH3
H3CO2C
Montmorillonite K10
H2O, 90 oC, air
OEt
X
EtO
N
H
N
H
Cl
X
1
2
Nifedipine (A)
Felodipine (B)
3
4
NO2
Scheme 1. Montmorillonite K-10 catalyzed one-pot green synthesis of 1,4-dihydropy-
ridines.
CF3
CO2CH3
O2N
H3CO2C
CO2(CH2)2N(CH3)CH2C6H5
organic transformations. Recently, we have reported an easy and
direct access to 2-substituted quinolines [25], via a montmorillonite
K-10 catalyzed MCR involving 3,4-disubstituted aniline, aldehydes
and ethyl-3,3-diethoxypropionate in water (Scheme 1). More
recently we have observed that the use of anilines containing mono
or 2,4-disubstitutions in place of 3,4-disubstituted aniline afforded
1,4-DHPs in good yields. Hereinwe report our results on convenient,
practical and green synthesis of 1,4-DHPs 4 (i.e. the target com-
pounds based on F, Fig. 2) in water in the presence of air (Scheme 1).
To the best of our knowledge only one similar method has been
reported involving the use of Yb(OTf)3 as a catalyst in non-aqueous
media to afford 1,4-DHPs in low to good (21e82%) yields [26].
N
H
N
H
BAY K 8644 (D)
Nicardipine (C)
Fig. 1. 1,4-dihydropyridine based drugs and bioactive molecules.
(DaxasÒ, Nycomed) has been launched in Europe recently for the
treatment of COPD. Notably, during clinical studies in addition to its
well-known anti-inflammatory effects roflumilast has shown
improvement of fasting blood glucose and hemoglobin A1C levels
in patients with type 2 diabetes mellitus indicating beneficial ef-
fects of PDE4 inhibitors in diabetic patients [19]. More recently,
possibility of the use of PDE4 inhibitors in combination with other
PDE inhibitors for mimicking Calorie Restriction (CR) thereby
treating the aging-related diseases has been implicated by the
discovery of resveratrolePDE link [20]. Thus, search for novel in-
hibitors of PDE4 possessing fewer side effects have received
a renewed interest both in academia and industrial organizations.
Several elegant and efficient methods have been reported for
the synthesis [21,22] of 1,4-DHPs the most popular one being the
classical Hantzsch synthesis [21]. Though effective many of these
reactions however involved the use of expensive and environ-
mentally harmful metal precursors and catalysts, elevated tem-
peratures and longer reaction time. Additionally, the Hantzsch
synthesis is not very convenient particularly for the synthesis of
2,6-unsubstituted DHPs possessing various functional groups. It is
therefore necessary to develop new methodologies amenable for
constructing functionalized 2,6-unsubstituted DHP framework
from readily available starting materials under mild conditions.
As an inexpensive catalyst, montmorillonite clay offers several
advantages over classical catalysts. For example, in addition to its
favorable acidic and non-corrosive properties, reactions catalyzed
by montmorillonite clay can be carried out under mild reaction
conditions affording high yields of products with selectivity [23,24].
Additionally, these reactions were often found to be operationally
simple. The montmorillonite clay therefore has found uses in several
2. Results and discussion
2.1. Chemistry
Like our earlier synthesis of quinolines we followed a similar
strategy to establish the optimal reaction conditions. Thus, the MCR
of aniline (1a), 3,4-diflourobenzaldehyde (2a) and ester (3) was
initially carried out in water in the presence of p-toluenesulfonic
acid (p-TSA) and air at 90 ꢀC when the corresponding dihydropyr-
idine 4a was isolated in 45% yield (entry 1, Table 1). The use of other
Lewis acids such as ZnCl2, TiCl4 and SnCl4 was examined but was
found to be less effective (entries 2e4, Table 1). The use of mont-
morillonite K-10 (50%) however improved the product yield to 85%
(entry 5, Table 1) and the reaction was completed within 5 h. The
reaction was then carried out with various compositions of mont-
morillonite K-10 (entries 6 and 7, Table 1), when montmorillonite
K-10 (50%) was found to be the most effective catalyst. The MCR did
not proceed in the absence of catalyst, indicating its key role in the
present reaction (entry 8, Table 1). Due to its reported recyclability
in other reactions montmorillonite K-10 was recovered from the
present MCR by simple filtration and reused. The product 4a was
isolated in 79, 77 and 75% yield after 1st, 2nd and 3rd recovery and
reuse of the catalyst respectively. Apart from water the present
MCR was also examined in other protic solvents such as EtOH and
n-BuOH (entries 9 and 10, Table 1) which were found to be less
effective in the present MCR. Based on these observations it was
evident that a combination of montmorillonite K-10 in water was
optimal for the preparation of 4a.
Having the optimized reaction conditions in hand we then
tested the generality and scope of the present MCR. A range of
anilines (1) and aldehydes (2) were employed under the optimized
reaction conditions and results are summarized in Table 2. Various
electron-donating groups e.g. F, Cl, Br and OMe present on the aryl
ring of aldehydes were well tolerated. The use of ethyl 2-oxoacetate
(2h) was also successful and afforded the desired dihydropyridine
in good yield (entry 8, Table 2). Aniline containing mono (entries
1e14, Table 2) and disubstitution (entry 15, Table 2) participated
well in the reaction. Notably, all these reactions were carried
out in open air as the methodology does not require the use of inert
or anhydrous atmosphere. This avoids the risk of pressure
R2
R6
R1
R4
R
N
CO2Et
EtO2C
R5
N
O
X
R7
R3
F
E
Fig. 2. Known dihydropyridines (E) for the potential treatment of COPD and 2,6-
unsubstituted 1,4-DHPs (F) as potential inhibitors of PDE4B.

T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
397
Table 1
Effect of reaction conditions on the MCR of 1a, 2a and 3.a
CO2Et
CHO
NH2
OMe
EtO2C
EtO
Catalyst
MeO
N
CO2Et
+
Solvent
heat, air
OEt
F
2a
3
1a
4a
Entry
Catalyst
Solvent
% Yieldb,c
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
p-TSA (2.5 mol%)
ZnCl2 (2.5 mol%)
TiCl4 (2.5 mol%)
SnCl4 (2.5 mol%)
Montmorillonite K-10 (50% w/w)
Montmorillonite K-10 (20% w/w)
Montmorillonite K-10 (0.5% w/w)
No catalyst
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
45
20
30
Trace
85 (79)d
30
12
0
Montmorillonite K-10 (50% w/w)
Montmorillonite K-10 (50% w/w)
EtOH
n-BuOH
40
45
a
All the reactions were carried out using 1a (1.2 mmol), 2a (1.34 mmol) and 3 (3 mmol) in a solvent at 90 ꢀC for 5 h in the presence of air.
With respect to the aniline 1a used.
Isolated yield.
b
c
d
Recovered Montmorillonite K-10 was used.
development often observed especially in a scale-up preparation
while potentially mitigating the adverse effects [28]. We therefore
evaluated some of the compounds synthesized initially for their
PDE4B inhibitory potential [29,30] in vitro using PDE4B enzyme
assay [31]. Rolipram [32] was used as a reference compound in this
assay. The results of selected compounds that showed significant
when performed in a closed reaction vessel. Moreover, in view of
superior product yields the present MCR seems to have advantages
over the previously reported Yb(OTf)3 mediated method carried out
in non-aqueous media. To demonstrate the utility of the present
MCR further structure elaboration of a representative compound 4c
was carried out under Heck conditions. The bromo derivative 4c
was reacted smoothly with methyl acrylate (5) to give the corre-
sponding alkene (6) in good yield (Scheme 2). All the compounds
synthesized were well characterized by spectral (NMR, IR and MS)
data. The characteristic CeH signal present at C-4 position of the
inhibition against PDE4B at 30
mM are summarized in Table 4. All
these compounds showed moderate to good inhibition of PDE4B
(Table 4) and the compounds 4b and 4l being the best among them.
As evident from Table 3 that a bulky group such as bromo at the p-
position of the C-4 aryl ring was beneficial for the activity (4a vs 4b)
whereas shifting of this group decreased the activity (4b vs 4c).
While replacing the bromo by chloro (4c vs 4d) or hydrogen (e.g. 4g)
DHP ring appeared in the region 4.27e4.95 d and 36.6e38.4 ppm in
1H and 13C NMR spectra, respectively (Table 3). The alkene 6 was
did not improve the activity a 3,4-difluorophenyl or 4-
identified as a trans-isomer as indicated by its 1H NMR signal at 6.41
methoxyphenyl moiety at C-4 enhanced the activity (e.g. 4e and
4f). Introduction of an additional OMe group increased the activity
further (4e vs 4o). The shifting of the OMe group from the o-position
of the N-aryl ring either decreased the activity (4b vs 4j) or did not
show any significant improvement (4a vs 4i, 4c vs 4k, 4g vs 4m). This
trend however was not observed in case of 4e and 4l indicating
different mode of interactions of 4b and 4l with the PDE4B enzyme.
Thus docking studies were performed using compounds 4b and 4l
to understand the nature of interactions of these molecules with
PDE4B. The glide scores and other parameters obtained after
docking of these molecules into the PDE4B protein are summarized
in Table 5. The data shown in Table 5 suggests that these molecules
bind well with PDE4B. The docking results of 4b with PDE4B protein
showed H-bonding interaction between one of the CO2Et group (the
carbonyl oxygen) of 4b and the histidine 278 moiety of the PDE4B
protein (Fig. 3). While similar H-bonding interaction was observed
in case of 4l involving one of the CO2Et moieties (the carbonyl ox-
ygen), the amino acid residue of the PDE4B protein however was
glutamine 443 in this case (Fig. 4). In a dose response study com-
pound 4b showed dose-dependent inhibition of PDE4B with an IC50
d
(d, J ¼ 16.4 Hz, 1H).
Mechanistically, the montmorillonite K-10 mediated MCR
leading to 2,6-unsubstituted 1,4-DHPs seemed to proceed via
a similar pathway reported earlier [26]. Briefly, the reaction pro-
ceeds via in situ generation of imine which after tautomerization to
enamine reacts with aldehyde to form the enamino alcohol. This on
subsequent dehydration followed by Michael-type annulation with
the enamine forms the tetrahydropyridine which undergoes
elimination of the aniline moiety to afford the desired product. It is
evident from our previous [25] and present study that the nature of
aniline used can change the course of the reaction. The partic-
ipation of the aryl ring of the aniline reactant in the reaction was
perhaps aided by the strong electron donating groups present on
the aryl ring that activated the o-position (with respect to the
amine group) leading to the formation of quinoline derivatives.
Nevertheless, being strongly acidic in nature montmorillonite K-10
facilitated the present MCR better than Yb catalyst thereby
affording relatively better yields of products.
2.2. Pharmacology
value >5.0
The compound 4b was also tested for its potential to inhibit PDE4D
in vitro when it showed 50% inhibition at 30 M compared to its 78%
inhibition of PDE4B at the same concentration (entry 2, Table 4).
Overall, the present series of 1,4-DHPs has been identified as a new
class of PDE4 inhibitors and therefore are of further interest.
mM compared to rolipram’s IC50 value w1.0 mM (Fig. 5).
Since recent studies have indicated that among the four sub-
types PDE4 e.g. A, B, C and D the PDE4B subtype is linked to in-
flammatory cell regulation [27] hence it was hypothesized that
inhibition of the PDE4B may provide a means to achieve efficacy
m

398
T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
Table 2
Synthesis of 1,4-dihydropyridine (4).a
Entry
Aniline (1)
Aldehyde (2)
Product (4)
Time (h)
Yield (%)b
F
CO2Et
NH2
OMe
OMe
MeO
F
OHC
OHC
1
5
85
N
N
CO2Et
2a
1a
4a
CO2Et
Br
Br
2
1a
6
83
CO2Et
2b
4b
Br
CO2Et
Br
OMe
3
1a
6
81
OHC
OHC
N
CO2Et
Cl
2c
4c
CO2Et
Cl
OMe
OMe
4
1a
7
73
N
N
CO2Et
2d
4d
CO2Et
F
F
F
5
1a
5
86
F
OHC
OHC
CO2Et
2e
4e
OMe
CO2Et
OMe
OMe
OMe
6
1a
10
72
N
N
CO2Et
2f
4f
CO2Et
OHC
7
1a
8
74
CO2Et
2g
4g

T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
399
Table 2 (continued )
Entry
Aniline (1)
Aldehyde (2)
Product (4)
Time (h)
Yield (%)b
CO2Et
CO2Et
O
OMe
OHC
2h
8
1a
10
69
N
OEt
CO2Et
4h
F
NH2
CO2Et
9
2a
7
89
N
CO2Et
OMe
1b
MeO
4i
Br
CO2Et
10
1b
2b
7
91
N
CO2Et
MeO
MeO
4j
Br
CO2Et
11
1b
2c
6
79
N
CO2Et
F
4k
CO2Et
F
12
1b
2e
6
89
N
CO2Et
MeO
MeO
4l
CO2Et
13
1b
2g
9
71
N
CO2Et
F
4m
CO2Et
NH2
N
14
2a
7
81
CO2Et
Cl
1c
Cl
(continued on next page)
4n

400
T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
Table 2 (continued )
Entry
Aniline (1)
Aldehyde (2)
Product (4)
Time (h)
Yield (%)b
F
F
CO2Et
NH2
15
2e
7
93
MeO
OMe
N
CO2Et
1d
MeO
OMe
4o
a
All the reactions were carried out using aniline 1 (1.2 mmol), aldehyde 2 (1.34 mmol) and ethyl-3,3-diethoxypropionate 3 (3.0 mmol) and montmorillonite K-10 (75 mg) in
water at 90 ꢀC.
b
Isolated yield.
3. Conclusions
chromatography was performed on silica gel (230e400 mesh) us-
ing distilled hexane, ethyl acetate, dichloromethane. 1H NMR and
13C NMR spectra were determined in CDCl3 solution by using 400
In conclusion, we have shown that montmorillonite K-10 medi-
ated one-pot three-component reaction of aniline, aryl aldehydes and
ethyl-3,3-diethoxypropionate in water can afford 2,6-unsubstituted
DHP in good yields depending on the nature of anilines employed.
The reaction does not require the use of inert or anhydrous atmo-
sphere and can be performed in the presence of air. Moreover, this
operationally simple methodology seemed to have advantages over
the previously reported Yb(OTf)3 mediated method carried out in
non-aqueous media in terms of product yields. The potential of this
green methodology has been demonstrated in further structure
elaboration of a compound synthesized via CeC bond forming re-
actions under Heck conditions. A range of 1,4-DHPs were synthesized
efficiently by using this straightforward method some of which
showed significant inhibition of PDE4B when tested in vitro. Docking
studies indicated that one of the ester moieties of the DHP used
played a key role in the interactions with the PDE4B protein through
H-bond. Overall, the DHP framework described here represent a new
template for the identification of novel inhibitors of PDE4 and the
synthetic methodology used could be useful in constructing library of
molecules related to DHP of potential pharmacological interest.
and 100 MHz spectrometers, respectively. Proton chemical shifts (
d)
are relative to tetramethylsilane (TMS,
d
¼ 0.00) as internal stan-
dard and expressed in ppm. Spin multiplicities are given as s (sin-
glet), d (doublet), t (triplet) and m (multiplet) as well as b (broad).
Coupling constants (J) are given in hertz. Infrared spectra were
recorded on an FT-IR spectrometer. Melting points were deter-
mined by using a melting point apparatus and are uncorrected. MS
spectra were obtained on a mass spectrometer.
4.1.2. General procedure for the preparation of dihydropyridine (4)
A mixture of montmorillonite K-10 (75 mg), an appropriate
aniline 1 (150 mg, 1.2 mmol), ethyl-3,3-diethoxypropionate (3)
(579 mg, 3 mmol) and an aldehyde (166 mg, 1.34 mmol) in pure
water (7.5 mL) was stirred at 90 ꢀC for the time mentioned in
Table 2. After completion of the reaction (indicated by TLC), the
mixture was cooled to room temperature and filtered. The filtrate
was extracted with EtOAc (3 ꢁ 5 mL). The organic layers were
collected, combined, dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel using ethyl acetateehexane to
give the desired product.
4. Experimental section
4.1. Chemistry
4.1.3. Diethyl 4-(4-fluorophenyl)-1-(2-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4a)
4.1.1. General methods
Off white solid; mp 174e176 ꢀC; IR (KBr) 2983, 1701, 1601, 1586,
Unless stated otherwise, reactions were performed under ni-
trogen atmosphere using oven dried glassware. Reactions were
monitored by thin layer chromatography (TLC) on silica gel plates
(60 F254), visualizing with ultraviolet light or iodine spray. Flash
1506, 1459, 1372, 1347, 1277, 1260, 1156, 1081 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
d
1.18 (t, J ¼ 6.8 Hz, 6H), 3.91 (s, 3H), 4.03e4.18 (m, 4H),
4.92 (s, 1H), 6.92e6.96 (m, 2H) 7.01e7.05 (m, 2H), 7.24e7.26 (m,
1H), 7.32e7.37 (m, 3H), 7.40e7.43 (m, 2H); 13C NMR (CDCl3,
MeO2C
Br
EtO2C
Pd(PPh3)2Cl2
EtO2C
MeO2C
+
MeO
MeO
Et3N, DMF
90-100°C, 4 h
81%
N
CO2Et
N
CO2Et
4c
5
6
Scheme 2. Structural elaboration of compound 4c.

T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
401
Table 3
Characteristic NMR and IR signals of 2,6-unsubstituted dihydropyridines (4).
Table 5
Glide scores and other parameters of compounds after docking with PDE4B.
Compound (4)
C-4 signal
1H NMR
C]O group
Compounds
GScore
LipophilicEvdW
PhobEn
HBond
Electro
13C NMR
IR
4b
4l
ꢂ6.79
ꢂ6.41
ꢂ4.78
ꢂ5.02
ꢂ0.6
ꢂ1.4
ꢂ1.08
ꢂ0.65
ꢂ0.32
ꢂ0.24
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4.92 (s, 1H)
4.91 (s, 1H)
4.91 (s, 1H)
4.93 (s, 1H)
4.91 (s, 1H)
4.87 (s, 1H)
4.93 (s, 1H)
4.27 (s, 1H)
4.94 (s, 1H)
4.92 (s, 1H)
4.92 (s, 1H)
4.93 (s, 1H)
4.95 (s, 1H)
4.94 (s, 1H)
4.90 (s, 1H)
36.7
37.0
37.3
37.1
36.7
36.6
37.3
38.4
36.9
37.0
37.5
37.0
37.4
37.0
36.7
1701, 1601
1706, 1693
1709, 1698
1707, 1685
1701, 1666
1701, 1674
1704, 1665
1709, 1665
1705, 1665
1707, 1687
1707, 1694
1707, 1688
1707, 1663
1707, 1693
1705, 1664
LipophilicEvdW ¼ Chemscore lipophilic term and fraction of total Van der Waals
energy. PhobEn ¼ Hydrophobic reward. HBond ¼ Reward for hydrogen bond.
Electro ¼ Electrostatic reward.
4.1.6. Diethyl 4-(3-chlorophenyl)-1-(2-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4d)
Yellow semi solid; IR (KBr) 2987, 1707, 1685, 1615, 1583, 1465,
1373, 1327, 1263, 1202, 1126, 1076 cmꢂ1; 1H NMR (CDCl3, 400 MHz)
d
1.18 (t, J ¼ 7.2 Hz, 6H), 3.91 (s, 3H), 4.02e4.21 (m, 4H), 4.93 (s, 1H),
100 MHz)
d 14.2 (2C), 36.7, 55.8, 60.0 (2C), 109.1 (2C), 112.2, 114.5
(2C), 114.7, 121.1, 126.0, 129.1, 130.1 (2C), 138.0 (2C), 142.6, 149.3,
162.7, 166.9 (2C); HRMS: m/z calcd for C24H25FNO5 (M þ 1)
426.1717; found 426.1708.
4.1.4. Diethyl 4-(4-bromophenyl)-1-(2-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4b)
Yellow solid; mp 211e213 ꢀC; IR (KBr) 2975, 1706, 1693, 1590,
1505, 1485, 1371, 1345, 1259, 1202, 1121, 1076 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
4.91 (s, 1H), 6.87e7.05 (m, 2H), 7.24e7.33 (m, 2H), 7.35e7.40 (m,
6H); 13C NMR (CDCl3, 100 MHz)
14.2 (2C), 37.0, 55.8, 60.0 (2C),
d
1.18 (t, J ¼ 6.8 Hz, 6H), 3.91 (s, 3H), 4.01e4.19 (m, 4H),
d
108.7 (2C), 112.2, 120.2, 121.1, 126.0, 129.1, 130.2 (2C), 130.9 (2C),
132.0, 138.1 (2C), 145.8, 154.0, 166.7 (2C); HRMS: m/z calcd for
C24H25BrNO5 (M þ 1) 486.0916; found 486.0914.
4.1.5. Diethyl 4-(3-bromophenyl)-1-(2-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4c)
Light yellow solid; mp 203e205 ꢀC; IR (KBr) 2978, 1709, 1698,
1600, 1587, 1506, 1472, 1350, 1272, 1260, 1123, 1080 cmꢂ1; 1H NMR
(CDCl3, 400 MHz)
d
1.19 (t, J ¼ 7.2 Hz, 6H), 3.95 (s, 3H), 4.02e4.21 (m,
4H), 4.91 (s, 1H), 7.01e7.04 (m, 2H), 7.11e7.14 (m, 1H), 7.24e7.33 (m,
2H), 7.35e7.41 (m, 4H), 7.62 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d
14.1 (2C), 37.3, 55.9, 60.1 (2C), 108.5 (2C), 112.1, 121.0, 122.2, 126.0,
127.2, 129.2, 129.4, 131.6 (2C), 131.9, 138.3 (2C), 149.0, 154.3, 166.7
(2C); HRMS: m/z calcd for C24H25BrNO5 (M þ 1) 486.0916; found
486.0923.
Table 4
Inhibition of PDE4B by compound 4 at 30
m
M.
Entry
Compounds
Average % inhibition
SD
1.
2.
3.
4.
5.
6.
7.
8.
4a
4b
4c
4d
4e
4f
4g
4i
4j
4k
4l
4m
4n
4o
33.41
78.36
37.27
30.35
45.99
59.21
29.94
40.23
35.11
42.01
78.71
39.32
44.24
69.17
2.86
2.56
1.81
1.12
1.07
1.25
2.62
3.31
2.11
1.99
2.24
3.43
1.51
3.16
9.
10.
11.
12.
13.
14.
SD ¼ standard deviation.
Fig. 3. Docking of 4b at the active site of PDE4B.

402
T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
6.93e7.05 (m, 2H), 7.14e7.18 (m, 1H), 7.24e7.33 (m, 3H), 7.36e7.43
(m, 3H), 7.58 (s, 1H); 13C NMR (CDCl3, 100 MHz)
14.0 (2C), 37.1,
d
55.5, 60.3 (2C), 108.5 (2C), 111.5, 116.1, 120.8, 126.3, 126.7 (2C), 128.3,
129.8, 130.1, 134.7, 139.6 (2C), 143.9, 148.9, 166.3 (2C); HRMS: m/z
calcd for C24H25ClNO5 (M þ 1) 442.1421; found 442.1405.
4.1.7. Diethyl 4-(3,4-difluorophenyl)-1-(2-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4e)
Off white solid; mp 194e195 ꢀC; IR (KBr) 2983, 1701, 1666, 1601,
1586,1466,1392,1328,1261,1202,1123,1080 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
4.91 (s,1H), 7.01e7.05 (m, 3H), 7.24e7.26 (m,1H), 7.27e7.32 (m, 2H),
7.33e7.36 (m, 3H); 13C NMR (CDCl3, 100 MHz)
14.1 (2C), 36.7, 55.7,
d
1.19 (t, J ¼ 7.2 Hz, 6H), 3.92 (s, 3H), 4.05e4.20 (m, 4H),
d
60.1 (2C),108.5 (2C),112.1,116.1,116.2,117.3,121.1,124.1,126.1,129.3,
138.2 (2C), 143.8, 148.9, 151.3, 154.3, 166.6 (2C); HRMS: m/z calcd for
C24H24F2NO5 (M þ 1) 444.1623; found 444.1625.
4.1.8. Diethyl 1-(2-methoxyphenyl)-4-(4-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4f)
Reddish brown oil; IR (KBr) 2987, 1701, 1674, 1596, 1507, 1463,
1373, 1362, 1275, 1202, 1127, 1083 cmꢂ1; 1H NMR (CDCl3, 400 MHz)
d
1.19 (t, J ¼ 7.2 Hz, 6H), 3.79 (s, 3H), 3.87 (s, 3H), 4.01e4.18 (m, 4H),
4.87 (s, 1H), 6.76e6.86 (m, 2H), 6.95e7.06 (m, 4H), 7.32e7.44 (m,
4H); 13C NMR (CDCl3, 100 MHz) 14.2 (2C), 36.6, 55.7 (2C), 60.1 (2C),
109.2 (2C), 112.1 (2C), 113.3 (2C), 124.0 (2C), 128.8 (2C), 131.8, 132.1,
139.6 (2C), 153.4, 157.9, 166.6 (2C); HRMS: m/z calcd for C25H28NO6
(M þ 1) 438.1850; found 438.1854.
4.1.9. Diethyl 1-(2-methoxyphenyl)-4-phenyl-1,4-dihydropyridine-
3,5-dicarboxylate (4g)
Off white solid; mp 165e167 ꢀC; IR (KBr) 2980, 1704, 1665, 1599,
1505,1454,1371,1349,1275,1260,1123,1080 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
4.93 (s, 1H), 7.01e7.04 (m, 2H), 7.14e7.17 (m, 1H), 7.21e7.38 (m, 6H),
7.45e7.47 (m, 2H); 13C NMR (CDCl3, 100 MHz)
14.1 (2C), 37.3, 55.8,
d
1.17 (t, J ¼ 7.2 Hz, 6H), 3.91 (s, 3H), 4.02e4.17 (m, 4H),
d
60.0 (2C), 109.2 (2C), 112.2, 121.0, 126.0, 126.6, 127.8 (2C), 128.4 (2C),
128.9, 132.1, 137.9 (2C), 146.7, 154.0, 167.0 (2C); HRMS: m/z calcd for
C24H26NO5 (M þ 1) 408.1811; found 408.1803.
4.1.10. Triethyl 1-(2-methoxyphenyl)-1,4-dihydropyridine-3,4,5-
tricarboxylate (4h)
Brown liquid; IR (KBr) 2985, 1709, 1665, 1573, 1465, 1373, 1263,
1126, 1073 cmꢂ1; 1H NMR (CDCl3, 400 MHz)
d
1.17 (t, J ¼ 6.8 Hz, 3H),
1.25 (t, J ¼ 7.2 Hz, 6H), 3.85 (s, 3H), 4.07 (q, J ¼ 6.8 Hz, 2H), 4.16e4.25
(m, 4H), 4.27 (s,1H), 6.96e7.01 (m, 2H), 7.18e7.20 (m,1H), 7.24e7.37
Fig. 4. Docking of 4l at the active site of PDE4B.
(m, 3H); 13C NMR (CDCl3, 100 MHz)
d
14.1 (3C), 38.4, 55.6, 59.9 (3C),
106.5 (2C), 111.5, 112.0, 120.9, 125.9, 128.7, 139.6 (2C), 153.4, 166.7,
Fig. 5. Dose response study of compounds 4b and rolipram.

T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
403
166.9 (2C); HRMS: m/z calcd for C21H26NO7 (M þ 1) 404.1689; found
(2C), 120.8, 126.3, 127.5, 130.5 (2C), 130.8, 135.6, 139.6 (2C), 141.9,
143.9, 158.2, 166.3 (2C); HRMS: m/z calcd for C23H22ClFNO4 (M þ 1)
430.1207; found 430.1201.
404.1683.
4.1.11. Diethyl 4-(4-fluorophenyl)-1-(4-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4i)
4.1.17. Diethyl 4-(3,4-difluorophenyl)-1-(2,4-dimethoxyphenyl)-
1,4-dihydropyridine-3,5-dicarboxylate (4o)
Off white solid; mp 171e173 ꢀC; IR (KBr) 2985, 1705, 1665, 1589,
1503,1463,1385, 1351, 1273,1256, 1143, 1086 cmꢂ1; 1H NMR (CDCl3,
Yellow solid; mp 186e188 ꢀC; IR (KBr) 2982, 1705, 1664, 1591,
400 MHz)
4.94 (s, 1H), 6.92e6.97 (m, 4H), 7.21e7.34 (m, 4H), 7.54e7.55 (m,
2H); 13C NMR (CDCl3, 100 MHz)
14.2 (2C), 36.9, 55.6, 60.2 (2C),
d
1.19 (t, J ¼ 6.8 Hz, 6H), 3.84 (s, 3H), 4.02e4.19 (m, 4H),
1512,1468,1371,1345,1269,1200,1132,1063 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
d
1.19 (t, J ¼ 7.2 Hz, 6H), 3.84 (s, 3H), 3.89 (s, 3H), 4.01e4.18
d
(m, 4H), 4.90 (s,1H), 6.50e6.56 (m, 2H), 7.01e7.06 (m,1H), 7.15e7.19
(m, 2H), 7.26e7.34 (m, 3H); 13C NMR (CDCl3, 100 MHz) 14.1 (2C),
36.7, 55.7 (2C), 60.0 (2C), 99.6, 104.4, 108.2 (2C), 116.2, 117.2, 124.1,
125.4, 127.1, 138.8 (2C), 143.9, 149.0, 151.3, 155.6, 160.6, 166.7 (2C);
HRMS: m/z calcd for C25H26F2NO6 (M þ 1) 474.1728; found 474.1717.
110.1 (2C), 114.6 (2C), 114.9 (2C), 122.8 (2C), 129.7 (2C), 132.1, 136.2
(2C), 142.2, 158.2, 162.7, 166.7 (2C); HRMS: m/z calcd for
C24H25FNO5 (M þ 1) 426.1717; found 426.1736.
4.1.12. Diethyl 4-(4-bromophenyl)-1-(4-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4j)
4.1.18. Preparation of (E)-diethyl 4-(3-(3-methoxy-3-oxoprop-1-
en-1-yl)phenyl)-1-(2-methoxyphenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (6)
Yellow solid; mp 207e209 ꢀC; IR (KBr) 2981, 1707, 1687, 1583,
1512, 1473, 1368, 1337, 1263, 1202, 1117, 1085 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
4.92 (s, 1H), 6.85e7.01 (m, 3H), 7.20e7.31 (m, 3H), 7.37e7.39 (m,
2H), 7.54e7.56 (m, 2H); 13C NMR (CDCl3, 100 MHz)
14.2 (2C), 37.0,
55.9, 60.1 (2C), 108.7 (2C), 112.2 (2C), 120.2, 121.1 (2C), 130.2 (2C),
130.9 (2C), 132.0, 138.1 (2C), 145.8, 154.0, 166.7 (2C); HRMS: m/z
calcd for C24H25BrNO5 (M þ 1) 486.0916; found 486.0938.
d
1.18 (t, J ¼ 6.8 Hz, 6H), 3.84 (s, 3H), 4.02e4.18 (m, 4H),
A
mixture of bromo-1,4-dihydropyridine 4c (200 mg,
0.41 mmol), methyl acrylate (5) (95 mg, 1.11 mmol), (PPh3)2PdCl2
(35 mg, 0.05 mmol), Et3N (196 mg, 1.94 mmol) in DMF (5 mL) was
stirred at room temperature for 15 min and then at 90 ꢀC for 4 h
under nitrogen (progress of the reaction was monitored by TLC).
After completion, the reaction mixture was cooled to room tem-
perature and filtered through celite bed. The filtrate was extracted
with ethyl acetate (3 ꢁ 5.0 mL). The organic layers were collected,
combined, dried over anhydrous Mg2SO4, filtered and concentrated
under vacuum. The residue was purified by column chromatogra-
phy; yellow semi solid; IR (KBr) 2975, 1710, 1698, 1615, 1593, 1508,
1483, 1372, 1264, 1226, 1132, 1073 cmꢂ1; 1H NMR (CDCl3, 400 MHz)
d
4.1.13. Diethyl 4-(3-bromophenyl)-1-(4-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4k)
Yellow solid; mp 189e191 ꢀC; IR (KBr) 2962, 1707, 1694, 1663,
1578, 1512, 1460, 1348, 1276, 1228, 1116, 1060 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
4.92 (s, 1H), 6.95e6.98 (m, 2H), 7.11e7.23 (m, 3H), 7.26e7.32 (m,
2H), 7.48 (s, 1H), 7.55 (s, 2H); 13C NMR (CDCl3, 100 MHz)
14.1 (2C),
d
1.20 (t, J ¼ 7.2 Hz, 6H), 3.84 (s, 3H), 4.04e4.23 (m, 4H),
d
1.17 (t, J ¼ 6.8 Hz, 6H), 3.79 (s, 3H), 3.94 (s, 3H), 4.02e4.18 (m, 4H),
d
4.96 (s, 1H), 6.41 (d, J ¼ 16.4 Hz, 1H), 7.03e7.06 (m, 2H), 7.26e7.40
37.5, 55.6, 60.2 (2C), 109.6 (2C), 114.9 (2C), 122.1, 122.9 (2C), 127.1,
129.4, 129.5, 131.5 (2C), 136.5 (2C), 148.5, 158.2, 166.6 (2C); HRMS:
m/z calcd for C24H25BrNO5 (M þ 1) 486.0916; found 486.0932.
(m, 6H), 7.50e7.52 (m, 1H), 7.66e7.71 (m, 2H); 13C NMR (CDCl3,
100 MHz)
d 14.1 (2C), 37.2, 51.5, 55.9, 60.0 (2C), 108.8 (2C), 112.1,
117.0, 121.0, 125.9, 126.3, 127.8, 128.6, 129.1, 130.6, 131.9, 134.0, 138.2
(2C), 145.4, 147.5, 154.0, 166.7 (2C), 167.5; HRMS: m/z calcd for
C28H30NO7 (M þ 1) 492.2022; found 492.2028.
4.1.14. Diethyl 4-(3,4-difluorophenyl)-1-(4-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4l)
Off white solid; mp 181e183 ꢀC; IR (KBr) 2981, 1707, 1688, 1598,
5. Pharmacology
1582, 1514, 1463, 1375, 1327, 1277, 1231, 1085 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
4.93 (s, 1H), 6.96e7.02 (m, 2H), 7.05e7.26 (m, 5H), 7.54e7.56 (m,
2H); 13C NMR (CDCl3, 100 MHz)
14.2 (2C), 37.0, 55.6, 60.3 (2C),
d
1.20 (t, J ¼ 7.2 Hz, 6H), 3.84 (s, 3H), 4.03e4.21 (m, 4H),
5.1. Materials and methods
d
5.1.1. Cells and reagents
109.5 (2C), 115.0 (2C), 116.4, 117.1, 122.9 (2C), 127.6, 136.4 (2C), 143.3,
150.2, 151.3, 155.6, 158.3, 166.5 (2C); HRMS: m/z calcd for
C24H24F2NO5 (M þ 1) 444.1623; found 444.1661.
HEK 293 and Sf9 cells were obtained from ATCC (Washington
D.C., USA). HEK 293 cells were cultured in DMEM supplemented
with 10% fetal bovine serum (Invitrogen Inc., San Diego, CA, USA).
Sf9 cells were routinely maintained in Grace’s supplemented me-
dium (Invitrogen) with 10% FBS. RAW 264.7 cells (murine macro-
phage cell line) were obtained from ATCC and routinely cultured in
RPMI 1640 medium with 10% fetal bovine serum (Invitrogen Inc.).
cAMP was purchased from SISCO Research Laboratories (Mumbai,
India). PDElight HTS cAMP phosphodiesterase assay kit was pro-
cured from Lonza (Basel, Switzerland).
4.1.15. Diethyl 1-(4-methoxyphenyl)-4-phenyl-1,4-
dihydropyridine-3,5-dicarboxylate (4m)
Off white solid; mp 157e158 ꢀC; IR (KBr) 2981, 1707, 1663, 1597,
1513,1478, 1375, 1340, 1275, 1250, 1156, 1078 cmꢂ1; 1H NMR (CDCl3,
400 MHz)
4.95 (s, 1H), 6.95e6.98 (m, 2H), 7.14e7.36 (m, 5H), 7.38e7.40 (m,
2H), 7.55e7.59 (m, 2H); 13C NMR (CDCl3, 100 MHz)
14.1 (2C), 37.4,
d
1.19 (t, J ¼ 6.8 Hz, 6H), 3.84 (s, 3H), 4.02e4.18 (m, 4H),
d
55.8, 60.1 (2C), 109.2 (2C), 114.9 (2C), 122.8 (2C), 126.4, 127.8 (2C),
128.8 (2C), 132.1, 137.9 (2C), 146.7, 154.0, 166.8 (2C); HRMS: m/z
calcd for C24H26NO5 (M þ 1) 408.1811; found 408.1835.
5.1.2. PDE4B protein production and purification
PDE4B cDNA was sub-cloned into pFAST Bac HTB vector (Invi-
trogen) and transformed into DH10Bac (Invitrogen) competent
cells. Recombinant bacmids were tested for integration by PCR
analysis. Sf9 cells were transfected with bacmid using Lipofect-
amine 2000 (Invitrogen) according to manufacturer’s instructions.
Subsequently, P3 viral titer was amplified, cells were infected and
48 h post-infection cells were lysed in lysis buffer (50 mM TriseHCl
pH 8.5, 10 mM 2-mercaptoethanol, 1% protease inhibitor cocktail
(Roche), 1% NP40). Recombinant His-tagged PDE4B protein was
purified as previously described elsewhere (Wang et al., 1997).
4.1.16. Diethyl 1-(3-chlorophenyl)-4-(4-fluorophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4n)
Yellow oil; IR (KBr) 2983,1707,1693,1599,1505, 1485,1362, 1373,
1283, 1202, 1137, 1086 cmꢂ1 1H NMR (CDCl3, 400 MHz)
; d 1.20 (t,
J ¼ 6.8 Hz, 6H), 4.04e4.21 (m, 4H), 4.94 (s,1H), 6.61 (s,1H), 6.84e6.99
(m, 2H), 7.04e7.14 (m, 2H), 7.24e7.42 (m, 3H), 7.52e7.58 (m, 2H); 13C
NMR (CDCl3, 100 MHz) 14.1 (2C), 37.0, 60.3 (2C), 108.2 (2C), 115.9

404
T.R. Reddy et al. / European Journal of Medicinal Chemistry 62 (2013) 395e404
Briefly, lysate was centrifuged at 10,000 rpm for 10 min at 4 ꢀC and
supernatant was collected. Supernatant was mixed with NieNTA
resin (GE Life Sciences) in a ratio of 4:1 (v/v) and equilibrated
with binding buffer (20 mM TriseHCl pH 8.0, 500 mM KCl, 5 mM
imidazole, 10 mM 2-mercaptoethanol and 10% glycerol) in a ratio of
2:1 (v/v) and mixed gently on rotary shaker for 1 h at 4 ꢀC. After
incubation, lysateeNieNTA mixture was centrifuged at 4500 rpm
for 5 min at 4 ꢀC and the supernatant was collected as the flow-
through fraction. Resin was washed twice with wash buffer
(20 mM TriseHCl pH 8.5, 1 M KCl, 10 mM 2-mercaptoethanol and
10% glycerol). Protein was eluted sequentially twice using elution
buffers (Buffer I: 20 mM TriseHCl pH 8.5, 100 mM KCl, 250 mM
imidazole, 10 mM 2-mercaptoethanol, 10% glycerol, Buffer II:
20 mM TriseHCl pH 8.5, 100 mM KCl, 500 mM imidazole, 10 mM
2-mercaptoethanol, 10% glycerol). Eluates were collected in four
fractions and analyzed by SDS-PAGE. Eluates containing PDE4B
protein were pooled and stored at ꢂ80 ꢀC in 50% glycerol until
further use.
by allowing sample ring conformations and sample nitrogens to
move to possible extent in docking. 10 poses were generated for
each ligand. The docking results are documented and analyzed.
Acknowledgements
The author (TRR) thanks Dr V. Dahanukar for his encouragement
and analytical group of DRL for spectral data.
Appendix A. Supplementary material
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.052.
References
[1] For an excellent review, see: C.-J. Li, L. Chen Chem. Soc. Rev. 35 (2006) 68e82.
[2] A. Dömling, I. Ugi, Angew. Chem. Int. Ed. 39 (2000) 3168e3210.
[3] K. Kumaravel, G. Vasuki, Curr. Org. Chem. 13 (2009) 1820e1841.
[4] R.A. Janis, P.J. Silver, D.J. Triggle, Adv. Drug Res. 16 (1987) 309e591.
[5] N. Baindur, A. Rutledge, D.J. Triggle, J. Med. Chem. 36 (1993) 3743e3746.
[6] A.M. van Rhee, J.L. Jiang, N. Melman, M.E. Olah, G.L. Stiles, K.A. Jacobson,
J. Med. Chem. 39 (1996) 2980e2989.
[7] R. Peri, S. Padmanabhan, A. Rutledge, S. Singh, D.J. Triggle, J. Med. Chem. 43
(2000) 2906e2914.
[8] D.J. Triggle, Cell. Mol. Neurobiol. 23 (2003) 293e303.
[9] R. Lavilla, J. Chem. Soc. Perkin 1 (2002) 1141e1156.
[10] C.O. Kappe, Eur. J. Med. Chem. 35 (2000) 1043e1052.
[11] M. Varache-Leme`bge, A. Nuhrich, V. Zemb, G. Devaux, P. Vacher, A.M. Vacher,
B. Dufy, Eur. J. Med. Chem. 31 (1996) 547e556.
[12] D. Alker, S.F. Campbell, P.E. Cross, R.A. Burges, A.J. Carter, D.G. Gardiner, J. Med.
Chem. 33 (1990) 585e591.
5.1.3. PDE4B enzymatic assay
The inhibition of PDE4B enzyme was measured using PDElight
HTS cAMP phosphodiesterase assay kit (Lonza) according to man-
ufacturer’s recommendations. Briefly, 10 ng of PDE4B enzyme was
pre-incubated either with DMSO (vehicle control) or compound for
15 min before incubation with the substrate cAMP (5 mM) for 1 h.
The reaction was halted with stop solution followed by incubation
with detection reagent for 10 min in dark. Luminescence values
(RLUs) were measured by a Multilabel plate reader (Perklin Elmer
1420 Multilabel counter). The percentage of inhibition was calcu-
lated using the following formula:
[13] M. Schramm, G. Thomas, R. Towart, G. Franckowiak, Nature 303 (1983)
535e537.
[14] H. Gielen-Haertwig, V.M.-J. Li, U. Rosentreter, K.-H. Schlemmer, S. Allerheili-
gen, L. Telan, L. Barfacker, J. Keldenich, M.F. Fitzgerald, K. Nash, B. Albrecht, D.
Meurer, US Patent Application US 7,230,017 B2, Jun. 12, 2007.
[15] A. Kodimuthali, S.S.L. Jabaris, M. Pal, J. Med. Chem. 51 (2008) 5471e5489.
[16] S.G. Dastidar, D. Rajagopal, A. Ray, Curr. Opin. Invest. Drugs 8 (2007) 364e372.
[17] R. Adepu, D. Rambabu, B. Prasad, C.L.T. Meda, A. Kandale, G.R. Krishna,
C.M. Reddy, L.N. Chennuru, K.V.L. Parsa, M. Pal, Org. Biomol. Chem. 10 (2012)
5554e5569.
% inhibition ¼ ½ðRUL of vehicle control ꢂ RUL of inhibitorÞ=
ꢁ ðRUL of vehicle controlÞꢃ ꢁ 100
[18] D. Spina, Br. J. Pharmacol. 155 (2008) 308e315.
[19] S.K. Field, Clin. Med. Insights Circ. Respir. Pulm. Med. 5 (2011) 57e70.
[20] J.H. Chung, Aging 4 (2012) 144e145.
6. Docking study
6.1. Method
[21] A. Hantzsch, Liebigs Ann. Chem. 215 (1882) 1e82.
[22] J. Moreau, A. Duboc, C. Hubert, J.-P. Hurvoisa, J.-L. Renaud, Tetrahedron Lett.
48 (2007) 8647e8650. and ref cited therein.
The docking studies were performed using Schrödinger 2011
software. The results were compared with the cocrystal ligand and
in vitro activities. The PDB ID 3O0J was used for the docking study.
The protein was prepared by giving preliminary treatment like
adding hydrogen, adding missing residues, refining the loop with
prime and finally minimized by using OPLS 2005 force field. The
search grid was generated by picking the cocrystal ligand upto 20 Å
search area. The hydroxyl groups of search area were allowed to
move.
All the molecules were minimized by using macromodule
application. We used 1000 iteration for minimization using OPLS
2005 force field and charges were also added from force field only.
The PRCG (PolakeRibier conjugate gradient) method was used for
minimization. All the molecules were docked by using glide XP
(extra precision) dock application. We performed flexible docking
[23] For a review, see: N. Kaur, D. Kishore J. Chem. Pharm. Res. 4 (2012) 991e1015
http://jocpr.com/vol4-iss2-2012/JCPR-2012-4-2-991-1015.pdf.
[24] L.J. Krstic, S. Sukdolak, S. Solujic, J. Serb. Chem. Soc. 67 (2002) 325e329.
[25] T.R. Reddy, L.S. Reddy, G.R. Reddy, K. Yarbagi, Y. Lingappa, D. Rambabu,
G.R. Krishna, C.M. Reddy, K.S. Kumar, M. Pal, Green Chem. 14 (2012)
1870e1872.
[26] S. Sueki, R. Takei, J. Abe, I. Shimizu, Tetrahedron Lett. 52 (2011) 4473e4477.
[27] S.-L.C. Jin, L. Lan, M. Zoudilova, M.J. Conti, Immunology 175 (2005)
1523e1531.
[28] P. Srivani, D. Usharani, E.D. Jemmis, G.N. Sastry, Curr. Pharm. Des. 14 (2008)
3854e3872.
[29] A. Robichaud, P.B. Stamatiou, S.L.C. Jin, N. Lachance, D. Mac Donald,
F. Laliberté, S. Liu, Z. Huang, M. Conti, C.C.J. Chan, Clin. Invest. 110 (2002)
1045e1052.
[30] S.L.C. Jin, M. Conti, Proc. Natl. Acad. Sci. U S A 99 (2002) 7628e7633.
[31] P. Wang, J.G. Myers, P. Wu, B. Cheewatrakoolpong, R.W. Egan, M.M. Billah,
Biochem. Biophys. Res. Commun. 234 (1997) 320e324.
[32] J. Demnitz, L. La Vecchia, E. Bacher, T.H. Keller, T. Muller, F. Schurch,
H.P. Weber, E. Pombo-Villar, Molecules 3 (1998) 107e119.