Novel imidazophenoxazine-4-sulfonamides: Their synthesis and evaluation as potential inhibitors of PDE4

Article abstract of DOI:10.1016/j.bmc.2013.01.023

A number of novel imidazophenoxazine-4-sulfonamides have been designed as potential inhibitors of PDE4. All these compounds were readily prepared via an elegant multi-step method involving the initial construction of 1-nitro-10H-phenoxazine ring and then fused imidazole ring as key steps. Some of these compounds showed promising PDE4B and D inhibition when tested in vitro and good interactions with these proteins in silico. Three of these compounds showed dose dependent inhibition of PDE4B with IC₅₀ value of 3.31 ± 0.62, 1.23 ± 0.18 and 0.53 ± 0.18 μM.

Full text of DOI:10.1016/j.bmc.2013.01.023

Bioorganic & Medicinal Chemistry 21 (2013) 1952–1963
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel imidazophenoxazine-4-sulfonamides: Their synthesis
and evaluation as potential inhibitors of PDE4
b
Seelam Venkata Reddy a, Gangula Mohan Rao a, Baru Vijaya Kumar a, , Chandana L. T. Meda ,
⇑
Girdhar Singh Deora b, K. Shiva Kumar b, Kishore V. L. Parsa b, Manojit Pal b,
⇑
a Medicinal Chemistry Laboratory, Research Centre, C.K.M. Arts and Science College, Warangal 506 006, Andhra Pradesh, India
b Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of novel imidazophenoxazine-4-sulfonamides have been designed as potential inhibitors of
PDE4. All these compounds were readily prepared via an elegant multi-step method involving the initial
construction of 1-nitro-10H-phenoxazine ring and then fused imidazole ring as key steps. Some of these
compounds showed promising PDE4B and D inhibition when tested in vitro and good interactions with
these proteins in silico. Three of these compounds showed dose dependent inhibition of PDE4B with IC50
Received 4 December 2012
Revised 11 January 2013
Accepted 12 January 2013
Available online 26 January 2013
value of 3.31 0.62, 1.23 0.18 and 0.53 0.18 lM.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Imidazophenoxazine
Sulfonamide
PDE4
Docking
1. Introduction
bition of PDE4 results in elevated levels of cAMP in the airway tis-
sues and cells thereby suppressing inflammatory cell functions.
This is supported by the fact that PDE4 inhibitors have been found
to be beneficial for the treatment of inflammatory and immunolog-
ical diseases including asthma and chronic obstructive pulmonary
disease (COPD). Notably, rolipram the first-generation PDE4 inhib-
itor showed adverse effects such as nausea and vomiting.5 More re-
cently, cardiovascular effects of PDE4 inhibitors have been
reported.6 While these dose-limiting side effects were reduced by
second-generation inhibitors like cilomilast7a (Ariflo) and roflumi-
last, their therapeutic index has delayed market launch so far.
While roflumilast (DaxasÒ, Nycomed) has been launched in Europe
for the treatment of COPD recently it is however, necessary to de-
vote a continuing effort in exploring new class of compounds for
their PDE4 inhibitory potential. Additionally, the improvement of
fasting blood glucose and hemoglobin A1C levels shown by roflu-
milast during its clinical studies in patients with type 2 diabetes7b
and the recent report7c of resveratrol-PDE link have generated re-
newed interest in the discovery and development of new PDE4
inhibitor. The design of our target compounds (E, Fig. 2) was per-
Due to their interesting pharmacological properties, compounds
containing the phenoxazine framework (A, Fig. 1) have attracted
considerable attention in medicinal chemistry. A number of com-
pounds based on A have been reported to be potent anti-prolifera-
tive agents.1 For example 5H-pyrido[2,3-a]phenoxazin-5-one (B,
Fig. 1) that belongs to this class has shown promising anti-prolifer-
ative activities when tested against human neoplastic cell lines.2 A
phenoxazine based dual PPAR agonist for example, DRF 2725 (C,
Fig. 1) has shown potent antihyperglycemic and lipid modulating
properties.3 In view of the pharmacological importance of phenox-
azine derivatives and our long standing interest in identification of
novel phosphodiesterase 4 (PDE4) inhibitors4 we became inter-
ested in assessing PDE4 inhibiting properties of small molecules
based on phenoxazine fused with an imidazole ring for example,
imidazo[4,5,1-kl]phenoxazine. In inflammatory and immune cells,
the inhibition of cellular responses, including the production
and/or release of proinflammatory mediators, cytokines, and active
oxygen species, is associated with elevated levels of cAMP. PDE4
exists in four different isoforms for example, PDE4A, B, C and D
and plays a key role in the hydrolysis of cAMP to AMP.5 Thus, inhi-
formed by incorporating
a phenoxazine ring (A) into the
benzo[d]imidazol based known inhibitors8a of PDE4 (D, Fig. 2). A
sulfonamide group was introduced at C-4 of the resulting imi-
dazo[4,5,1-kl]phenoxazine moiety with the anticipation that this
group might induce anti-inflammatory8b as well as favorable drug
like properties8c within the molecule. Herein we report imi-
dazo[4,5,1-kl]phenoxazine-4-sulfonamide as a new template for
⇑
Corresponding authors. Tel.: +91 8008098787; fax: +91 40 6657 1581 (B.V.K.);
tel.: +91 40 6657 1500; fax: +91 40 6657 1581 (M.P.).
E-mail addresses: baruvijayakumar@yahoo.com (B.V. Kumar), manojitpal@re-
diffmail.com (M. Pal).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.023

S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
1953
O
O
O
N
O
COOH
N
H
N
H
OCH2CH3
(CH2)2
N
O
A
B
C
Figure 1. The phenoxazine framework (A) and related bioactive molecules B and C.
synthesis of phenoxazine derivatives starting from 2-aminophenol
and substituted difluorobenzene has been reported.10 Introduction
of sulfonic acid group at C-3 position of the phenoxazine nucleus
led to the construction of p-aminobenzenesulfonic acid pharmaco-
phore in phenoxazine nucleus. It was reported11 that the synthetic
applications of phenoxazine appeared to be problematic due to the
insoluble nature of the molecule. Therefore it was planned to intro-
duce a nitro group at an initial stage to increase the solubility. The
synthon potassium-4-chloro-3,5-dinitrobenzene sulfonate,12 used
to build heterocyclic nuclei in the earlier work13 has been used
in the present synthesis. Thus synthesis of our target compounds
that is, 1-aryl imidazo[4,5,1-kl]phenoxazine-4-sulfonamides (7)
was carried out starting from potassium salt of 1-nitro-10H-phen-
oxazine-3-sulfonate (3)14 (Scheme 1). Previously, there was only
one report15 in which 1-amino phenoxazine was treated with for-
mic acid to construct the imidazo fused phenoxazine ring. The key
starting material 3 prepared (Scheme 1) by the condensation of 2-
aminophenol (1) with potassium salt of 4-chloro-3,5-dinitroben-
zenesulfonate (2) was treated with excess of phosphorous oxychlo-
ride in a 1:7 molar ratio to get the corresponding sulfonyl chloride
(4). This was subsequently treated with aqueous ammonia in THF
to afford the 1-nitro-10H-phenoxazine-3-sulfonamide (5). On
reduction with Raney Ni the compound 5 afforded 1-amino-10H-
phenoxazine-3-sulfonamide (6) which finally treated with a range
of aromatic aldehydes to give the target compounds 7 (Scheme 1).
OMe
N
N
N
R
R
NH
N
H
O
O
RHN
O
D
E
A
Figure 2. Design of imidazophenoxazine based new inhibitors (E) of PDE4.
the discovery of novel inhibitors of PDE4. To the best of our knowl-
edge this is the first example of disclosing PDE4 inhibitors based on
this pharmacophore.
2. Results and discussion
2.1. Chemistry
While several synthesis9 of substituted phenoxazines have been
attempted earlier incorporation of two substituents like nitro and
sulfonic acid in a single step followed by subsequent functional
group modifications however remained unexplored. Recently, the
SO3-K+
OH O2N
+
SO3-K+
O
SO2Cl
O
c
a
b
N
H
N
H
NH2
Cl
NO2
NO2
NO2
1
2
3
4
O
N
O
SO2NH2
SO2NH2
O
SO2NH2
e
d
N
H
N
H
6
N
NO2
NH2
R
5
7
R
% yield
64
7a
7b
7c
7d
7e
7f
7g
7h
7i
phenyl
3-methoxy4-hydroxy phenyl
3-hydroxy phenyl
53
60
4- methoxy phenyl
4-hydroxy phenyl
52
60
2-hydroxy phenyl
60
4-diethylamino-2-hydroxyphenyl
3-nitrophenyl
50
71
2-bromophenyl
60
7j
7k
7l
3-bromophenyl
60
2-chlorophenyl
70
5-bromo-2-fluorophenyl
53
7m 2,3-dichlorophenyl
52
7n
7o
7p
4-chlorophenyl
70
4-dimethylaminophenyl
2,6-dichlorophenyl
51
64
Scheme 1. Reagents and conditions: (a) NaOH, EtOH, 3 h, 83%; (b) POCl3, reflux, 3 h, 84%; (c) aq ammonia, THF, 0 °C, 30 min, 80%; (d) Raney Ni, NH2NH2, MeOH, reflux, 30 min,
85%; (e) RCHO, DMF, 100 °C, 48 h.

1954
S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
O
SO3-K+
O
N
-
SO3 K+
O
N
SO2Cl
c
d
b
a
7a
3
N
H
N
N
NH2
Ph
Ph
8
10
9
Scheme 2. Reagents and conditions: (a) Raney Ni, NH2NH2, MeOH, reflux, 30 min, 77%; (b) PhCHO, DMF, 100 °C, 24 h, 52%; (c) POCl3, reflux, 3 h; (d) aq ammonia, THF, 0 °C,
30 min, 50%.
One of the target compounds 7a was also prepared via an alter-
native route (Scheme 2). Thus, the compound 3 was reduced to the
potassium salt of 1-amino-10H-phenoxazine-3-sulfonate (8) using
Raney Ni, which was treated with benzaldehyde to give the potas-
sium salt of 1-phenylimidazo[4,5,1-kl]phenoxazine-4-sulfonates
(9). On treating with POCl3 the compound 9 provided the corre-
sponding chloro derivative 10 which on treatment with aqueous
ammonia in THF afforded the compound 7a.16
the o-BrC6H4 and m-BrC6H4 group at C-1 was well tolerated for
example, compounds 7i and 7j (Table 1, entries 9 and 10). While
o-ClC6H4 group at C-1 decreased the activity marginally for example,
compound 7k (Table 1, entry 11) a 2-F-5-BrC6H3 or p-ClC6H4 or 2,6-
dichloro phenyl at the same position was well tolerated (Table 1,
entries 12–14). Based on their promising inhibitory properties dose
response studies were carried out using most active compounds 7f,
7j and 7l. All of them showed dose dependent inhibition of PDE4
with IC50 value of 3.31 0.62, 1.23 0.18 and 0.53 0.18
respectively (Figs. 3–5) in compared to rolipram’s IC50 value of
0.941 0.24 M (Fig. 6). Thus the compound 7l was identified as
lM,
2.2. Pharmacology
l
Having synthesized a range of 1-substituted imidazo[4,5,1-
kl]phenoxazine-4-sulfonamides many of these compounds were
tested initially for their PDE4B inhibitory properties in vitro at
the most potent compound in this series. To assess the other subtype
inhibitory potential of 7 few selected compounds were tested
against PDE4D when compounds 7f, 7g, 7h, and 7l showed 88.4%,
16.9%, 68.6%, and 85.3% inhibition, respectively.
30 l
M using PDE4B enzyme assay17 (Table 1). Rolipram18 was used
as a reference compound in this assay. Except 7g (along with 7m and
7o) all other compounds showed significant inhibition of PDE4B and
compounds 7f, 7j and 7l showed superior inhibitions in compared to
2.3. Docking studies
other molecules when tested at 30
l
M (Table 1). The initial com-
In order to understand the nature of interactions of these mol-
ecules with PDE4B docking studies were carried out using com-
pounds 7f, 7j and 7l. The XP (extra precision) docking was
performed for all the molecules using glide module of Schrödinger
2011. The glide scores and other parameters obtained after docking
of these molecules into the PDE4B protein are summarized in Ta-
ble 2. The data shown in Table 2 suggests that these molecules bind
well with PDE4B. The interaction of compound 7f with the PDE4B
protein (Fig. 7) was mainly contributed by (i) a H-bonding between
the amino group of 7f and His-278, (ii) a H-bonding between the
pound 7a showed good inhibition of PDE4B which though was not
affected by the addition of OH and OMe substituents to the C-1 ben-
zene ring for example, compound 7b (Table 1, entry 2) but improved
upon addition of m-OH group to the C-1 benzene ring for example,
compound 7c (Table 1). However, a p-MeOC6H4 moiety at C-1 de-
creased the activity for example, compound 7d (Table 1, entry 4)
whereas a p-HOC6H4 or o-HOC6H4 moiety at C-1 restored the activity
for example, compound 7e and 7f (Table 1, entries 5 and 6). A bulkier
group that is, p-(Et2N)-o-(HO)C6H3 at C-1 was not tolerated for
example, compound 7g (Table 1, entry 7) and a m-NO2C6H4 moiety
at C-1 decreased the activity (Table 1, entry 8). Interestingly, both
hydroxyl group of 7f and Asp-392 and (iii) two
P–P stacking inter-
actions between the benzoimidazole moiety and His234 of the pro-
tein. Similarly, the interaction of compound 7j with the PDE4B
protein (Fig. 8) was contributed by (i) H-bonding between the
Table 1
NH2-group of 7j and Thr-345 as well as Asp-392, (ii)
interaction between the central 1,4-oxazine ring and tyrosine
(Tyr233) and (iii) stacking interaction between the phenyl
P–P stacking
Inhibition of PDE4B by compound 7 at 30 lM
O
SO2NH2
P–P
group of 7j and phenylalanine (Phe446). The interaction of com-
pound 7l with the PDE4B protein (Fig. 9) was mainly contributed
by (i) a H-bonding between the amino group of 7l and Asp-275
N
N
R
and (ii)
P–P stacking interaction between aromatic ring
7
systems of 7l and Tyr233, His234 and Phe446. Overall, the present
Entry Compounds R =
Average %
inhibition
SD
imidazophenoxazine-4-sulfonamides showed good interactions
1
2
7a
7b
Phenyl
3-Methoxy4-hydroxy
phenyl
79.65
82.43
1.16
1.48
3
4
5
6
7
7c
7d
7e
7f
3-Hydroxy phenyl
4-Methoxy phenyl
4-Hydroxy phenyl
2-Hydroxy phenyl
4-Diethylamino-2-
hydroxyphenyl
87.58
66.53
88.80
89.28
19.66
0.54
4.16
0.92
3.31
1.43
7g
8
9
7h
7i
7j
7k
7l
7n
7p
3-Nitrophenyl
66.81
81.02
89.69
76.32
92.86
80.35
80.23
2.56
1.17
1.23
0.86
0.53
1.32
2.15
2-Bromophenyl
3-Bromophenyl
2-Chlorophenyl
5-Bromo-2-fluorophenyl
4-Chlorophenyl
2,6-Dichlorophenyl
10
11
12
13
14
SD = standard deviation.
Figure 3. Dose dependent inhibition of PDE4B by compound 7f.

S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
1955
Table 2
Glide scores and other parameters of compounds after docking with PDE4B
Entry
Compound
Dock score
E-1a
E-2b
E-3c
E-4d
1
2
3
7f
7j
7l
ꢀ6.18
ꢀ7.84
ꢀ8.19
ꢀ2.95
ꢀ3.82
ꢀ3.99
ꢀ1.4
0
ꢀ0.22
ꢀ0.59
ꢀ2.88
ꢀ2.82
ꢀ1.05
ꢀ0.97
ꢀ0.88
a
Chemscore lipophilic pair term and fraction of the total protein–ligand vdw
energy reward.
b
Hydrophobic enclosure reward.
Electrostatic reward.
Rewards for hydrogen bonding interaction between ligand and protein.
c
d
P–P stacking with Tyr-159 and hydrogen bonding with Asp-318
and Thr-271 was observed (see Figs. in Supplementary data).
Figure 4. Dose dependent inhibition of PDE4B by compound 7j.
3. Conclusions
In conclusion, a number of novel imidazophenoxazine-4-sulfon-
amides have been designed and synthesized as potential inhibitors
of PDE4. All these compounds were readily prepared via a multi-
step method starting from potassium-4-chloro-3,5-dinitro ben-
zene sulfonate involving the construction of 1-nitro-10H-phenoxa-
zine ring and then fused imidazole ring as key steps. Some of these
compounds showed promising PDE4B and D inhibition when
tested in vitro and good interactions with these proteins in silico.
The docking studies indicated that the central phenoxazine ring
and sulfonamide moiety played a key role in these interactions.
In a dose response study three compounds showed dose dependent
inhibition of PDE4B with IC50 value of 3.31 0.62, 1.23 0.18 and
0.53 0.18 lM. Overall, the imidazophenoxazine-4-sulfonamide
framework presented here could be a new template for the identi-
fication of small molecule based novel inhibitors of PDE4.
Figure 5. Dose dependent inhibition of PDE4B by compound 7l.
4. Experimental
4.1. Chemistry
with PDE4B protein where the central phenoxazine ring and sul-
fonamide moiety played a key role in these interactions. Notably,
the observed dissimilar orientation of binding of 7f compared to
7j and 7l was possibly aided by the strong H-bonding between
the hydroxyl group of 7f and Asp-392 of the PDE4B protein. Dock-
ing of all these molecules into the PDE4D suggested that they bind
well with this protein (see Supplementary data) which correlated
the results of their PDE4D inhibitions in vitro. Thus, the amino
group of sulfonamide moiety of 7f interacted with Asp-201 and
318 and the hydroxyl group of 7f formed an H-bond with Asn
4.1.1. General methods
Unless stated otherwise, reactions were performed under nitrogen
atmosphere. Reactions were monitored by thin layer chromatography
(TLC) on silica gel plates (60 F254), visualizing with ultraviolet light or
iodine spray. Flash chromatography was performed on silica gel (60–
120 mesh) using hexane, ethyl acetate, dichloromethane, methanol.
1H NMR and 13C NMR spectra were determined in DMSO-d6 solution
by using 400 and 100 MHz spectrometers, respectively. Proton chem-
ical shifts (d) are relative to tetramethylsilane (TMS, d = 0.00) as inter-
nal standard and expressed in ppm. Spin multiplicities are given as s
(singlet), d (doublet), t (triplet) and m (multiplet) as well as b (broad).
Coupling constants (J) are given in hertz. Infrared spectra were re-
cordedona FT-IRspectrometer. Melting pointsweredetermined using
melting point apparatus and are uncorrected. MS spectra were ob-
tained on a mass spectrometer.
321. Aromatic rings of molecules 7j participated in
P–P stacking
with Tyr-159, His-160 and Phe-372. The amino group of same mol-
ecule formed a hydrogen bond with Thr-271. In case of molecule 7l
4.1.2. Potassium-4-chloro-3,5-dinitrobenzenesulfonate (2)
SO3-K+
O2N
Cl
NO2
Chlorobenzene (50 ml) was added to a mixture of fuming sulfuric
acid (260 ml) and sulfuric acid (60 ml) at 70 °C. The mixture was
stirred at same temperature for 1 h. Potassium nitrate (50 g) was
added portion wise to the reaction mixture for about 15 min, and
then fuming sulfuric acid (130 ml) was added followed by 2 more
Figure 6. Dose dependent inhibition of PDE4B by rolipram.

1956
S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
Figure 7. Docking of 7f at the active site of PDE4B.
portions of potassium nitrate (50 + 50 g) portion wise. The reaction
mass was stirred at 130 °C for 1 h, cooled to room temperature,
poured in excess crushed ice and allowed to stand for 12 h. The solid
was filtered and washed with cold water (3 ꢁ 200 ml), dried and
washed three times with hot toluene (3 ꢁ 200 ml) and dried, yield
56% (80 g) of pure product; mp 298–300 °C (lit.12 290 °C).
To a solution of sodium hydroxide (7.5 g, 0.187 mol) in
ethanol (500 ml) was added 2-aminophenol (17 g, 0.156
mol) and potassium-4-chloro-3,5-dinitrobenzenesulfonate (50 g,
0.156 mol). The reaction mixture was refluxed for 1 h. Then a
solution of sodium hydroxide (3.75 g) in water (5 ml) was added
to the reaction mass and refluxed for two more hours. Completion
of the reaction was monitored by TLC. The reaction mass was cooled
to room temperature and the solid was filtered and washed twice
with ethanol (2 ꢁ 200 ml) and dried, yield 83% (45 g); mp >300 °C;
m/z (CI) 307 (M-K, 100); 1H NMR (400 MHz, DMSO-d6) d 9.45 (s,
1H, NH), 7.73 (s, 1H), 7.20 (dd, J = 2 and 6.8 Hz, 1H,), 7.01–6.9 (m,
4.1.3. Potassium-1-nitro-10H-phenoxazine-3-sulfonate (3)
O
SO3-K+
1H), 6.94–6.36 (m, 3H); IR (KBr, cmꢀ1
1052, 745.
) vmax: 3341, 1531, 1203,
N
H
NO2

S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
1957
Figure 8. Docking of 7j at the active site of PDE4B.
4.1.4. 1-Nitro-10H-phenoxazine-3-sulfonylchloride (4)
and yield 8 g (84%); mp 204 °C (decomposition) and the product
was immediately used for the next step.
O
SO2Cl
4.1.5. 1-Nitro-10H-phenoxazine-3-sulfonamide (5)
N
H
O
SO2NH2
NO2
Potassium-1-nitro-10H-phenoxazine-3-sulfonate
(10 g)
in
N
H
POCl3 (70 ml) was refluxed for 3 h. Completion of the reaction
was monitored by TLC. The excess of POCl3 was removed by distil-
lation, the crude cherry red coloured solid was poured in crushed
ice and filtered, washed with water, cold methanol and dried,
NO2
To a solution of aqueous ammonia (10 ml) in tetrahydrofuran
(50 ml) was added 1-nitro-10H-phenoxazine-3-sulfonylchloride

1958
S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
Figure 9. Docking of 7l at the active site of PDE4B.
(8 g) at 0 °C and the resulting reaction mixture was stirred for
30 min at same temperature. Completion of the reaction was mon-
itored by TLC. The excess of solvent was removed by distillation
under reduced pressure and the crude solid was diluted with water
and extracted with ethyl acetate (300 ml). The organic layer was
collected, washed with water (300 ml), dilute HCl (100 ml), water
(100 ml) and saturated sodium chloride solution (100 ml) and
dried over anhydrous sodium sulfate. The organic layer was fil-
tered and concentrated under reduced pressure, yield 80% (6.0 g);
mp 282–283 °C; m/z (CI) 306 (M-1, 100); 1H NMR (400 MHz,
DMSO-d6) d 9.75 (s, 1H), 7.91 (s, 1H), 7.43 (s, 2H), 7.20 (dd,
J = 2.2 and 8.0 Hz, 1H), 7.12 (s, 1H), 6.89–6.72 (m, 3H); IR (KBr,
To a suspension of 1-nitro-10H-phenoxazine-3-sulfonamide (5)
(3.9 g) in methanol was added Raney Ni (2.4 g) and hydrazine hy-
drate (4 ml). The resulting reaction mixture was refluxed for
30 min. Completion of the reaction was monitored by TLC. The cat-
alyst was removed by filtration and the filtrate was concentrated
under reduced pressure, yield 85% (3.0 g); off white solid; mp
176 °C (decomposition); m/z (CI) 276 (M-1, 100); 1H NMR
(400 MHz, DMSO-d6) d 7.56 (s, 1H), 7.01 (s, 2H), 6.77–6.75 (m,
1H), 6.74 (s, 1H), 6.70 (d, J = 4 Hz, 2H), 6.55–6.53 (m, 1H), 6.37 (s,
1H), 5.12 (s, 2H); IR (KBr, cmꢀ1
) vmax 3385, 3316, 3268, 1605,
1583, 1522, 1504, 1451, 1424, 1337, 1315, 1278, 1235, 1145,
1113, 1080, 1038, 752.
cmꢀ1
) vmax 3388, 3335, 3255, 1575, 1536, 1506, 1406, 1337,
1291, 1226, 1146, 1093, 936, 876, 756
4.1.7. General procedure for the synthesis of 1-
(aryl)imidazo[4,5,1-kl]phenoxazine-4-sulfonamides (7)
To a solution of 1-amino-10H-phenoxazine-3-sulfonamide (6,
7 mmol) in dimethylformamide (40 ml) was added aryl aldehyde
(10 mmol). The resulting reaction mixture was stirred at 100 °C
for 48 h. Completion of the reaction was monitored by TLC. Then
the reaction mass was cooled to room temperature and poured
in cold water and the solid was filtered and dried, purified by col-
umn chromatography using 3% methanol in dichloromethane.
4.1.6. 1-Amino-10H-phenoxazine-3-sulfonamide (6)
O
SO2NH2
N
H
NH2

S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
1959
4.1.8. 1-Phenylimidazo[4,5,1-kl]phenoxazine-4-sulfonamide (7a)
4.1.11. 1-(4-Methoxyphenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7d)
O
S
O
O
N
O
S
O
O
N
NH2
NH2
N
N
Compound 7a was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.4 g, 5 mmol) and benzaldehyde (0.64 g,
6 mmol) in DMF according to the general procedure as an ash col-
oured solid; yield 64% (1.2 g); mp 316–317 °C; m/z (CI) 364 (M+1,
100); 1H NMR (400 MHz, DMSO-d6) d 7.81 (d, J = 6.8 Hz, 2H), 7.68–
7.62 (m, 4H), 7.39 (s, 2H), 7.27 (d, J = 8 Hz, 1H), 7.18 (t, J = 8 Hz,
1H,), 7.13 (s, 1H), 6.94 (t, J = 8 Hz, 1H), 6.89 (d, J = 8 Hz, 1H); 13C
NMR (100 MHz, DMSO-d6) d 150.2, 144.8, 141.7, 140.8, 130.8,
130.2, 129.5, 129.0, 128.8, 127.6, 126.1, 124.9, 124.4, 118.5,
H3CO
Compound 7d was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (2.0 g, 7.2 mmol) and 4-methoxybenzalde-
hyde (1.5 g, 10 mmol) in DMF according to the general procedure
as an off white solid; yield 52% (1.5 g); mp 302–303 °C; m/z (CI)
392 (M-1, 100); 1H NMR (400 MHz, DMSO-d6)
d 7.73 (d,
J = 8.8 Hz, 2H), 7.66 (s, 1H), 7.39 (s, 2H, SO2NH2), 7.23 (d,
J = 8.0 Hz, 1H), 7.18–7.13 (m, 3H), 7.12 (s, 1H), 6.99–6.93 (m, 2H),
3.89 (3H); 13C NMR (100 MHz, DMSO-d6) d 161.0, 150.4, 145.0,
141.9, 141.6, 140.9, 131.1, 127.6, 126.2, 125.1, 124.5, 122.2,
116.1, 111.2, 102.4; IR (KBr, cmꢀ1
) vmax 3344, 3024, 1662, 1494,
1454, 1380, 1336, 1299, 1267, 1148, 1096, 751.
118.5, 116.2, 114.3, 111.0, 102.3, 55.4; IR (KBr, cmꢀ1
) vmax 3385,
4.1.9. 1-(4-Hydroxy-3-methoxyphenyl)imidazo[4,5,1-
kl]phenoxazine-4-sulfonamide (7b)
3116, 1664, 1613, 1584, 1495, 1378, 1301, 1229, 1208, 1128,
1079, 750.
O
O
O
N
S
4.1.12. 1-(4-Hydroxyphenyl)imidazo[4,5,1-kl]phenoxazine-4-
NH2
sulfonamide (7e)
O
O
O
S
N
NH2
N
N
HO
OCH3
Compound 7b was prepared by using 1-amino-10H-phenoxazine-
3-sulfonamide (6) (1.4 g, 5 mmol) and 4-hydroxy-3-methoxybenzal-
dehyde (1.12 g, 7.5 mmol) in DMF according to the general procedure
as an off white solid, yield 53% (1.23 g); mp 294–296 °C; m/z (CI) 408
(M-1, 100); 1H NMR (400 MHz, DMSO-d6) d 9.77 (s, 1H), 7.64 (s, 1H),
7.38 (s, 2H), 7.34 (d, J = 2 Hz, 1H), 7.26–7.16 (m, 3H), 7.114 (s, 1H),
7.075–6.981 (m, 3H), 3.80 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d
150.8, 148.9, 147.6, 144.9, 141.8, 141.6, 140.8, 127.5, 126.1, 125.2,
124.5, 123.5, 122.7, 120.6, 118.4, 116.4, 115.6, 113.4, 111.8, 102.3,
HO
Compound 7e was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 4-hydroxybenzalde-
hyde (0.99 g, 8.1 mmol) in DMF according to the general
procedure as an off white solid, yield 60% (1.2 g); mp 323–
325 °C; m/z (CI) 378 (M-1, 100); 1H NMR (400 MHz, DMSO-d6)
d 10.16 (s, 1H), 7.63–7.60 (m, 3H), 7.37 (s, 2H), 7.26–7.18 (m,
2H), 7.10 (s, 1H), 7.02–6.97 (m, 4H); IR (KBr, cmꢀ1
) vmax 3323,
3243, 3117, 1663, 1588, 1497, 1452, 1382, 1308, 1266, 1151,
1102, 795.
55.8; IR (KBr, cmꢀ1
) vmax 3343, 3064, 1662, 1499, 1459, 1424,
1328, 1303, 1235, 1151, 1032, 936, 875, 787, 760.
4.1.10. 1-(3-Hydroxyphenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7c)
4.1.13. 1-(2-Hydroxyphenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7f)
O
O
O
O
O
N
S
O
N
S
NH2
NH2
N
N
OH
OH
Compound 7f was prepared by using 1-amino-10H-
phenoxazine-3-sulfonamide (6) (1.4 g, 5.4 mmol) and 2-hydroxy-
benzaldehyde (0.98 g, 8.1 mmol) in DMF according to the
general procedure as an off white solid, yield 60% (1.2 g); mp
308–310 °C; m/z (CI) 378 (M-1, 100); 1H NMR (400 MHz,
DMSO-d6) d 10.25 (s, 1H), 7.66 (s, 1H), 7.53–7.50 (m, 2H), 7.38
(s, 2H), 7.25–7.23 (m, 1H), 7.18–6.95 (m, 5H), 6.82 (d,
) vmax 3329, 3236, 3119, 1661,
1572, 1463, 1463, 1392, 1318, 1266, 1151, 1102, 1023, 962,
853,789, 744, 667.
Compound 7c was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.4 g, 5 mmol) and 3-hydroxy benzalde-
hyde (0.98 g, 8.1 mmol) in DMF according to the general
procedure as an off white solid, yield 60% (1.2 g); mp 273–276 °C;
m/z (CI) 378 (M-1, 100); 1H NMR (400 MHz, DMSO-d6) d 9.94 (s,
1H, OH), 7.66 (s, 1H), 7.43 (t, J = 8.0 Hz, 1H,), 7.38 (s, 2H, SO2NH2),
7.26 (d, J = 7.6 Hz, 1H), 7.19–7.12 (m, 4H), 7.05 (dd, J = 1.6 and
J = 7.6 Hz, 1H); IR (KBr, cmꢀ1
8.0 Hz, 1H,), 6.98 (m, 2H); IR (KBr, cmꢀ1
1662, 1582, 1493, 1453, 1382, 1308, 1266, 1163,1151, 1102, 755.
) vmax 3324, 3246, 3115,

1960
S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
4.1.14. 1-(4-(Diethylamino)-2-hydroxyphenyl)imidazo[4,5,1-
4.1.17. 1-(3-Bromophenyl)imidazo[4,5,1-kl]phenoxazine-4-
kl]phenoxazine-4-sulfonamide (7g)
sulfonamide (7j)
O
O
O
O
O
S
O
N
S
NH2
NH2
N
N
N
OH
N
Br
Compound 7j was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 3-bromobenzalde-
hyde (1.5 g, 8.1 mmol) in DMF according to the general
procedure as an off white solid; yield 60% (1.4 g); mp 310–
312 °C; m/z (CI) 440 (M-2, 100); 1H NMR (400 MHz, DMSO-d6) d
7.69 (s, 1H), 7.49 (t, J = 8.8 Hz, 1H,), 7.34 (s, 2H), 7.24 (d,
J = 8.8 Hz, 1H,), 7.12–7.08 (m, 4H), 7.05 (d, J = 8.0 Hz, 1H), 6.98
Compound 7g was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 4-diethylamino-2-
hydroxybenzaldehyde (1.2 g, 6.4 mmol) in DMF according to the
general procedure as an off white solid; yield 50% (1.2 g); mp
280–282 °C; m/z (CI) 449 (M-1, 100); 1H NMR (400 MHz, DMSO-
d6) d 9.69 (1H), 7.65 (s, 1H), 7.35 (s, 2H), 7.31 (d, J = 7.6 Hz, 1H),
7.19–7.12 (m, 4H), 6.51–6.4 (m, 3H), 3.4 (q, J = 7.2 Hz, 4H), 1.0 (t,
(m, 2H); IR (KBr, cmꢀ1
) vmax 3342, 3029, 1663, 1483, 1455, 1307,
J = 7.2 Hz, 6H); IR (KBr, cmꢀ1
) vmax 3332, 3233, 3117, 1661, 1562,
1262, 1192, 1136, 1083, 837, 756, 657.
1473, 1389, 1321, 1257, 1151, 1102, 1015, 755.
4.1.18. 1-(2-Chlorophenyl)imidazo[4,5,1-kl]phenoxazine-4-
4.1.15. 1-(3-Nitrophenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7k)
sulfonamide (7h)
O
O
O
N
S
O
O
O
S
NH2
NH2
N
N
N
Cl
Compound 7k was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 2-chlorobenzalde-
hyde (1.14 g, 8.1 mmol) in DMF according to the general
procedure as an off white solid; yield 70% (1.4 g); mp 327–
330 °C; m/z (CI) 396 (M-1, 100); 1H NMR (400 MHz, DMSO-d6)
d 7.82–7.73 (m, 3H), 7.71 (s, 1H), 7.66–7.64 (m, 1H), 7.40 (s,
2H), 7.29–7.21 (m, 1H), 7.21–7.19 (m, 2H), 6.96 (t, J = 8.0 Hz,
NO2
Compound 7h was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.45 g, 5.2 mmol) and 3-nitrobenzalde-
hyde (1.18 g, 7.8 mmol) in DMF according to the general
procedure as an off white solid, yield 71% (1.5 g); mp 332–
335 °C; m/z (CI) 407 (M-1, 100); 1H NMR (400 MHz, DMSO-d6) d
8.67 (s, 1H), 8.52 (d, J = 9.6 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.94
(t, J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.41 (s, 2H), 7.30–7.17 (m, 3H),
1H), 6.45 (d, J = 7.8 Hz, 1H); IR (KBr, cmꢀ1
) vmax 3342,
3029, 1663, 1483, 1455, 1307, 1262, 1192, 1136, 1083, 837,
756, 657.
6.97–6.89 (m, 2H); IR (KBr, cmꢀ1
) vmax 3344, 3024, 1654, 1540,
1491, 1350, 1304, 1195, 1077, 1036, 734.
4.1.19. 1-(5-Bromo-2-fluorophenyl)imidazo[4,5,1-
kl]phenoxazine-4-sulfonamide (7l)
4.1.16. 1-(2-Bromophenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7i)
O
O
O
O
O
N
S
O
N
S
NH2
NH2
N
N
Br
F
Br
Compound 7l was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.4 g, 5 mmol) and 5-bromo-2-florobenz-
aldehyde(1.12 g, 7.5 mmol) in DMF according to the general
procedure as a light brown solid; yield 53% (1.23 g); mp 315–
317 °C; m/z (CI) 458 (M-1, 100); 1H NMR (400 MHz, DMSO-d6) d
8.07–7.99 (m, 1H), 7.99–7.96 (m, 1H), 7.71 (s, 1H), 7.55 (t,
J = 9.6 Hz, 1H), 7.42–7.31 (m, 2H), 7.29–7.21 (m, 2H), 7.17 (s, 1H),
Compound 7i was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 2-bromobenzalde-
hyde (1.2 g, 6.4 mmol) in DMF according to the general
procedure as an off white solid, yield 60% (1.4 g); mp 310–
312 °C; m/z (CI) 440 (M-2, 100); 1H NMR (400 MHz, DMSO-d6) d
7.94–7.92 (m, 1H), 7.80–7.77 (m, 1H), 7.71–7.66 (m, 3H), 7.40 (s,
2H), 7.29–7.27 (m, 1H), 7.21–7.16 (m, 2H), 6.95 (t, J = 8.0 Hz, 1H),
7.04 (t, J = 7.2 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H); IR (KBr, cmꢀ1
) vmax
6.41 (d, J = 8.0 Hz, 1H); IR (KBr, cmꢀ1
1483, 1476, 1339, 1234, 1179, 1137, 1085, 847, 766, 647.
) vmax 3343, 3069, 1662,
3342, 3032, 1665, 1497, 1459, 1329, 1301, 1225, 1188, 1080,
1038, 737, 690, 679.

S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
1961
4.1.20. 1-(2,3-Dichlorophenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7m)
(s, 6H); IR (KBr, cmꢀ1
1389, 1321, 1257, 1151, 1102, 1090, 1015, 928, 866, 765, 684.
)
vmax 3332, 3233, 3117, 1662, 1562, 1473,
4.1.23. 1-(2,6-Dichlorophenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7p)
O
S
O
O
N
NH2
O
O
S
N
O
NH2
Cl
N
N
Cl
Cl
Compound 7m was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 2,3-dichlorobenzal-
dehyde (1.42 g, 8.1 mmol) in DMF according to the general
procedure as an ash colour solid, yield 52% (1.2 g); mp 316–
320 °C; m/z (CI) 430 (M-1, 100); 1H NMR (400 MHz, DMSO-d6) d
7.65 (s, 1H), 7.38 (s, 2H), 7.32 (s, 1H), 7.30–7.25 (m, 3H), 7.15 (s,
Cl
Compound 7p was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 2,6-dichlorobenzal-
dehyde (1.13 g, 6.4 mmol) in DMF according to the general
procedure as an off white solid; yield 64% (1.5 g); mp 288–
290 °C; m/z (CI) 430 (M-1, 100); 1H NMR (400 MHz, DMSO-d6) d
7.62–7.60 (m, 2H), 7.36 (s, 2H), 7.26–7.24 (m, 1H), 7.19–7.16 (m,
2H), 7.09 (s, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H);
1H), 7.01–6.92 (m, 3H); IR (KBr, cmꢀ1
) vmax 3344, 3024, 1662,
1499, 1484, 1380, 1336, 1299, 1267, 1158, 1096, 955, 821, 785,
725.
IR (KBr, cmꢀ1
) vmax 3344, 3024, 1662, 1494, 1454, 1380, 1336,
1299, 1267, 1148, 1096, 823, 765, 741,720.
4.1.21. 1-(4-Chlorophenyl)imidazo[4,5,1-kl]phenoxazine-4-
sulfonamide (7n)
4.1.24. Potassium-1-amino-10H-phenoxazine-3-sulfonate (8)
O
O
O
N
SO3-K+
S
O
NH2
N
H
N
NH2
To a solution of potassium 1-nitro-10H-phenoxazine-3-sulfo-
nate (3) (5 g) in methanol (200 ml) was added Raney Ni (5 g) and
hydrazine hydrate (2 ml). The resulting mixture was stirred for
30 min at 65 °C. Completion of the reaction was monitored by
TLC. The catalyst was removed by filtration under reduced pressure
and concentrated under reduce pressure obtained as a light ash
colour solid; yield 77% (3.50 g); mp >300 °C; m/z (CI) 315 (M-1,
100); 1H NMR (400 MHz, DMSO-d6) d 7.32 (s, 1H), 6.82–6.65 (m,
Cl
Compound 7n was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.5 g, 5.4 mmol) and 4-chlorobenzalde-
hyde (1.140 g, 8.1 mmol) in DMF according to the general
procedure as an off white solid; yield 70% (1.4 g); mp 335–
337 °C; m/z (CI) 396 (M+1, 100); 1H NMR (400 MHz, DMSO-d6) d
7.86–7.84 (m, 2H), 7.73–7.67 (m, 3H), 7.39 (s, 2H), 7.29–7.27 (m,
1H), 7.20 (t, J = 7.2 Hz, 1H), 7.14 (s, 1H), 7.00 (t, J = 8.2 Hz, 1H),
1H), 6.64–6.45 (m, 4H), 6.21(s, 1H), 4.78 (s, 2H); IR (KBr, cmꢀ1
) vmax
3338, 1611, 1500, 1442, 1417, 1329, 1188, 1108, 1085, 1044, 848.
6.90 (d, J = 7.6 Hz, 1H); IR (KBr, cmꢀ1
1483, 1455, 1307, 1262, 1192, 1136, 756.
) vmax 3344,, 3037, 1663,
4.1.25. Potassium-1-phenylimidazo[4,5,1-kl]phenoxazine-4-
sulfonate (9)
4.1.22. 1-(4-Dimethylaminophenyl)imidazo [4,5,1-
kl]phenoxazine-4-sulfonamide (7o)
SO3 K+
-
O
N
O
O
O
N
S
N
NH2
Ph
N
To a solution of potassium-1-amino-10H-phenoxazine-3-sulfo-
nate (8) (3.0 g, 9.43 mmol) in DMF (80 ml) was added benzalde-
hyde (1.0 g, 9.43 mmol) and the resulting reaction mixture was
stirred for 24 h at 100 °C. Completion of the reaction was moni-
tored by TLC. The solvent was removed from the reaction under re-
duced pressure, then the crude solid was triturated with ethyl
acetate, filtered and purified by column chromatography; yield
52% (2 g), mp >300 °C; m/z (CI) 365 (M-K, 100); 1H NMR
(400 MHz, DMSO-d6) d 7.79 (d, 2H, J = 7.9 Hz), 7.64–7.59 (m, 3H),
7.42 (s, 1H), 7.22 (d, 1H, J = 8.0 Hz), 7.12 (t, 1H, J = 8.0 Hz), 6.91–
N
Compound 7o was prepared by using 1-amino-10H-phenoxa-
zine-3-sulfonamide (6) (1.4 g, 5 mmol) and 4-diethylaminobenzal-
dehyde (1.56 g, 10 mmol) in DMF according to the general
procedure as an off white solid; yield 51% (1.5 g); mp 260–
262 °C; m/z (CI) 405 (M-1, 100); 1H NMR (400 MHz, DMSO-d6) d
7.62–7.60 (m, 3H), 7.36 (s, 2H), 7.26–7.24 (m, 1H), 7.19–7.16 (m,
2H), 7.09 (s, 1H), 7.02–6.98 (m, 1H), 6.88 (d, J = 8.2 Hz, 2H), 3.04
6.83 (m, 3H); IR (KBr, cmꢀ1
1195, 1078, 1039, 700.
) vmax 3492, 1661, 1494, 1455, 1302,

1962
S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
4.1.26. 1-Phenylimidazo[4,5,1-kl]phenoxazine-4-
sulfonylchloride (10)
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 ꢀ80 °C in 50% glycerol until further use.
O
N
SO2Cl
5.1.3. PDE4 enzymatic assay
The inhibition of PDE4 enzyme was measured using PDElight
HTS cAMP phosphodiesterase assay kit (Lonza) according to man-
ufacturer’s recommendations. Briefly, 10 ng of in house purified
PDE4B1 or 0.5 ng commercially procured PDE4D2 enzyme was
pre-incubated either with DMSO (vehicle control) or compound
N
Ph
Potassium-1-phenylimidazo[4,5,1-kl]phenoxazine-4-sulfonate
(9) (2 g) was added to POCl3 (14 ml) and refluxed for 3 h. Comple-
tion of the reaction was monitored by TLC. Excess of POCl3 was re-
moved by distillation and the crude solid was poured in excess
crushed ice and the solid was filtered and used immediately for
the next step.
for 15 min before incubation with the substrate cAMP (5 lM) for
1 h. The reaction was halted with stop solution and reaction mix
was incubated with detection reagent for 10 min in dark. Dose re-
sponse studies were performed at 9 different concentrations rang-
ing from 100 lM to 0.01 lM. Luminescence values (RLUs) were
measured by a Multilabel plate reader (Perklin Elmer 1420 Multi-
label counter).The percentage of inhibition was calculated using
the following formula and the IC50 values were determined by a
nonlinear regression analysis from dose response curve using
Graphpad Prism software (San Diego, USA). IC50 values are ex-
pressed as mean SD.
4.1.27. Alternative preparation of 1-Phenylimidazo[4,5,1-
kl]phenoxazine-4-sulfonamide (7a)
To a solution of aqueous ammonia (5 ml) in tetrahydrofuran
(20 ml) was added 1-phenylimidazo [4,5,1-kl]phenoxazine-4-sul-
fonylchloride (10) at 0 °C. The resulting reaction mixture was stir-
red for 30 min at same temperature. Completion of the reaction
was monitored by TLC and the excess solvent was removed under
reduced pressure, the crude solid was acidified with dilute hydro-
chloric acid and the solid was filtered and dried. Compound is
matching with the prepared by another method, yield 50% (0.8 g).
%Inhibition ¼ ½ðRLU of vehicle control
ꢀ RLU of inhibitorÞ=ðRLU of vehicle controlÞꢂ
ꢁ 100
5. Pharmacology
6. Docking study
6.1. With PDE4B
5.1. Materials and methods
5.1.1. Cells and reagents
Docking simulations of molecules were performed using Schro-
dinger software suite (Maestro, version 9.2).19 The Protein (PDE4B)
for docking studies was retrieved from protein data bank with PDB
ID: 3O0J.20 The protein was prepared by giving preliminary treat-
ment 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 co-crystal li-
gands and extended up to 20 Å. The hydroxyl groups of search area
were kept flexible during grid generation process.
Sf9 cells were obtained from ATCC (Washington DC, USA) and
were routinely maintained in Grace’s supplemented medium
(Invitrogen) with 10% FBS. cAMP was purchased from SISCO Re-
search Laboratories (Mumbai, India). PDElight HTS cAMP phospho-
diesterase assay kit was procured from Lonza (Basel, Switzerland).
PDE4B1 clone was procured from OriGene Technologies (Rockville,
MD, USA). PDE4D2 enzyme was purchased from BPS Bioscience
(San Diego, CA, USA).
5.1.2. PDE4B protein production and purification
6.2. With PDE4D
PDE4B1 cDNA was sub-cloned into pFAST Bac HTB vector (Invit-
rogen) 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 Tris–HCl
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.17 Briefly, lysate was
centrifuged at 10,000 rpm for 10 min at 4 °C and supernatant
was collected. Supernatant was mixed with Ni-NTA resin (GE Life
Sciences) in a ratio of 4:1 (v/v) and equilibrated with binding buffer
(20 mM Tris–HCl 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, lysate–Ni–
NTA 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 Tris–HCl 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 Tris–
HCl pH 8.5, 100 mM KCl, 250 mM imidazole, 10 mM 2-mercap-
toethanol, 10% glycerol, Buffer II: 20 mM Tris–HCl pH 8.5,
Docking simulations of molecules were performed using Schro-
dinger software suite (Maestro, version 9.2).19 The Protein coordi-
nates (PDE4D) for docking studies was retrieved from protein data
bank with PDB ID: 1XOR.20 The protein was prepared by giving
preliminary treatment like adding hydrogen, adding missing resi-
dues, refining the loop with prime and finally minimized by using
OPLS 2005 force field. The search grid was generated by picking the
co-crystal ligands and extended up to 20 Å. The hydroxyl groups of
search area were kept flexible during grid generation process.
All molecules were minimized by using MacroModel21 applica-
tion. Molecules were docked by using glide XP (extra precision)
docking mode.22 We performed flexible docking by allowing sam-
ple ring conformations and sample nitrogens to move to possible
extent in docking. The docking results are shown in tables and
figures.
Acknowledgments
One of the authors (B.V.K.) is thankful to UGC, New Delhi, India
for Major Research Project (F. No. 35-151/2008). The authors are
thankful to Principal and Management of C.K.M. Arts and Science

S. V. Reddy et al. / Bioorg. Med. Chem. 21 (2013) 1952–1963
1963
5. Kodimuthali, A.; Jabaris, S. S. L.; Pal, M. J. Med. Chem. 2008, 51, 5471.
6. Rao, Y. J.; Xi, L. Acta Pharmacol. Sin. 2009, 30, 1.
7. (a) Dastidar, S. G.; Rajagopal, D.; Ray, A. Curr. Opin. Invest. Drugs 2007, 85, 364;
(b) Field, S. K. Clin Med. Insights Circ. Respir. Pulm. Med. 2011, 5, 57; (c) Chung, J.
H. Aging 2012, 4, 144.
8. (a) Regan, J.; Bruno, J.; Mc Garry, D.; Poli, G.; Hanney, B.; Bower, S.; Travis, J.;
Sweeney, D.; Miller, B.; Souness, J.; Djuric, S. Bioorg. Med. Chem. Lett. 1998, 8,
2737; (b) Roberts, D. E.; Curd, J. G. Arthritis Rheum. 1990, 33, 1590; (c) Lajiness,
M. S.; Vieth, M.; Erickson, J. Curr. Opin. Drug. Disc. Dev. 2004, 7, 470.
9. (a) Bernthsen, A. Ber. Dtsh. Chem. Ges. 1887, 20, 942; (b) Kehrmann, F. Ann.
Chem. 1902, 322, 1; (c) Kehrmann, F.; Neil, A. A. Ber. Dtsch. Chem. Ges. 1914, 47,
3102; (d) Cullinane, N. M.; Davey, H. G.; Padfield, H. J. H. J. Chem. Soc. 1934, 716;
(e) Gilman, H.; Moore, L. O. J. Am. Chem. Soc. 1957, 79, 3485.
College for encouragement and obliged to Dr. D. Mohan Rao,
SYMED Laboratories Ltd, Hyderabad for analytical support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.023.
References and notes
10. Prinz, H.; Chamasmani, B.; Vogel, K.; Beohm, K. J.; Aicher, B.; Gerlach, M.;
Geunther, E. G.; Amon, P.; Ivanov, I.; Meuller, K. J. Med. Chem. 2011, 54, 4247.
11. (a) Motohashi, N. Yakugaku Zasshi 1983, 103, 364; (b) Shimamoto, T.; Tomoda,
A.; Ishida, R. Clin. Cancer Res. 2001, 7, 704.
1. Suzuki, F.; Hashimoto, K.; Ishihara, M.; Westman, G.; Samuelsson, K.; Kawase,
M.; Motohashi, N.; Sakagami, H. Anticancer Res. 2007, 27, 4233.
2. Bolognese, A.; Correale, G.; Manfra, M.; Lavecchia, A.; Mazzoni, O.; Novellino,
E.; Barone, V.; La Colla, P.; Loddo, R. J. Med. Chem. 2002, 45, 5217.
3. Lohray, B. B.; Lohray, V. B.; Bajji, A. C.; Kalchar, S.; Poondra, R. R.; Padakanti, S.;
Chakrabarti, R.; Vikramadithyan, R. K.; Misra, P.; Juluri, S.; Mamidi, N. V.;
Rajagopalan, R. J. Med. Chem. 2001, 44, 2675.
12. Gunstone, F. D.; Tucker, S. H. J. Appl. Chem. 1952, 2, 204.
13. (a) Kumar, B. V.; Rathore, H. G. S.; Reddy, V. M. J. Indian Chem. 1982, 21B, 1126;
(b) Kumar, B. V.; Reddy, V. M. Indian Drugs 1985, 23, 94; (c) Kumar, B. V.; Reddy,
V. M. J. Indian Chem. 1985, 24B, 1098; (d) Rao, V. M.; Kumar, B. V.; Reddy, M. M.;
Reddy, V. M. Indian Drugs 1988, 25, 304.
14. Ullmann, F.; Engl, G.; Wosnessensky, N.; Kuhn, E.; Herre, E. Justus Liebig Ann.
Chem. 1909, 366, 79.
4. (a) Reddy, G. R.; Reddy, T. R.; Joseph, S. C.; Reddy, K. S.; Reddy, L. S.; Kumar, P.
M.; Krishna, G. R.; Reddy, C. M.; Rambabu, D.; Kapavarapu, R.; Lakshmi, C.;
Meda, T.; Priya, K. K.; Parsa, K. V. L.; Pal, M. Chem. Commun. 2011, 47, 7779; (b)
Kodimuthali, A.; Gupta, R.; Parsa, K. V. L.; Prasunamba, P. L.; Pal, M. Lett. Drug
Des. Disc. 2010, 7, 402; (c) Kumar, K. S.; Kumar, S. K.; Sreenivas, B. Y.; Gorja, D.
R.; Kapavarapu, R.; Rambabu, D.; Krishna, G. R.; Reddy, C. M.; Rao, M. V. B.;
Parsa, K. V. L.; Pal, M. Bioorg. Med. Chem. 2012, 20, 2199; (d) Pal, S.; Durgadas, S.;
Nallapati, S. B.; Mukkanti, K.; Kapavarapu, R.; Lakshmi, C.; Parsa, K. V. L.; Pal, M.
Bioorg. Med. Chem. Lett. 2011, 21, 6573; (e) Kumar, P. M.; Kumar, K. S.;
Mohakhud, P. K.; Mukkanti, K.; Kapavarapu, R.; Parsa, K. V. L.; Pal, M. Chem.
Commun. 2012, 48, 431; (f) Kumar, P. M.; Kumar, K. S.; Meda, C. L. T.; Reddy, G.
R.; Mohakhud, P. K.; Mukkanti, K.; Krishna, G. R.; Reddy, C. M.; Rambabu, D.;
Kumar, K. S.; Priya, K. K.; Chennubhotla, K. S.; Banote, R. K.; Kulkarni, P.; Parsa,
K. V. L.; Pal, M. Med. Chem. Commun. 2012, 3, 667; (g) Adepu, R.; Rambabu, D.;
Prasad, B.; Meda, C. L. T.; Kandale, A.; Krishna, G. R.; Reddy, C. M.; Chennuru, L.
N.; Parsa, K. V. L.; Pal, M. Org. Biomol. Chem. 2012, 10, 5554; (h) Kumar, T. B.;
Sumanth, Ch.; Vaishaly, S.; Rao, M. S.; Sekhar, K. B. C.; Meda, C. L. T.; Kandale,
A.; Rambabu, D.; Krishna, G. R.; Reddy, C. M.; Kumar, K. S.; Parsa, K. V. L.; Pal, M.
Bioorg. Med. Chem. Lett. 2012, 22, 5639.
15. Shirai, H.; Hayazaki, T. Yakugaku Zasshi 1970, 90, 588.
16. This route is amenable for the preparation of sulfonamide derivatives
possessing various substituents on the sulfonamide nitrogen depending on
the amines used in place of aqueous ammonia in the last step. We thank the
reviewer for pointing out this.
17. Wang, P.; Myers, J. G.; Wu, P.; Cheewatrakoolpong, B.; Egan, R. W.; Billah, M. M.
Biochem. Biophys. Res. Commun. 1997, 234, 320.
18. Demnitz, J.; LaVecchia, L.; Bacher, E.; Keller, T. H.; Muller, T.; Schurch, F.;
Weber, H. P.; Pombo-Villar, E. Molecules 1998, 3, 107.
19. Maestro, version 9.2, Schrodinger, LLC: New York, NY, 2012.
20. Card, G. L.; England, B. P.; Suzuki, Y.; Fong, D.; Powell, B.; Lee, B.; Luu, C.;
Tabrizizad, M.; Gillette, S.; Ibrahim, P. N.; Artis, D. R.; Bollag, G.; Milburn, M. V.;
Kim, S.-H.; Schlessinger, J.; Zhang, K. Y. J. Structure 2004, 12, 2233.
21. MacroModel, version 9.9, Schrödinger, LLC: New York, NY, 2011.
22. Glide, version 5.7, Schrodinger, LLC: New York, NY, 2012.