Synthesis of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole moiety: Their in vitro evaluation against PDE4B

Article abstract of DOI:10.1016/j.bmcl.2013.12.117

A number of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole or benzofuran moieties were synthesized by using Pd/C-Cu mediated coupling-cyclization strategy as a key step. The o-iodoanilides or o-iodophenol were coupled with 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one using 10%Pd/C-CuI-PPh₃ as a catalyst system and Et₃N as a base to give the target compounds. All the synthesized compounds were tested for their PDE4B inhibitory potential in vitro using a cell based cAMP reporter assay. Some of them showed fold increase of the cAMP level when tested at 30 μM. A representative compound showed encouraging PDE4B inhibitory properties that were supported by its docking results.

Full text of DOI:10.1016/j.bmcl.2013.12.117

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1166–1171
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 2H-1,3-benzoxazin-4(3H)-one derivatives containing
indole moiety: Their in vitro evaluation against PDE4B
a
b
c
c
Raja Mohan Rao , Bethala Jawahar Luther , Chekuri Sharmila Rani , Namburi Suresh ,
d
a
c,
⇑
a,
⇑
Ravikumar Kapavarapu , Kishore V. L. Parsa , Mandava V. Basaveswara Rao , Manojit Pal
a
Dr. Reddys Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India
Department of Chemistry, Krishna University, Krishna Dist., Andhra Pradesh, India
Doctoral Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole or benzofuran moieties were
synthesized by using Pd/C–Cu mediated coupling-cyclization strategy as a key step. The o-iodoanilides
or o-iodophenol were coupled with 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one
Received 24 November 2013
Revised 26 December 2013
Accepted 28 December 2013
Available online 7 January 2014
using 10%Pd/C–CuI–PPh
synthesized compounds were tested for their PDE4B inhibitory potential in vitro using a cell based cAMP
reporter assay. Some of them showed fold increase of the cAMP level when tested at 30 M. A represen-
3 3
as a catalyst system and Et N as a base to give the target compounds. All the
l
Keywords:
tative compound showed encouraging PDE4B inhibitory properties that were supported by its docking
results.
2H-1,3-Benzoxazin-4(3H)-one
Indole
Benzofuran
Pd/C
Ó 2014 Elsevier Ltd. All rights reserved.
PDE4
While asthma affects ꢀ300 million people worldwide at pres-
isozymes, for example, PDE1 to PDE11. Inhibition of the PDE4
effectively elevates the intracellular cAMP levels, thereby activat-
ing specific protein phosphorylation cascades that in turn inhibits
the release of inflammatory mediators such as cytokines [tumor
ent, chronic obstructive pulmonary disease (COPD) is projected
1
to be the 3rd leading cause of death globally by 2020. Inhibitors
of phosphodiesterase 4 (PDE4) are considered as beneficial to treat
1
,2
COPD or asthma. However, the first-generation (e.g., rolipram) as
well as second generation PDE4 inhibitors (e.g., cilomilast and rof-
necrosis factor-
a
(TNF-a), interleukin-2 (IL-2), interleukin-12
(IL-12), leukotriene B4 (LTB4), interferon-
c
(IFN- )], as well as acti-
c
1
,2
3
4
lumilast) suffered from side effects like nausea and emesis.
vation of inflammatory cells. The cAMP specific PDE4 isozymes
(which require a divalent metal ion, for example, Zn for catalysis)
are encoded by four genes (A-D) that give rise to four isoforms,
Ò
Encouragingly, roflumilast (Daxas , Nycomed) has been launched
in Europe for the treatment of chronic bronchitis in 2009 and in
US (Daliresp, Forest Lab) for exacerbations during COPD in 2012.
Nevertheless, it is desirable to devote continued efforts towards
identification of newer class of PDE4 inhibitors having fewer side
effects.
5
for example, PDE4A to PDE4D. Since knockout mice studies have
revealed that PDE4B ablation suppresses TNF-a production, hence
inhibition of PDE4B has been proposed to be beneficial for the
development of more effective anti-inflammatory drugs to combat
with COPD and asthma.
In the inflammatory cells cAMP (cyclic adenosine monophos-
phate) plays the role of a negative regulator of the primary activat-
ing pathways such as cytokine release by T-cells. Levels of cAMP on
the other hand are regulated by cAMP-specific phosphodiesterases
Due to their wide range of pharmacological properties 2-substi-
tuted indoles have been explored as a number of potential
6
7
therapeutic agents including PDE4 inhibitors. The 2H-benzo[e]
[1,3]oxazin-4(3H)-one nucleus on the other hand has been found
(
PDE) isozyme for example, PDE4 predominantly expressed in
1,2
8,9
inflammatory and immune cells in addition to brain. PDEs the
super family of enzymes (that hydrolyze the phosphodiester bond
of cAMP and cGMP) can be subdivided into 11 different groups or
to be integral part of several bioactive agents. Thus, combination
of both in a single entity via linking them through an appropriate
linker should provide a new framework from which the molecules
derived might show interesting pharmacological activities.
Prompted by this idea we designed the template C (when X = NR
or O) from A and B. Initially, a number of virtual molecules derived
from C were tested in silico against several drug targets including
⇑
Corresponding authors. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail addresses: vbrmandava@yahoo.com (M.V.B. Rao), manojitpal@rediffmail.
com (M. Pal).
0
960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.117

R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171
1167
PDE4B without performing their actual chemical synthesis.
Accordingly, the compound D emerged as a virtual hit against
PDE4B in this study. Docking studies (Fig. 2, see also Fig. S-1 in
ESI) [performed using Chemical Computing Group’s Molecular
Operating Environment (MOE) software 2008.10 Version, ‘DOCK’
application module, see the ESI] indicated that compound D uti-
lized the conserved active binding site with its benzooxazinone
carbonyl and N-methanesulfonyl oxygen participated in H-bond-
ing interactions with the PDE4B protein. The carbonyl oxygen
formed H-bond with the His234 of metal binding pocket and sul-
fonyl oxygen formed H-bond with the Gln443 of Q pocket in the
active site. These interactions were similar to that of the known
inhibitor rolipram (see Fig. S-2 in ESI). Indeed, the dock score of
compound D (À19.79 K cal/mol) was comparable with that of roli-
pram (À24.62 K cal/mol). The compound D has also shown an
arene-arene interaction with the Phe446 residue of PDE4B
Several elegant and attractive methods11–13 have been reported
for the construction of indole ring including the transition metal
mediated methods of which the palladium catalyzed reactions
gained particular attention. As a less expensive catalyst system
the use of Pd/C–CuI–PPh
3
has also gained considerable interest
14
for the efficient synthesis of various heterocyclic structures
1
5,16
including indoles.
Compared to other Pd catalysts Pd/C is sta-
ble, easy to handle and separable from the product and is recycla-
1
4
ble. The use of Pd/C catalyzed reaction therefore is advantageous.
All these features prompted us to explore a Pd/C mediated con-
struction of indole ring as a key step for the synthesis of our target
compounds C and D (or 3) (Scheme 1).
The starting alkyne that is, 3-{2-(prop-2-ynyloxy)ethyl}-2H-
benzo[e][1,3]oxazin-4(3H)-one (2) required for the synthesis of
our target compound 3 was prepared according to a reported
8
procedure as shown in Scheme 2. Thus, the methyl salicylate 5
(
Fig. 2, see also Fig. S-1 in ESI). Encouraged by these observations
obtained from 4 was converted to 2-hydroxy-N-(2-hydroxyethyl)
benzamide (6) which was then treated with paraformaldehyde
under acidic conditions followed by the hydrolysis of the interme-
diate formed to give 3-(2-hydroxyethyl)-2H-benzo[e][1,3]oxazin-
4(3H)-one (7). The subsequent propargylation of 7 afforded the
desired terminal alkyne 2 that was used for the coupling-
cyclization with a range of o-iodoanilides and o-iodophenol (1) in
we then decided to synthesize a library of small molecules based
on C and screen them against PDE4B in vitro. As part of our ongo-
1
0
ing effort on the identification of novel PDE4 inhibitors we now
report the results of our recent study. To the best of our knowl-
edge the in vitro pharmacological properties of these 2H-1,3-benz-
oxazin-4(3H)-one derivatives (C) containing indole or benzofuran
moieties have not been explored earlier.
3 3
the presence of 10% Pd/C, PPh , CuI and Et N in MeOH (Table 1).
group
N
O
O
O N
2
A
N
N
O
O
O
O
O
X
O
N
S
group
O
O
N
X = NR, O
C
D
B
Figure 1. Design of novel and potential bioactive molecules C (and D) from A and B.
Figure 2. Binding mode of D in the PDE4B (PDB code-1XMY).

1
168
R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171
O
R
O
O
1
0% Pd/C, PPh3
R
I
O
CuI, Et N,
O
N
+
N
3
X
O
X
H
MeOH,reflux
4-6h
3
2
1
R = Me, Cl, H, CF , I,
X = NSO Me, O
3
2
,
COCH , F, NO2 CN.
3
Scheme 1. Pd/C-mediated synthesis of 3-[(indol-2-ylmethoxy)ethyl]-2H-1,3-benzoxazin-4(3H)-one derivatives (3).
O
O
O
OH
(CH
HCl, CHCl
3
O) , AcOH
Oxalyl chloride
MeOH
2 n
H N
OH
2
OH
OMe
OH
N
H
8
9
0o
0%
C
170o
70%
OH
Na CO ,CH OH
2 3 3
O0 C -RT
40%
OH
O
C
6
4
5
O
OH
Br
N
O
N
O
K CO
2
3
O
7
Acetone
rt, 5-6 h
2
7
2%
Scheme 2. Synthesis of 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one (2).8
Table 1
Synthesis of 3-[(indol-2-ylmethoxy)ethyl]-2H-1,3-benzoxazin-4(3H)-ones/3-[(benzofuran-2-ylmethoxy)ethyl]-2H-1,3-benzoxazin-4(3H)-one (3) (Scheme 1)a
Entry
o-Iodoanilide/o-iodophenol (1)
Time (h)
Product (3)
Yieldb (%)
I
O
N
NH
S
O
O
O
O
1
4
65
N
S
1a
O
O
3
a
Cl
I
O
N
NH
S
O
Cl
O
O
O
2
3
4
4.5
62
68
64
N
S
1
b
O
O
3
b
I
O
N
NH
S
O
c
O
O
O
4
N
S
O
1
O
3
c
F3C
I
O
N
NH
S
O
F3C
O
O
O
5
N
S
1d
O
O
3
d

R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171
1169
Table 1 (continued)
Entry
o-Iodoanilide/o-iodophenol (1)
Time (h)
Product (3)
Yieldb (%)
I
I
O
N
NH
S
O
I
O
O
O
5
6
7
8
9
4
60
64
69
61
70
N
S
1e
O
O
3
e
O
I
O
F
N
O
O
NH
S
O
O
4.5
5.5
6
O
N
O
S
1
f
O
f
3
I
O
N
NH
S
O
F
O
O
O
N
S
O
3
1
g
O
g
O N
I
O
N
2
NH
S
O
O N
O
O
2
O
N
1h
O
S
O
3
h
NC
I
O
N
NH
S
NC
O
O
O
6
O
N
1
i
O
S
O
3
i
I
O
N
OH
1
0
4
O
O
69c
1j
O
3
j
a
All the reactions were performed using o-iodoanilide (1, 2 mmol), alkyne (2, 2 mmol), 10% Pd/C (0.02 mmol), CuI (0.02 mmol), PPh
3
(0.04 mmol) and Et
3
N (0.5 mL) in
MeOH (5.0 mL) at refluxing temp under a nitrogen atmosphere.
b
Isolated yield.
c
o-Iodophenol (1j, 2 mmol) was used in place of o-iodoanilide.
The presence of various groups for example, Me, F, Cl, I, CF
3
, COMe,
compound 1 and (iii) intramolecular cyclization of the resulting
NO
2
and CN (entries 1–9, Table 1) on the anilide ring were toler-
internal alkyne. Thus, in the initial step an active Pd(0) species is
generated via a Pd leaching process into the solution [from the
minor portion of the bound palladium (Pd/C)] followed by interac-
1
4
ated as the reactions proceeded well in all these cases affording
the desired indoles 3 in acceptable yields. The use of o-iodophenol
(1j) in place of o-iodoanilide however afforded the corresponding
3
tions with the phosphine ligands. The dissolved Pd(0)–PPh com-
benzofuran derivative 3j (entry 10, Table 1) following the
coupling-cyclization pathway.
plex then produces the organo-Pd(II) species E-1 via oxidative
addition with 1. The E-1 then undergoes trans organometallation
with copper acetylide generated in situ from CuI and terminal al-
kyne 2 followed by reductive elimination of Pd(0) to afford the
internal alkyne E-2. The E-2 thus formed subsequently undergoes
Mechanistically (Scheme 3), the one-pot synthesis of compound
3
3
under the catalysis of Pd/C–CuI–PPh seemed to proceed via (i)
the generation of actual catalytic species, (ii) alkynylation of iodo

1
170
R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171
Pd/C
Pd
leaching
Generation
of actual
catalytic
species
Precipitation of
Pd on C at the end
of catalytic cycle
Pd in
solution
B
1
R
PPh3
R
I
BH+I-
Cu
3
Pd(0)-PPh3
complex in
solution
X
BH+
X
CuI, B
BH+I-
CuI, BH+
The catalytic cycle
B = Et N)
Intramolecular
cyclization
(
3
B
R
Pd
I
R
2
BH+
+
BH+
Pd(0)
BH+
X
CuI/Et
3
N
Cu
X
E-1
E-2
R
Pd
X
BH+
Scheme 3. Proposed mechanism for the Pd/C-Cu catalyzed one-pot synthesis of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole or benzofuran moieties (3).
Cu-mediated 5-exo-dig ring closure in an intramolecular fashion to
give the product (3). Overall, the catalytic cycle seemed to work in
solution rather than on the surface and at the end of the reaction
Pd was re-precipitated on the charcoal surface.
1
0
8
6
4
2
All the synthesized compounds were tested for their PDE4B
1
7
inhibitory potential in vitro. In a cell based cAMP reporter assay
fold increase of the cAMP level caused by the test compounds over
forskolin control was determined. The known inhibitor rolipram
was used as a reference compound in this assay. Among the com-
pounds tested indole derivatives 3b, 3d, 3g, 3i and the benzofuran
derivative 3j showed fold increase when tested at 30 lM (Fig. 3).
However, the compound 3h showed >5 fold increase over control
when tested at the same concentration which is comparable to
the rolipram’s >7 fold increase in the same assay. It is evident that
the presence of an electron withdrawing group for example, Cl, CF
F, NO or CN at the C-5 of the indole ring seemed to be favorable for
the activity and the NO group was found to be the most effective
3
,
2
0
0
2
.001 0.01
0.1
[3h]µM
1
10
100
among them. Notably, an acetyl group at the same position (e.g.,
compound 3f) was found to be detrimental for activity perhaps
due to its bulkier size than other electron withdrawing groups
tested. In contrast the benzofuran derivative showed significant
fold increase in spite of not having any electron withdrawing group
at the C-5 position of the ring. Nevertheless, the compound 3h was
identified as the best among compounds tested. In a dose response
study compound 3h showed dose-dependent fold increase of the
cAMP level (Fig. 4) indicating its potential as an inhibitor. The ob-
served PDE4B inhibitory properties of compound 3h (or compound
Figure 4. Dose-dependent fold increase of the cAMP level by compound 3h (PDE4B
HEK 293 cell based reporter assay).
D, Fig. 1) was also supported by the docking studies as presented in
Fig. 2 that showed good binding of 3h with the PDE4B.
In conclusion,
a number of 2H-1,3-benzoxazin-4(3H)-one
derivatives containing indole or benzofuran moieties were initially
designed as a novel class of bioactive compounds. In silico studies
of these compounds against a number of drug target indicated that
they could be explored as potential inhibitors of PDE4. Accordingly,
compounds were synthesized by using Pd/C-Cu mediated coupling-
cyclization strategy as
o-iodophenol were coupled with 3-{2-(prop-2-ynyloxy)ethyl}-2H-
benzo[e][1,3]oxazin-4(3H)-one using 10%Pd/C–CuI–PPh as
catalyst system and Et N as a base to give the target compounds.
a key step. The o-iodoanilides or
3
a
3
All the synthesized compounds were tested for their PDE4B inhibi-
tory potential in vitro using a cell based cAMP reporter assay. Some
of them showed fold increase of the cAMP level when tested at
30 lM. A representative compound 3h showed encouraging PDE4B
inhibitory properties and was supported by its docking results
obtained prior to its synthesis. Overall, the newly designed
Figure 3. PDE4B HEK 293 cell based reporter screen of compound 3 (Y-axis: fold
elevation of cAMP over forskolin control).

R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171
1171
framework presented here seems to provide a useful basis for the
development of novel future PDE4 inhibitors for the potential
treatment of asthma and COPD.
8. Benedini, F.; Bertolini, G.; Cereda, R.; Dona, G.; Gromo, G.; Levi, S.; Mizrahi, J.;
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Acknowledgments
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