Synthesis and pharmacological evaluation of N-substituted 2-(2-oxo-2H-chromen-4-yloxy)propanamide as cyclooxygenase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.082

A series of novel N-substituted 2-(2-oxo-2H-chromen-4-yloxy)propanamide derivatives were synthesized via converting the readily available 4-hydroxy coumarin to the corresponding ethyl 2-(2-oxo-2H-chromen-4-yloxy)propanoate followed by hydrolysis and then reacting with different substituted amines. The molecular structures of two representative compounds, that is, 3 and 5l were confirmed by single crystal X-ray diffraction study. All the compounds synthesized were evaluated for their cyclooxygenase (COX) inhibiting properties in vitro. The compound 5i showed balanced selectivity towards COX-2 over COX-1 inhibition and good docking scores when docked into the COX-2 protein.

Full text of DOI:10.1016/j.bmcl.2012.08.082

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6745–6749
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological evaluation of N-substituted
2-(2-oxo-2H-chromen-4-yloxy)propanamide as cyclooxygenase inhibitors
D. Rambabu a, , Naveen Mulakayala a, , Ismail a, K. Ravi Kumar a, G. Pavan Kumar b, Chaitanya Mulakayala c,
Chitta Suresh Kumar c, Arunasree M. Kalle a, M. V. Basaveswara Rao d, Srinivas Oruganti a, Manojit Pal a,
⇑
a Institute of Life Sciences, University of Hyderabad Campus, Hyderabad 500 046, India
b Department of Chemistry, Acharya Nagarjuna University, Guntur, AP 522510, India
c Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515003, India
d Department of Chemistry, Krishna University, Machilipatnam, AP 521001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2012
Revised 20 July 2012
Accepted 21 August 2012
Available online 31 August 2012
A series of novel N-substituted 2-(2-oxo-2H-chromen-4-yloxy)propanamide derivatives were synthe-
sized via converting the readily available 4-hydroxy coumarin to the corresponding ethyl 2-(2-oxo-2H-
chromen-4-yloxy)propanoate followed by hydrolysis and then reacting with different substituted
amines. The molecular structures of two representative compounds, that is, 3 and 5l were confirmed
by single crystal X-ray diffraction study. All the compounds synthesized were evaluated for their
cyclooxygenase (COX) inhibiting properties in vitro. The compound 5i showed balanced selectivity
towards COX-2 over COX-1 inhibition and good docking scores when docked into the COX-2 protein.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Coumarin
Cyclooxygenase
X-ray
Docking
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most
extensively used therapeutics worldwide for the treatment of
inflammation, pain, fever, and for the prevention of thrombosis.
The mechanisms of their pharmacological effects are based on
inhibition of the catalytic domain of cyclooxygenase enzymes by
sterically hindering the entrance of arachidonic acid as their phys-
iological binder. This results in a reduced production of prostaglan-
dins and thromboxanes, which contribute as important autocrine
and paracrine mediators in many physiologic and pathophysiologic
responses.1–4
Cyclooxygenases (COXs) are membrane-bound heme proteins
which exist in two distinct isoforms, a constitutive form (COX-1)
and an inducible form (COX-2). Both COX-1 and COX-2 share the
same substrates, produce the same products and catalyze the same
reaction using identical catalytic mechanisms. The X-ray crystal
structure suggests that both the proteins are very similar in their
tertiary conformation.5,6 Their binding pocket and catalytic site
are nearly identical. The COX-1 enzyme is responsible for main-
taining homeostasis (gastric and renal integrity) and normal pro-
duction of eicosanoids. The COX-2 is mainly found in brain and
kidney while being virtually absent in most other tissues. However,
COX-2 expression is significantly upregulated under various acute
and chronic inflammatory conditions. It is also well documented
that COX-2 is over expressed in numerous human cancers such
as colorectal, gastric, and breast cancer.7
Recognition of the importance of COX-2 in inflammation and
carcinogenesis has prompted discovery of various COX-2 selective
inhibitors over the last two decades. A large number of compounds
have been synthesized and investigated as selective inhibitors of
COX-2 and many of them belong to the diaryl heterocyclic class
of compounds, for example, celecoxib, rofecoxib, valdecoxib, etor-
icoxib, SC57666 etc. (Fig. 1). 8a While celecoxib is still in the market
other inhibitors are either withdrawn or not launched due to their
possible adverse side effects. For example refocoxib was with-
drawn based on the fact that its uses may be associated with an in-
creased risk of cardiovascular side effect.8b Similarly, lumiracoxib
(prexige) was hold back due to concerns that it may cause liver
failure.8c However, the lack of common reason for withdrawal of
these inhibitors suggest that the possible risk associated with their
uses may be drug-specific rather than class-specific. Incidentally,
all the inhibitors withdrawn are known to be highly selective to-
wards COX-2 compared to celecoxib. It is therefore unclear that
which degree of COX-2 selectivity should be considered as safe.
It appeared that developing moderately selective inhibitors rather
than those possessing high selectivity might be a more balanced
approach. Nevertheless, the identification of new inhibitors based
on a novel scaffold possessing structural features other than that
of the known inhibitors could be beneficial and desirable for the
potential treatment of inflammatory diseases.
⇑
Corresponding author. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail address: manojitpal@rediffmail.com (M. Pal).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.082

6746
D. Rambabu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6745–6749
SO2NH2
H2NO2S
H2NO2S
H3CO2S
H3CO2S
F
H3C
N
N
N
Cl
H3C
H3C
O
O
N
N
O
CF3
Celecoxib
Valdecoxib
Figure 1. Examples of known COX-2 inhibitors.
Rofecoxib
SC57666
Etoricoxib
1,1,3,3-tetramethyluronium hexafluorophosphate]. A variety of
alkyl/(hetro)aryl amines were employed to prepare a range of
target amides that are shown in Table 1. All the compounds syn-
thesized were mixture of enantiomers whereas the compound 5h
was a mixture of diastereomers. All these compounds were charac-
terized by 1H and 13C NMR as well as MS spectra. Additionally, the
molecular structure of the intermediate 3 and a representative
compound 5l were established unambiguously by single crystal
X-ray diffraction study (Figs. 3 and 4).11,12
Less
CH3
susceptible
to enzymatic
hydrolysis
C-7 to C-4
NHAr
CH3
O
O
shift with
modifications
O
O
O
O
O
O
B
A
NHAr
All the compounds synthesized were evaluated for their COX
inhibiting potential and selectivity by using biochemical COX
(COX-1 and COX-2) enzyme based assay. The COX-1 enzyme
was isolated from Ram seminal vesicles whereas the recombinant
human COX-2 was expressed in insect cell expression system.
These enzymes were purified by employing conventional chro-
matographic techniques. Enzymatic activities of COX-1 and
COX-2 were measured according to the method reported earlier,13
with slight modifications using a chromogenic assay based on the
oxidation of N,N,N,N,-tetra methyl-p-phenylene diamine (TMPD)
during the reduction of PGG2 to PGH2.14,15 The known non-selec-
tive inhibitor indomethacin and COX-2 inhibitor celecoxib was
used as reference compounds in this assay. The IC50 values deter-
mined for all the compounds along with their selectivity (COX-2/
COX-1 ratio) are listed in Table 2. While most of the compounds
showed COX inhibiting properties in vitro only three of them
however were found to be COX-2 selective. While it was not clear
that which structural features were particularly responsible for
COX-2 inhibition the data presented in Table 2 however indicated
that the size of the amide side chain perhaps played a key role.
The size of this amide moiety seemed to be favorable for com-
pounds 5b, 5h and 5i that showed COX-2 selectivity (entry 2, 8
and 9, Table 2). The compound 5i was found to have better activ-
ity than 5b and 5h. It is better than indomethacin though inferior
to the diaryl heterocyclic class celecoxib in terms of selectivity.
Notably, while celecoxib is still in the market its uses in patients
having heart disease (or those who have a risk of developing
heart disease) are restricted. The use of paracetamol or certain
NSAIDs such as non-selective inhibitor naproxen have been sug-
gested to be safer choices in these patients. The compound 5i
Figure 2. The design of 4-substituted coumarin derivative (B) from the known
7-substituted analogue (A).
Our long standing interest in the identification of new COX-2
inhibitors9 prompted us to explore the use of coumarin framework
for the design of non archetype inhibitors. We were particularly
encouraged by the recent report on docking studies of 7-substituted
coumarin derivatives (A, Fig. 2) with COX-2 enzyme and their
promising anti-inflammatory activities.10 These compounds con-
taining (hetero)aryl amine connected through a linker at C-7 of
the coumarin ring were shown to interact mainly with Arg 44
amino acid, which was thought to be involved when COX-2 was
inhibited. We anticipated that a similar group at C-4 instead of at
C-7 of the coumarin ring (B, Fig. 2) would maintain the interaction
with COX-2. Moreover, an amide moiety (i.e., –CONHAr of B)
possessing a
better pharmacokineticstabilityratherthananester(i.e., –OCOCH2–
of A) containing a -amino moiety (Fig. 2). Herein we report our
a-methyl group was thought to provide possibility of
a
preliminaryresults on the synthesis and COX-2 inhibitingproperties
of 4-oxyalkyl substituted coumarin derivatives which to the best of
our knowledge were not explored as inhibitors of COX earlier.
The synthesis of our target compounds
B is outlined in
Scheme 1. Thus 4-hydroxy coumarin on reaction with ethyl
2-bromopropanoate yielded ethyl 2-(2-oxo-2H-chromen-4-yloxy)
propanoate (3), which on hydrolysis using NaOH solution afforded
the key starting material 2-(2-oxo-2H-chromen-4-yloxy)propanoic
acid (4). Further amidation of the compound 4 with different
amines yielded N-substituted-2-(2-oxo-2H-chromen-4-yloxy)pro-
panamide 5 (Table 1). This reaction was carried out in DMF using
the coupling reagent HATU [2-(7-aza-1H- benzotriazole-1-yl)-
O
O
O
O
O
O
O
O
b
a
OEt
+
Br
O
O
O
O
OH
OEt
OH
1
2
3
4
c
O
H
N
O
Alkyl/
(Het)aryl
O
5
Scheme 1. Reagents and conditions: (a) K2CO3, acetone, reflux, 5 h, 98%; (b) NaOH then HCl, EtOH, 2 h, reflux, 99%; (c) amine, HATU, DMF, room temp, 12 h.

D. Rambabu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6745–6749
6747
Table 1
Preparation of coumarin derivatives (5) from 2-(2-oxo-2H-chromen-4-yloxy)propanoic acid (4) (Scheme 1)a
Entry
Amine
Product (5)
Yieldb (%)
Cl
O
O
O
Cl
1
92
O
H2N
H2N
N
H
5a
O
O
O
O
2
90
N
H
5b
5c
5d
5e
F
F
O
O
O
O
F
F
H2N
3
4
5
86
95
87
N
H
O
O
O
O
O
O
H2N
N
H
NC
CF3
O
O
O
O
CF3
NC
H2N
N
H
O
O
O
O
HN
HN
6
7
8
9
86
84
81
N
N
5f
O
O
O
O
O
O
5g
MeO2C
H2N
O
O
O
CO2Me
O
N
H
5h
5i
O
O
O
O
O
NH
O
90
N
NH2
H
NH
(continued on next page)

6748
D. Rambabu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6745–6749
Table 1 (continued)
Entry
Amine
Product (5)
Yieldb (%)
O
O
O
O
O
O
O
N
N
10
11
92
O
H2N
H2N
N
H
5j
O
O
O
O
N
N
85
N
H
5k
NH2
O
O
O
O
CH3
NH
CH3
12
87
CH3
CH3
5l
a
All the reactions were carried out using compound 4 (1 mmol) and an appropriate amine (1.1 mmol) in the presence of HATU (1.1 mmol) in DMF (3.0 mL) for 12 h at room
temp.
b
Isolated yield.
Figure 3. ORTEP representation of the compound 3 (thermal ellipsoids are drawn at
50% probability level).
Figure 4. ORTEP representation of the compound 5l (thermal ellipsoids are drawn
at 50% probability level).
therefore was of further interest as the moderate or balanced
selectivity displayed by this compound might be beneficial from
the view point of adverse side effects shown by rofecoxib or
lumiracoxib.
Leu376, Leu377, Tyr390, Phe393 and Ile394 residues. In a similar
docking studies the binding site of celecoxib was found to be
formed by Phe188, Gln189, His193, Asp348, Phe349, Tyr371, Trp373
,
To understand the interaction of compound 5i with COX-2
enzyme, in silico docking studies were carried out (see ESI). The
docking analysis was performed to identify the key amino acid res-
idues involved in making interactions between the compound 5i
and the human COX-2 model. The compound 5i was docked suc-
cessfully in the human COX-2 homology model, which showed
good docking scores, that is, À9.48 kcal/mol. The docking program
AutoDock computes binding energy, RMSD and inhibition constant
(Ki) with respect to the docked molecule. The AutoDock computed
binding energy; RMSD and Ki values along with H-bond interacting
residues are presented in Table 3. The binding 3D conformation of
human COX-2 with compound 5i is shown in Figure 5. It was ob-
served that the interaction of 5i with human COX-2 occurred in a
Ser516, Gly519 and Ile520 residues indicating a different binding
pocket for celecoxib which perhaps justify the differences in poten-
cies of celecoxib and 5i in COX-2 inhibition.
In summary, a series of novel N-substituted 2-(2-oxo-2H-chro-
men-4-yloxy)propanamide derivatives were designed and ex-
plored as potential inhibitors of COX. These compounds were
synthesized from readily available 4-hydroxy coumarin via a sim-
ple and straightforward three step method. The molecular struc-
tures of two representative compounds were confirmed by single
crystal X-ray diffraction study. All the compounds synthesized
were evaluated for their cyclooxygenase (COX) inhibiting proper-
ties in vitro. The compound 5i showed balanced selectivity towards
COX-2 over COX-1 inhibition and good docking scores when
distinct site, formed by Phe188, Gln189, His193, Tyr371, Trp373
,

D. Rambabu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6745–6749
6749
Table 2
Acknowledgments
In vitro COX inhibition by N-substituted 2-(2-oxo-2H-chromen-4-yloxy)propanamide
derivatives (5)
The authors are thankful to the management of Institute of Life
Sciences for providing necessary facilities. D.R. thanks DST for
awarding a fellowship. N.M. thanks DST SERB (SR/FT/CS-141/
2010) for awarding the project and C.M. thanks CSIR for awarding
fellowship.
Entry
Compound
COX inhibition IC50
(
l
M)
COX-2/COX-1
Selectivity ratio
COX-1
COX-2
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
4.61
0
74.91
13.19
16.83
38.71
50.25
0
16.24
—
10.26
17.12
5.23
—
1.64
2.26
9.60
13.73
5.03
0
Supplementary data
69.94
8.25
13.90
—
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.082.
9
2.98
4.60
9.64
3.33
0.0067
15.0
1.02
0.33
2.74
5.43
2.53
7.16
0.0028
10
11
12
13
14
12.64
52.39
8.43
0.048
0.042
References and Notes
Indomethacin
Celecoxib
1. Kurumbail, R. G.; Kiefer, J. R.; Marnett, L. J. Curr. Opin. Struct. Biol 2001, 11, 752.
2. Marnett, L. J. Curr. Opin. Chem. Biol 2000, 4, 545.
3. Fitzpatrick, F. A. Curr. Pharm. Des. 2004, 10, 577.
4. Wang, M. T.; Honn, K. V.; Nie, D. Cancer Metastasis Rev. 2007, 26, 525.
5. Xu, X. C. Anticancer Drugs 2002, 13, 127.
6. Jiménez, P.; García, A.; Santander, S.; Piazuelo, E. Curr. Pharm. Des. 2007, 13,
2261.
Table 3
Docking results of compound N-(1-oxo-1,2-dihydroisoquinolin-5-yl)-2-(2-oxo-2H-
7. (a) Yasojima, K.; Tourtellotte, W. W.; McGeer, E. G.; McGeer, P. L. Neurology
2001, 57, 952; (b) Nivsarkar, M.; Banerjee, A.; Padh, H. Pharmacol. Rep. 2008, 60,
692; (c) Herschman, H. R. Biochim. Biophys. Acta 1996, 1299, 125; (d) Vane, J. R.;
Botting, R. M. Inflamm. Res. 1998, 47, S78; (e) Katori, M.; Majima, M. Inflamm.
Res. 2000, 49, 367; (f) Kawamori, T.; Rao, C. V.; Seibert, K.; Reddy, B. S. Cancer
Res. 1998, 58, 406.
chromen-4-yloxy)propanamide onto human COX-2
Compound
Binding energy
(kcal/mol)
Estimated inhibition
constant (Ki) (nm)
RMSD
(Å)
5i
À9.48
13.2
0.56
8. (a) Ramalho, T. C.; Rocha, M. V.; da Cunha, E. F.; Freitas, M. P. Expert Opin. Ther.
Pat. 2009, 19, 1193; (b) Mukherjee, D. M.; Nissen, S. E.; Topol, E. J. JAMA 2001,
286, 954; c Withdrawal of Market Authorization for Prexige http://www.hc-
sc.gc.ca/ahc-asc/media/advisories-avis/_2007/2007_141-eng.php; retrieved on
June 5, 2012.
9. (a) Shafi, S.; Alam, M. M.; Mulakayala, N.; Mulakayala, C.; Vanaja, G.; Kalle, A.
M.; Pallu, R.; Alam, M. S. Eur. J. Med. Chem. 2012, 49, 324; (b) Pal, M.; Madan, M.;
Padakanti, S.; Pattabiraman, V. R.; Kalleda, S.; Vanguri, A.; Mullangi, R.; Mamidi,
N. V. S. R.; Casturi, S. R.; Yeleswarapu, K. R. J. Med. Chem. 2003, 46, 3975; (c) Pal,
M.; Veeramaneni, V. R.; Nagabelli, M.; Kalleda, S. R.; Misra, P.; Casturi, S. R.;
Yeleswarapu, K. R. Bioorg. Med. Chem. Lett. 2003, 13, 1639; (d) Pal, M.;
Veeramaneni, V. R.; Kumar, S.; Vangoori, A.; Mullangi, R.; Misra, P.; Rajjak, S. A.;
Lohray, V. B.; Casturi, S. R.; Yeleswarapu, K. R. Lett. Drug Des. Disc. 2005, 2, 329;
(e) Khanna, S.; Madan, M.; Vangoori, A.; Banerjee, R.; Thaimattam, R.; Basha, S.
K. J. S.; Ramesh, M.; Casturi, S. R.; Pal, M. Bioorg. Med. Chem. 2006, 14, 4820.
10. Sandhya, B.; Giles, D.; Mathew, V.; Basavarajaswamy, G.; Abraham, R. Eur. J.
Med. Chem. 2011, 46, 4696.
11. Crystal data of 3: Molecular formula = C14H14O5, formula weight = 262.25,
Crystal system = Monoclinic, space group = P2(1)/n, a = 11.699 (4) Å, b = 8.056
(3)Å, c = 14.034 (5) Å, V = 1301.6 (8) Å3, T = 296(2) K, Z = 4, Dc = 1.339 mg mÀ3
,
l
(Mo-K
a
) = 0.09 mmÀ1
,
5053 reflections measured, 1659 independent
(I)], R1_obs = 0.040, Goodness
reflections, 1057 observed reflections [I > 2.0
r
of fit = 0.91. Crystallographic data (excluding structure factors) for 3 have been
deposited with the Cambridge Crystallographic Data Center as supplementary
publication number CCDC 871213.
12. Crystal data of 5l: Molecular formula = C24H29NO4, formula weight = 395.48,
ꢀ
Crystal system = Monoclinic, space group = P1, a = 7.324 (6) Å, b = 11.5596
(9) Å, c = 13.115 (10) Å, V = 1017.67 (14) Å3, T = 296(2) K, Z = 2, Dc =
1.291 mg mÀ3
,
l
(Mo-K
a
) = 0.09 mmÀ1
,
16,879 reflections measured, 4436
(I)], R1_obs =
independent reflections, 3891 observed reflections [I > 2.0
r
0.038, Goodness of fit = 0.90. Crystallographic data (excluding structure
factors) for 5l have been deposited with the Cambridge Crystallographic Data
Center as supplementary publication number CCDC 871214.
13. Copeland, R. A.; Williams, J. M.; Giannaras, J.; Nurnberg, S.; Covington, M.;
Pinto, D.; Pick, S.; Trzaskos, J. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11202.
14. (a) Egan, R. W.; Paxton, J., Jr.; Kuehl, F. A. J. Biol. Chem. 1976, 251, 7329; (b)
Pagels, W. R.; Sachs, R. J.; Marnett, L. J.; Dewitt, D. L.; Day, J. S.; Smith, W. L. J.
Biol. Chem. 1983, 258, 6517.
Figure 5. Docking of compound 5i into the human COX-2.
15. Briefly, the assay mixture containing Tris HCl buffer (100 mM, pH 8.0), hematin
(15 mM), EDTA (3 mM), enzyme (100 mg COX-1 or COX-2) and the test
compound was pre-incubated at 25 °C for 1 min and then the reaction was
initiated by the addition of arachidonic acid and TMPD, in total volume of 1 mL.
The enzyme activity was determined by estimating the velocity of TMPD
oxidation for the first 25 s of the reaction by following the increase in
absorbance at 603 nm. A low rate of nonenzymatic oxidation observed in the
absence of COX-1 and COX-2 was subtracted from the experimental value
while calculating the percent inhibition.
docked into the COX-2 protein. Overall, the coumarin framework
presented here could be an attractive template for the identifica-
tion of novel cyclooxygenase inhibitors and the corresponding
synthetic strategy described could be useful for generating diver-
sity based library of small molecules of potential pharmacological
interest.