Synthesis, anti-inflammatory, ulcerogenic and cyclooxygenase activities of indenopyrimidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.12.088

Objective of the present work was to evaluate the anti-inflammatory, ulcerogenicity and cyclooxygenase activity of indenopyrimidine derivatives. Anti-inflammatory activity of the tested compounds is investigated by carrageenan-induced rat paw edema assay.

Full text of DOI:10.1016/j.bmcl.2015.12.088

Accepted Manuscript
Synthesis, anti-inflammatory, ulcerogenic and cyclooxygenase activities of in-
denopyrimidine derivatives
Santosh S. Undare, Navanath J. Valekar, Ajinkya A. Patravale, Dattatraya. K.
Jamale, Sunil S. Vibhute, Laxman S. Walekar, Govind B. Kolekar, M.B.
Deshmukh, Prashant V. Anbhule
PII:
S0960-894X(15)30401-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.088
BMCL 23449
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 July 2015
2 December 2015
25 December 2015
Please cite this article as: Undare, S.S., Valekar, N.J., Patravale, A.A., Jamale, a.K., Vibhute, S.S., Walekar, L.S.,
Kolekar, G.B., Deshmukh, M.B., Anbhule, P.V., Synthesis, anti-inflammatory, ulcerogenic and cyclooxygenase
activities of indenopyrimidine derivatives, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/
10.1016/j.bmcl.2015.12.088
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Synthesis, anti-inflammatory, ulcerogenic and cyclooxygenase activities of
indenopyrimidine derivatives
Santosh S. Undarea, Navanath J. Valekara, Ajinkya A. Patravalea, Dattatraya. K. Jamaleb, Sunil
S. Vibhuteb, Laxman S. Walekara, Govind B. Kolekara, M. B. Deshmukha and Prashant V.
Anbhulea*
a Medicinal Chemistry Research Lab., Department of Chemistry, Shivaji University, Kolhapur,
416004, M.S., India
b Shri Shivaji Mahavidyalaya, Barshi, M.S., India
__________________________________________________________________________
*Corresponding Author
E-mail: pvanbhule@gmail.com
Tel.: +91 231 260 9169; fax: +91 231 2692333
1

Abstract
Objective of the present work was to evaluate the anti-inflammatory, ulcerogenicity and
cyclooxygenase activity of indenopyrimidine derivatives. Anti-inflammatory activity of the
tested compounds is investigated by carrageenan-induced rat paw edema assay. Compounds A1,
A6, A7 and A12 exhibit the comparable anti-inflammatory activity (79.33-81.33 %) to the
standard drug diclofenac sodium (85.33%), while A6, A7, A9, A12 and A14 show better ulcer
index than the reference standard diclofenac sodium. To rationalize the anti-inflammatory
activity, docking experiments are performed to study the ability of these compounds to bind into
the active site of COX-2 enzyme.
Keywords Indenopyrimidine; anti-inflammatory; anti-ulcerogenic; COX-2; molecular docking
2

Non-steroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of pain and
inflammation and arthritis.1 Prostaglandins are produced within the body by the enzyme
cyclooxygenase which can exist in cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)
forms. COX-1 and COX-2 catalyze arachidonic acid to its own prostaglandins but they differ in
their physiological functions.2-5 COX-1 plays an important role in physiological functions while
pro-inflammatory COX-2 present at the site of inflammation and also in central nervous system
which is mainly responsible for inflammation.6
Most of the presently used NSAIDs exert their anti-inflammatory activity by inhibition of
both isoforms of cyclooxygenase enzymes.7-9 Some of the reported nonselective NSAIDs allied
with side effects such as gastric ulcer, gastrointestinal bleeding and cardiovascular effects.10-13
The selective COX-2 inhibitors such as Rofecoxib and Valdecoxib are withdrawn from market
because of their cardiovascular side effects.14 Therefore, there is need to develop safer and
selective COX-2 inhibitors to overcome the problems associated with current drugs.
Pyrimidine heterocycles plays an important role in diversity oriented synthesis,
combinatorial synthesis and medicinal chemistry because of their noteworthy various
pharmacological and biological properties15-26 and are highly studied for their anti-inflammatory
activities.27-28
In view of the facts mentioned above and in continuation of our ongoing efforts towards
the development of new bioactive molecule,29 we evaluated in vivo assessment of anti-
inflammatory, ulcerogenicity and in vitro COX-2/COX-1 selectivity study of indenopyrimidine
derivatives for the development of non-ulcerogenic anti-inflammatory candidates. On the basis
of the docking score, seven compounds were selected and screened for their anti-inflammatory,
ulcerogenicity and cyclooxygenase activity.
3

Recently, we have successfully reported the synthesis and anti-cancer activity against
breast cancer cell lines of indenopyrimidine derivatives. Based on the docking study of the said
compounds, we have decided to explore the anti-inflammatory activity of the same compounds.
The compounds used here are synthesized by our own method30 and fully characterized by
spectroscopic techniques. In fact, sixteen compounds have been synthesized, including six
compounds are novel one as shown in Scheme 1 and given in table 1.
<Scheme 1>
< Table 1>
In the Pharmacological study, all the synthesized compounds were docked into the active
site of cyclooxygenase enzyme for the indication of credible anti-inflammatory activity. We have
investigated anti-inflammatory, ulcerogenicity and cyclooxygenase activity of some selected
derivatives of 2-amino-4-phenyl-5H-indeno [1, 2-d] pyrimidine-5-one.
Anti-inflammatory activities of selected seven compounds were evaluated by
carrageenan- induced rat paw edema assay.31-32 The compounds were tested at 25 and 50 mg/kg
in albino Wistar rats and compared with reference drug diclofenac at 30 mg/kg. The tested
compounds showed anti-inflammatory activity ranging from 78.00 to 81.33% inhibition as
compared to diclofenac (85.33%) and presented in Table 2. Among the tested compounds of 2-
amino-4-phenyl-5H-indeno [1, 2-d] pyrimidine-5-one derivatives, A1, A7 and A12 showed best
activity (79.33-80.66 %). The compound A6 showed highest activity (81.33%). The results also
showed that there is no significant effect of the substituent present on the phenyl ring at fourth
position. The animal studies were approved by the Institutional Animal Ethics Committee
(IAEC) of SCAN research Laboratory, Bhopal, (MP) constituted for the purpose of control and
4

supervision of experimental animals by Ministry of Environment and Forests, Government of
India, New Delhi, India.
< Table 2>
After evaluating the anti-inflammatory activity, ulcerogenic activity was screened by
reported method.33-34 The tested compounds and Diclofenac sodium were given orally to rats and
they were sacrificed for the evolution of ulcerogenic activity. The stomach was removed, opened
along the greater curvature, washed with distilled water and finally rinsed gently with normal
saline. The number of mucosal damage for each stomach was examined using magnifying lens
for the macroscopically visible lesions. It was counted and their ulcerogenic severity was
assessed according to the grading system. The ulcer index of A1, A6, A7, A9, A11, A12 and
A14 compounds was calculated and presented in Table 3 and figure 1.
All the tested compounds showed reduction in ulcerogenic activity ranging from 2.80 ±
0.14 to 3.00 ± 0.16 as compared to standard drug Diclofenac 5.70 ± 0.17. Ulcerogenic effect was
compared to a classical NSAID, diclofenac sodium. Results revealed that compounds A6, A7,
A12 and A14 shows good ulcerogenic activity than diclofenac sodium. Compound A9 having
three methoxy substituent showed maximum reduction in ulcerogenic activity (2.80 ± 0.14).
< Table 3>
< Figure 1>
The ability of the test compounds A1, A6, A7, A9, A11, A12 and A14 to inhibit COX-1
and COX-2 was assayed using cyclooxygenase ovine inhibitor screening kit (catalogue number
560131, Cayman chemical, Ann Arbor, MI, USA) by the method of Gierse et al.35 The ratio of
IC50 of COX-2 to IC50 of COX-1 (COX-2/COX-1) of the above compounds showed that
5

compounds A7, A9, A11, A12 and A14 are selective COX-2 inhibitor with a ratio of 0.70, 0.69,
0.70, 0.69 and 0.68 respectively Table 4.
< Table 4>
Significant anti-inflammatory activity of synthesized compounds prompted us to perform
molecular docking studies of compounds A1, A6, A7, A9, A11, A12 and A14 to understand the
ligand–protein interactions and cyclooxygenase-2 (COX-2) selectivity in detail. Automated
docking analysis of the synthesized derivatives was carried out using biopredicta module of the
V Life MDS 4.3. The crystal structure of COX-2 (PDB: 1CX2) and COX-1 (PDB: 2OYE) were
used for docking study. As per the docking study with 1CX2 the following results can be drawn.
A6 is the active compound in the series showed five hydrogen bond interactions with ALA 527
(1.8A0), VAL 523 (2.2A0), HIS 90 (1.8A0, 2.2A0, 2.0A0) and two aromatic interactions TYR 348
(5A0) and PHE 518 (5.2A0). All the active molecules A1, A6, A7, A9, A11, A12, and A14
showed one common interaction with the hydrogen of the NH2 of Arg 513 with a bond distance
of 1.9, 2.2, 2.4, 1.7, 2.4, 1.9 and 1.8 A0 respectively, while A12 showed hydrogen bond
interactions with LEU 352 (2.4 A0), ARG 513 (1.9 A0) and aromatic interactions with HIS 90
(4.4 A0). Docking results are a clear indication that substitution on the phenyl ring does not
contribute significantly towards the binding of the molecules with COX-II. Derivatives with the
free amino groups at R1 are showing significant interaction with the COX-II which also helps to
two phenyl ring to accommodate parallel conformation with respect to the heterocyclic amino
acids like HIS 90 which are located on the upper half of the receptor to form aromatic
interactions. Pharmacophoric identification of the synthesized derivatives indicated four point
pharmacophore consisting of three hydrogen bond accepter and two aromatic features which
6

show synthesized derivatives, are binding with the COX-II receptor via the creation of hydrogen
bonds and aromatic interaction.
< Figure 2>
< Figure 3>
Synthesized molecules were screened for the drug likeness using Lipinski ‘Rule of Five’
and oral absorption of the lead molecules was also calculated using Molinspiration
Chemoinformatics server. All the seven molecules screened are having acceptable drug like
properties and none of them is violating the Lipinski rule. A6, A12 and A14 molecules are
having good % oral absorption greater than 85 %, which shows these molecules can be orally
active. Table 5
<Table 5>
The tested indenopyrimidine derivatives showed better anti-inflammatory activity with minimum
ulcerogenic activity than the Diclofenac sodium. Also, the ratio of COX-2/COX-1 suggests the
selectivity of compounds towards COX-2. The compound A14 was a potent, selective inhibitor
to COX-2 than COX-1.
Acknowledgement
The author Santosh S. Undare is thankful to UGC (WRO), Pune for the award of Teacher
fellowship under XII plan. One of the author (PVA) sincerely acknowledges the Principal,
Bharati Vidyapeeth, College of Pharmacy, Kolhapur for providing docking analysis.
7

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10

Index of Tables and Figures
Table 1 Synthesis of indenopyrimidine derivatives
Table 2 Effect of compounds on paw edema induced by carrageen in rats
Table 3 Ulcer index of tested compounds of the most active compounds and diclofenac sodium
Figure1 Determination of ulcer index- diclofenac-induced gastric ulceration in rats
Table 4 COX-2/COX-1 ratio of tested compounds and Celecoxib
Figure 2 Docking of compounds into the active site of COX-2
Figure 3 Pharmacophoric hypothesis of the synthesized compounds
Table 5 Pharmacokinetic properties for good oral bioavailability of selected compounds
Scheme 1 General reaction for the synthesis of indenopyrimidine derivatives
11

Table 1 Synthesis of indenopyrimidine derivatives
0
Entry
R
R1
Time (hrs)
(hrs)
7
Yield
( % )
80
mp C
Obs.
Lit
A1
A2
H
NH2
C6H5
C6H5
C6H5
C6H5
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
89-91
202-204
>300
90
3,4-(OCH3)2
3,4,5-(OCH3)3
4-N(CH3)2
4-NO2
8
75
A3
8.10
8
78
A4
80
210-212
260-262
>300
A5
7.5
8
83
A6
4-NO2
84
>300
272
178
240
A7
3-NO2
7.4
8.2
8.3
8.4
7.5
8.2
8.3
8.2
8.4
8.1
86
271-273
177-179
238-240
260
A8
3,4-(OCH3)2
3,4,5-(OCH3)3
4-N(CH3)2
4-Cl
82
A9
80
A10
A11
A12
A13
A14
A15
A16
81
86
242-244
207-209
99-101
168-171
185-187
193-195
244
210
100
4-Br
83
4-OCH3
80
3-OCH3
79
Pyridine-3-yl
Thiophen-2-yl
81
186
194
78
12

Table 2 Effect of compounds on paw edema induced by carrageen in rats
Treatment
Dose (mg/kg)
Mean differences in
Paw Volume (ml)
Percentage of
Inhibition (%)
Control
Diclofenac
A1
0.1 ml of 1% (w/v)
--
1.50 ± 0.05
0.97± 0.05*
1.10 ± 0.05
1.04 ± 0.06*
1.09 ± 0.05
1.03± 0.04*
1.11 ± 0.06
1.06 ± 0.05*
1.12 ± 0.07
1.07 ± 0.07*
1.11 ± 0.06
1.08± 0.07*
1.10 ± 0.08
1.05± 0.06*
1.12± 0.06
1.08± 0.04*
30
25
50
25
50
25
50
25
50
25
50
25
50
25
50
85.33
76.66
80.66
77.33
81.33
76.00
79.33
75.33
78.66
76.00
78.00
76.66
80.00
75.33
78.00
A6
A7
A9
A11
A12
A14
Each data suggests Mean ± SEM (n=6). One-way ANOVA using Dunnett’s test is applied for
statistical analysis, treatment groups compared with Control group.
Significant at* p < 0.01, compared to control group.
13

Table 3 Ulcer index of tested compounds of the most active compounds and diclofenac sodium
Treatment
Dose (mg/kg)
No. of animals
with ulcer
5/5
Ulcer
Index
Diclofenac
A1
100
50
50
50
50
50
50
50
5.70 ± 0.17
4/5
4/5
4/5
4/5
4/5
4/5
4/5
3.00 ± 0.16
2.90 ± 0.15
2.90 ± 0.16
2.80 ± 0.14
3.00 ± 0.17
2.90 ± 0.15
2.90 ± 0.15
A6
A7
A9
A11
A12
A14
Each data suggests Mean ± SEM (n=6). One-way ANOVA using Dunnett’s test is applied for
statistical analysis, Treatment groups compared with Control group.
Significant at* p < 0.01, compared to control group.
14

Figure1 Determination of ulcer index- diclofenac-induced gastric ulceration in rats
15

Table 4 COX-2/COX-1 ratio of tested compounds and Celecoxib
Treatment
Celecoxib
A1
Dose (mg/kg)
COX-2/COX-1
0.04
100
25
50
25
50
25
50
25
50
25
50
25
50
25
0.44
0.60
A6
0.40
0.66
A7
0.45
0.70
A9
0.47
0.69
A11
A12
A14
0.48
0.70
0.50
0.69
0.41
16

50
0.68
17

Figure 2 Docking of compounds into the active site of COX-2
18

Figure 3 Pharmacophoric hypothesis of the synthesized compounds
19

Table 5 Pharmacokinetic properties for good oral bioavailability of selected compounds
H-
H-Donor Rotatable
Entry
MW
acceptor count
count
bond
count
4
XlogP
% oral
absorption
--
Diclofenac
sodium*
318.13
3
1
1.45
A1
A6
273.29
318.29
318.29
363.37
377.44
352.19
303.32
2
5
5
5
2
2
3
1
1
1
1
0
1
1
2
3
3
8
5
2
4
2.721
2.593
2.593
2.61
82.0520
87.8446
69.4285
69.4285
75.6799
85.2364
85.2364
A7
A9
A11
A12
A14
5.125
3.517
2.684
*Reference no: 38-39.
20

Scheme 1 General reaction for the synthesis of indenopyrimidine derivatives
R
CHO
O
NH
H2O + EtOH
NaOH, heat
O
R1
+
+
N
NH2
R
O
N
R1
3a: R1= Ph
3b: R1= NH2
4
2
1
21

Grapical Abstract
The synthesized indenopyrimidine derivatives were screened for their anti-inflammatory,
ulcerogenic and cyclooxygenase activities.
22