Synthesis, characterization and evaluation of topical antiinflammatory activity of dimethyl 4-oxo-2,6-diphenylcyclohexane-1,1-dicarboxylate

Article abstract of DOI:10.14233/ajchem.2013.13013

An attempt was made to synthesize a topical preparation of an active compound that has a potent antiinflammatory activity. Dimethyl 4- oxo-2,6-diphenylcyclohexane-1,1-dicarboxylate (II) was prepared by aldol condensation of benzaldehyde and acetone followed by Michael addition of dimethyl malonate. The optimum concentration of the compound was determined by comparing the antiinflammatory effect of ointment preparations at different concentrations. The antiinflammatory effects were studied by using carrageenan-induced paw edema method in rat and xylene induced rat ear edema. Good effect was observed with ointment containing 4 % and 5 % of the compound II concentration. The results showed that the drug had an obvious antiinflammatory effect as an external preparation and the activity is comparable to that of the standard ointment.

Full text of DOI:10.14233/ajchem.2013.13013

Asian Journal of Chemistry; Vol. 25, No. 5 (2013), 2366-2368
http://dx.doi.org/10.14233/ajchem.2013.13013
Synthesis, Characterization and Evaluation of Topical Antiinflammatory
Activity of Dimethyl 4-Oxo-2,6-diphenylcyclohexane-1,1-dicarboxylate
MAZIN NADHIM MOUSA AL UGLA
Department of Pharmaceutical Chemistry and Pharmacognosy, College of Pharmacy, University of Basrah, Basrah, Iraq
Corresponding author: E-mail: malugla@yahoo.com
(Received: 4 November 2011;
Accepted: 5 November 2012)
AJC-12361
An attempt was made to synthesize a topical preparation of an active compound that has a potent antiinflammatory activity. Dimethyl 4-
oxo-2,6-diphenylcyclohexane-1,1-dicarboxylate (II) was prepared by aldol condensation of benzaldehyde and acetone followed by Michael
addition of dimethyl malonate. The optimum concentration of the compound was determined by comparing the antiinflammatory effect
of ointment preparations at different concentrations. The antiinflammatory effects were studied by using carrageenan-induced paw edema
method in rat and xylene induced rat ear edema. Good effect was observed with ointment containing 4 % and 5 % of the compound II
concentration. The results showed that the drug had an obvious antiinflammatory effect as an external preparation and the activity is
comparable to that of the standard ointment.
Key Words: Antiinflammatory, Carrageenan-induced edema, Xylene induced edema.
inflammation process causing pain. The inhibitory activities
of non-steroidal antiinflammatory drug ointment on inflamma-
tory responses were almost the same as those obtained by oral
administration of such non-steroidal antiinflammatory drug
and more potent than those of steroidal ointment3. So, the aim
of this study is to develop a compound that have antiinflam-
matory effect, applied topically to reduce the side effects.
INTRODUCTION
Inflammation is a localized protective response charac-
terized by injury or destruction of tissues, which serve to
destroy, dilute or sequester both the injurious agent and
injured tissue.
Antiinflammatory agents are used in the treatment of
inflammation. There are two main types of antiinflammatory
agents; glucocorticosteroids and non-steroidal antiinflamma-
tory drugs. The non-steroidal antiinflammatory drugs is so
named because they do not belong to the steroidal groups and
ceases discomfort by blocking the pathway of cyclo-oxygenase
enzyme that creates prostaglandins and lessen the pain in diffe-
rent parts of the body. While the steroids reduces the inflam-
mation by binding to cortisol receptors.
EXPERIMENTAL
Synthesis of (1E, 4E)-1,5-diphenylpenta-1.4-dien-3-one
(I): In a 100 mL beaker, place 10 mL of 3 M sodium hydroxide
solution, 16 mL of 95 % ethanol and 2.12 g (20 mmol) of
benzaldehyde, then add 0.58 g (10 mmol) of acetone to the
reaction mixture and shake the mixture. The benzaldehyde,
initially insoluble, goes into solution, resulting in a clear, pale-
yellow solution. After a minute it suddenly becomes cloudy
and a yellow precipitate product forms. Continue to shake the
beaker for the next 10 min. Remove the liquid from the beaker
and add 30 mL of water and shake it vigorously. Remove the
wash liquid and wash the crystals twice with 20-mL portions
of water and then filtration by vacuum, drying and recrystallized
from hot ethanol. The yield was ca. 78 % and the melting
point was 111-112 ºC.4
Many therapeutic agents from synthetic source have
advantages and disadvantages, which limits their usefulness
on long term basis. Similarly, non-steroidal antiinflammatory
drugs also show many side effects includes stomach ulcers,
bleeding ulcers, kidney dysfunction, constipation, dizziness
and headaches1. Thus, the application of the drug on the
affected site or the nearby area is reasonable, since drugs
produce effects after they reach the affected site. Topical non-
steroidal anti inflammatory drugs are applied on the skin in
the form of ointment, gel, cream or spray in the region where
pain is exist2. They have to penetrate the skin, enter tissues or
joints and at high concentration to have an effect on the
IR (KBr, νmax, cm-1): 3051.1 ( Ar. C-H stretching), 3029.9
(C-H stretching), 1649 (C=O stretching), 1591, 1496 (Ar. C=C
stretching), 883 (Ar. C-H out of plane).

Vol. 25, No. 5 (2013)
Synthesis andAntiinflammatoryActivity of Dimethyl 4-Oxo-2,6-diphenylcyclohexane-1,1-dicarboxylate 2367
O
O
CHO
O
NaOH
NaOH
2
+
H3C
CH3
H3C
O
O
Compound I
O
OCH3
OCH3
O
O
Compound II
O
H3C
TABLE-2
EFFECT OF TOPICAL APPLICATION OF THE FORMULATION ON CARRAGEENAN INDUCED PAW EDEMA IN RATS
Change in paw volume (in mL) after drug administration (Mean SEM)
Percentage inhibition of edema
Compound
1 h
2 h
3 h
4h
1 h
-
2 h
-
3 h
-
4h
-
Control
Standard
F1
0.71 0.02
0.42 0.03**
0.58 0.04
0.54 0.02*
0.52 0.01*
0.49 0.02**
0.47 0.04**
0.77 002.
0.43 0.04**
0.60 0.01
0.57 0.01*
0.55 0.01*
0.53 0.02**
0.50 0.04**
0.71 0.02
0.44 0.04**
0.57 0.05
0.53 0.04
0.51 0.04*
0.48 0.04**
0.47 0.04**
0.60 0.02
0.40 0.03**
0.49 0.03
0.48 0.04
0.48 0.04
0.47 0.04
0.46 0.04
40
19
24
27
30
34
44
22
26
28
31
35
38
21
26
29
33
34
33
19
20
20
22
24
F2
F3
F4
F5
SEM- Standard error mean, *P < 0.05; **P < 0.01
Synthesis of dimethyl 4-oxo-2,6-diphenylcyclohexane-
1,1-dicarboxylate (II): Dissolve 1 g (4.46 mmol) of compound
I in methanol, to which add dimethyl malonate 0.6 g (4.5
mmol) and 3 mL 25 % sodium hydroxide solution and reflux
for 5 min, then left for it over night, cool and collect the
product by vacuum filtration and recrystallized from ethanol
and water. The yield was about 81 % and the melting point
was 292-295 ºC.
IR (KBr, νmax cm-1): 3047 ( Ar. C-H stretching), 2873
(C-H stretching CH3), 2851 (C-H stretching CH2) 1735 (ester
C=O), 1672 (keton C=O), 1587, 1501 (Ar. C=C stretching),
1370 (C-H bending CH3), 1105 (C-O stretching), 879 (Ar. C-
H out of plane).
1H NMR (DMSO): δ 7.39 (4H), δ 7.31 (4H), δ 7.26 (2H),
δ 4,34 (2H), δ 3.66 (6H), δ 2,61 (4 2H), C22H22O5 (m.w. 366):
Elemental analysis (%) (Calcd.) C, 72.12; H, 6.05; (Found) C,
72.3; H, 6.09.
Method of preparation of ointment5: Ointment was
prepared by melting together petrolatum, polyethylene glycol-
3000, propylene glycol and menthol on a hot plate (70 ºC).
The compound II was dissolved in it under stirring and cooled.
Various compositions of ointment formulations were mentioned
in the Table-1.
ment (0.1 % diclofenac sodium in ointment base). Remaining
groups were treated with formulated ointment (F1-F5). 0.2 g
of ointment was applied to the plantar surface of the hind paw
by gentle rubbing 50 times with index finger. After 30 min,
0.1 mL of 1 % w/v of carrageenan was injected in the plantar
region of the left paw of rats. The paw volumes were noted for
1, 2, 3 and 4 h after carrageenan injection. The results were
tabulated in Table-2. The percentage edema inhibition by the
drug was calculated by the formula:
Percentage edema inhibition = [1-( Vt / Vc)] × 100 %
where, Vt: is edema volume in drug treated group. Vc: is edema
volume in the control group.
TABLE-1
COMPOSITION USED IN THE STUDY
TO PREPARE THE OINTMENTS
Ingredients (g)
Tested compound
Polyethyleneglycol-3000
Propylene glycol
Menthol
F1
0.1
0.6
0.4
0.5
q.s.
F2
0.2
0.6
0.4
0.5
q.s.
F3
0.3
0.6
0.4
0.5
q.s.
F4
0.4
0.6
0.4
0.5
q.s.
F5
0.5
0.6
0.4
0.5
q.s.
Petroleum
to 10 to 10 to 10 to 10 to 10
SEM-Standard error mean, *P < 0.05; **P < 0.01
Antiinflammatory activity
Xylene induced rat ear edema: The effect of the product
on acute edema was assessed by using xylene induced ear
edema in rats as described by Dkhil et al.7. The animals were
divided into six groups each of six animals. 50 µL of xylene
was applied to the anterior and posterior surfaces of the two
ears under light ether anesthesia. After 15 mins, 0.1 g of the
formulated ointment (F1-F5) were applied on the right ears of
five groups,the sixth group receive 0.1 g diclofenac sodium
Carrageenan-induced rat paw edema: Antiinflammatory
activity was carried out using carrageenan-induced rat paw
edema6. The rats weighing between 150-200 g were selected
randomly excluding pregnant female rats for the test.Animals
were divided into different groups with six animals in each
group. One group of rats were treated with ointment base which
served as control, other group was treated with standard oint-

2368 Al Ugla
Asian J. Chem.
(standard). The left ear was considered as control and receive
ointment base only. After 4 h, xylene application rats were
sacrificed and both ears were removed. Ear lobes were punched
out in circular disc using metal punch (6 mm diameter) and
weighed. The difference in the weight of discs from right
treated and left untreated was calculated and was used as
measure of edema. The results were tabulated in Table- 3. The
level of percentage inhibition was calculated using the formula:
Percentage inhibition (%) = [(control-treated)/control] × 100%
Statistical analysis: The results were expressed in mean
SEM. The data from experiments were analyzed separately
using one-way Anova followed by Dunnett test was used
to determine significant difference between the groups and
p < 0.05 was considered significant.
Carrageenan-induced paw edema in rat and xylene induced
rat ear edema has been accepted as a useful phlogistic tool for
investigating antiinflammatory agents. It is suggested that there
are biphasic effects in carrageenan-induced edema. The early
hyperemia results from the release of histamine and serotonin,
the delayed phase of carrageenan-induced edema results
mainly from the potentiating effects of bradykinin on mediator
release and also from prostaglandins which produce edema
after the mobilization of leukocytes8.
The edema was reached its highest thickness 4 h after the
application of the stimulus9.
The compound II promoted a significant and dose-
dependent inhibition of xylene induced skin inflammation.
Topically applied xylene resulted in activation of pro-inflam-
matory mediators that promoted the manifestation of several
inflammatory parameters similar to some skin disorders10.
Using this model, compounds that inhibit this process can be
target in the search for new therapeutic strategies.
TABLE-3
EFFECT OF TOPICAL APPLICATION OF THE FORMULATION
ON XYLENE INDUCED RATS EAR EDMA MODEL
Treatment groups
Ear edema (mg)
(Mean SEM)
8.33 0.61
3.50 0.22
7.31 0.55
6.52 0.32
5.47 0.37
4.13 0.28
3.95 0.23
Inhibition (%)
Conclusion
Control
Standard
F1
-
From this result, it is concluded that topical preparation
containing 5 % possesses good antiinflammatory activity which
can be used for the treatment of topical inflammation.
57.98
12.2
F2
21.7*
34.3*
50.4**
52.6**
F3
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F4
F5
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vol. 2, no.3, pp. 1855-1860, July-Sept (2010).
SEM- Standard error mean, *P < 0.05; **P < 0.01
RESULTS AND DISCUSSION
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3. J.H. Vaile and P. Davis, Drugs, 56, 783 (1998).
Statistical analysis showed that, the edema inhibition was
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effect was more for the formulations containing 4 % and 5 %
of the tested compound.
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Antiinflammatory activity was carried out for the formu-
lated compounds using carrageenan-induced rat paw edema
method and xylene induced rat ear edema.