Synthetically modified bioisosteres of salicyl alcohol and their gastroulcerogenic assessment versus aspirin: Biochemical and histological correlates

Article abstract of DOI:10.1007/s00210-013-0941-5

The present study was conducted to synthesize nitrogen containing derivatives of salicyl alcohol and to investigate in vivo their ulcerogenic potential in comparison with aspirin in rats. The compounds [4-(2-hydroxybenzyl) morpholin-4-iumchloride (I)] and [1,4-bis(2-hydroxybenzyl) piperazine-1,4-diium chloride (II)] were synthesized and their chemical structures were characterized using spectral data. In our previous study (Ali et al., Afr J Pharm Pharmacol 7:585-596, 2013), both compounds showed anti-inflammatory, antinociceptive, and antipyretic properties in standard animal models and a greater binding affinity for cyclooxygenase-2 versus cyclooxygenase-1 in molecular docking and dynamics analysis. For in vivo studies, animals were randomly divided into four groups. The synthetic compounds (both at 100 or 150 mg/kg), aspirin (150 mg/kg), or saline vehicle was administered orally, once daily for 6 days and then tested for ulcerogenic activity. At the end of the procedure, gastric juice and tissues were collected and subjected to biochemical and histological analyses. The results of the study revealed that in the case of the aspirin-treated group, there was a significant increase in gastric juice volume, free acidity, total acidity, and ulcer score and a decrease in gastric pH. Moreover, histological examination of the gastric mucosa of the aspirin-treated group indicated morphological changes while neither of the synthetic compounds showed any significant ulcerogenic or cytotoxic properties. The results of the present study suggest that both compounds are free from ulcerogenic side effects and may represent a better alternative to aspirin.

Full text of DOI:10.1007/s00210-013-0941-5

Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:281–290
DOI 10.1007/s00210-013-0941-5
ORIGINAL ARTICLE
Synthetically modified bioisosteres of salicyl alcohol
and their gastroulcerogenic assessment versus aspirin:
biochemical and histological correlates
Gowhar Ali & Fazal Subhan & Nazar Ul Islam &
Nasir Ullah & Robert D. E. Sewell & Muhammad Shahid &
Ikhtiar Khan
Received: 24 August 2013 /Accepted: 13 November 2013 /Published online: 29 November 2013
#
Springer-Verlag Berlin Heidelberg 2013
Abstract The present study was conducted to synthesize
nitrogen containing derivatives of salicyl alcohol and to in-
vestigate in vivo their ulcerogenic potential in comparison
with aspirin in rats. The compounds [4-(2-hydroxybenzyl)
morpholin-4-iumchloride (I)] and [1,4-bis(2-hydroxybenzyl)
piperazine-1,4-diium chloride (II)] were synthesized and their
chemical structures were characterized using spectral data. In
our previous study (Ali et al., Afr J Pharm Pharmacol 7:585–
596, 2013), both compounds showed anti-inflammatory,
antinociceptive, and antipyretic properties in standard animal
models and a greater binding affinity for cyclooxygenase-2
versus cyclooxygenase-1 in molecular docking and dynamics
analysis. For in vivo studies, animals were randomly divided
into four groups. The synthetic compounds (both at 100 or
150 mg/kg), aspirin (150 mg/kg), or saline vehicle was ad-
ministered orally, once daily for 6 days and then tested for
ulcerogenic activity. At the end of the procedure, gastric juice
and tissues were collected and subjected to biochemical and
histological analyses. The results of the study revealed that in
the case of the aspirin-treated group, there was a significant
increase in gastric juice volume, free acidity, total acidity, and
ulcer score and a decrease in gastric pH. Moreover, histolog-
ical examination of the gastric mucosa of the aspirin-treated
group indicated morphological changes while neither of the
synthetic compounds showed any significant ulcerogenic or
cytotoxic properties. The results of the present study suggest
that both compounds are free from ulcerogenic side effects
and may represent a better alternative to aspirin.
.
Keywords Bioisosteric synthesis [4-(2-Hydroxybenzyl)
.
morpholin-4-ium chloride] [1,4-bis(2-Hydroxybenzyl)
.
.
piperazine-1,4-diium chloride] Acetyl salicylic acid
.
Ulcerogenicity Histology
Introduction
Studies on the pharmacological mechanism(s) underlying non-
steroidal anti-inflammatory drugs (NSAIDs) have established
an inhibitory link with cycloxygenase (COX) enzyme activity
(Amir and Shikha 2004; Smith et al. 1998). There are three
isoforms of the COX enzyme (Dannhardt and Kiefer 2001;
Marnett and Kalgutkar 1999), namely, COX-1, COX-2, and
COX-3; the latter is a splice variant of COX-1 not thought to be
expressed in humans (Warner and Mitchell 2004).
:
:
G. Ali F. Subhan (*) M. Shahid
Department of Pharmacy, University of Peshawar,
Peshawar 25120, Pakistan
Pain, inflammation, and pyrexia frequently occur in com-
bination, implicating common mediating pathways. So, it is
not surprising that inhibitors of COX enzymes are used to treat
these clinical disorders. COX-1 has a housekeeping or
gastroprotective function while COX-2 is inducible and is
predominantly involved in inflammation, pyrexia, and pain
(Almansa et al. 2003; Habeeb et al. 2001). The majority of the
NSAIDs either inhibit all forms of COX enzyme
nonselectively (Tapiero et al. 2002) or have greater affinity
for COX-1 compared to COX-2 (Jackson and Hawkey 1999).
This is thought to be the origin of serious adverse effects such
e-mail: fazal_subhan@upesh.edu.pk
N. U. Islam
Sarhad University of Sciences and Information Technology,
Peshawar 25000, Pakistan
R. D. E. Sewell
Cardiff School of Pharmacy and Pharmaceutical Sciences,
Cardiff University, Cardiff CF10 3NU, UK
:
:
N. U. Islam N. Ullah I. Khan
Institute of Chemical Sciences, University of Peshawar,
Peshawar 25120, Pakistan

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as peptic ulcer, perforation, and gastrointestinal bleeding
(Bulbena et al. 1993; Paulus and Harold 1988; Singh and
Rosen 1998) associated with the use of NSAIDs, and this
imposes a major limitation of their therapeutic application
(Tammara et al. 1994).
Currently, peptic ulcer is a major global health problem and
the search for novel plant-derived or synthetic NSAID mole-
cules devoid of ulcerogenic propensity is an active area of
investigation for biomedical researchers (Tammara et al.
1994). Certain biochemical actions of drugs are clinically
advantageous in the diagnosis or treatment of disorders, yet
others may be detrimental giving rise to adverse effects.
Hence, alterations in chemical structure are necessary to en-
hance beneficial properties.
Methods and materials
Chemicals
Bovine serum albumin and acetyl salicylic acid were obtained
from Sigma. The starting materials were either purchased from
Aldrich and Sigma or prepared by standard procedures avail-
able in the literature. All other chemicals used were of analytical
grade and acquired commercially from local sources.
Chemistry
General
A range of biochemical activities have been reported for
glycosides and it is principally the genin (aglycone) moiety
which is responsible for these actions (Robyt 1998; Salga
et al. 2011; Yoon et al. 2004). Salicin (2-(hydroxymethyl)
phenyl-β-D-glucopyranoside) is a glycoside chemically related
to aspirin which may be hydrolyzed to yield salicyl alcohol
(2-hydroxy methyl phenol or saligenin) (Table 1). Salicin has
been reported to be an anti-rheumatic, anti-inflammatory, and
antipyretic pro-drug with no propensity to induce gastric damage
(Akao et al. 2002; Yoon et al. 2004). These properties of salicin
which is devoid of a –COOH group prompted the bioisosteric
synthesis of new compounds lacking this group with a targeted
pharmacological profile. Evidence regarding anesthetic as well
as antipyretic activities of salicin have been reported (Charles
and Tony 1956; Machagan 1876). The compounds under cur-
rent study are bioisosteres derived from salicyl alcohol, via
incorporation of pharmacologically important morpholine and
piperazine moieties. Other compounds comprising such moie-
ties inhibit eicosanoid pathways and exhibit varied pharmaco-
logical properties including antiproliferative activities (Berardi
et al. 2008; Coonrod et al. 2001; Nozawa et al. 2007). In this
regard, the piperazine moiety has been proposed as a critical core
for novel drug design (Bali et al. 2010) and morpholine substi-
tution in heterocyclics has yielded both anti-inflammatory and
antinociceptive properties (Panneerselvam et al. 2009).
Bearing in mind the concept of salicin as a prodrug and the
potential for pharmacological properties of compounds with
morpholine or piperazine substitution, we performed the
bioisosteric synthesis of compounds I [4-(2-hydroxybenzyl)
morpholin-4-iumchloride] and II [1,4-bis(2-hydroxybenzyl)
piperazine-1,4-diiumchloride] (Table 1, Fig. 1) and evaluated
their ulcerogenic propensity.
In light of this, it is noteworthy that we have recently reported
these compounds to possess significant antinociceptive, anti-
inflammatory, and anti-pyretic activities comparable to aspirin
in animal models which were supported by molecular docking
and dynamics simulation studies (Ali et al. 2013). Consequently,
we now wished to establish the gastrointestinal tolerability of
these compounds in comparison with aspirin.
Infrared spectra (IR) were determined on a Nicolet 380
thermoscientific FTIR. Mass spectra were recorded on a JEOL
MSRoute spectrometer and nuclear magnetic resonance (NMR)
spectra scanned on a BRUKER 300-MHz spectrophotometer
using D2O as a solvent. Melting points (mp) of compounds were
determined using a Gallenkamp Melting Point apparatus. The
chemical shifts were presented as parts per million (δ ppm)
using tetramethylsilane (TMS) as an internal standard.
Synthesis
Compound I [4-(2-hydroxybenzyl)morpholin-4-ium chloride]
and compound II [1,4-bis(2-hydroxybenzyl)piperazine-1,4-
diium chloride] were synthesized (Fig. 1) using salicylaldehyde
as a starting material according to the following synthetic
protocol. A mixture of NaBH4 (10 mmol), salicylaldehyde
(10 mmol), and boric acid (10 mmol) was milled with mortar
and pestle until thin layer chromatography exhibited entire
vanishing of the starting material. The reaction mixture was
quenched with a 10-mL solution of 1 N HCl and extracted with
ethyl acetate. The ethyl acetate layer was separated and dried
over sodium sulfate. On evaporation of the solvent, pure salicyl
alcohol was obtained, which was used in further reactions
without purification.
Thionyl chloride (1.19 g, 10 mmol) was slowly added to a
solution of salicyl alcohol (1.24 g, 10 mmol) in dichlorometh-
ane at room temperature. After the addition of thionyl chlo-
ride, the reaction mixture was refluxed for 2 h. The reaction
mixture was cooled to 0 °C and amine (10 mmol) was slowly
added to it. The product was precipitated as a white powder.
The product was filtered, washed with dichloromethane, and
kept under vacuum for 24 h. Their chemical structures were
confirmed from IR, 1H and 13C NMR, and MS data.
4-(2-Hydroxybenzyl) morpholin-4-ium chloride
(compound I)
Yield 56.0 %; mp 140 °C; IR (neat): 3,391, 3,048, 2,944, 1,604,
1
1,432, 1,130, 793; H NMR (300 MHz, D2O): δ 7.32–6.95

Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:281–290
283
Table 1 Chemical structures of salicin, salicyl alcohol, compounds I and II, and acetyl salicylic acid
Chemical structures
Salicin
Salicyl alcohol
Compound (I)
[4-(2-hydroxybenzyl) morpholin-4-ium chloride]
Compound (II)
[1,4-bis(2-hydroxybenzyl)piperazine-1,4-diium chloride]
Acetyl salicylic acid
(m, 4H), 4.69 (s, 2H), 3.87 (t, J =5.1 Hz, 4H), 3.22 (t, J =5.1Hz,
4H); 13C NMR (75 MHz, D2O): δ 45.7 (2C), 53.6, 66.2 (2C),
118.4, 123.1, 123.1, 134.7, 135.6, 159.3; MS (m/z): M+ 193,
146, 134, 107, 86, 76. CHN analysis required for
C11H16ClNO2: C, 57.52; H, 7.02; N, 6.10. Found: C, 57.32;
H, 6.88; N, 5.84.
119.2 (2C), 123.2 (2C), 134.2 (2C), 134.9 (2C), 158.2 (2C);
MS (m/z): M+ 298, 278, 192, 107, 85, 56. CHN analysis
required for C18H24Cl2N2O2: C, 58.23; H, 6.52; N, 7.54.
Found: C, 57.97; H, 6.43; N, 7.39.
Pharmacology
1,4-bis(2-Hydroxybenzyl)piperazine-1,4-diium chloride
(compound II)
Animals and animal husbandry
Sprague–Dawley rats of either sex (150–200 g) were used in
the study. The animals were bred in the Animal House and
Bioassay Laboratories of the Department of Pharmacy, Uni-
versity of Peshawar. Animals were kept in standard cages with
free access to standard laboratory food and water available ad
Yield 62.3 %; mp 230 °C (decomposition); IR (neat): 3,479, 3,
007, 2,926, 2,783, 1,620, 1,433, 1,139; 1H NMR (300 MHz,
D2O): δ 7.29–6.91 (m, 8H), 4.69 (s, 4H), 3.49 (s, 8H); 13C
NMR (75 MHz, D2O): δ 43.0 (4C), 50.8 (2C), 118.4 (2C),

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Fig. 1 Synthesis of compound I
[4-(2-hydroxybenzyl)morpholin-
4-ium chloride] and
compound II [1,4-bis
(2-hydroxybenzyl)piperazine-
1,4-diium chloride]
libitum. Experiments were conducted between 8:00 am and
4:00 pm, on a 12 h/12 h light/dark cycle with temperature
maintained at 22 2 °C.
Drug administration and surgical procedure
Test compounds and aspirin in their respective doses were
dissolved in normal saline vehicle and administered orally
(p.o.) by feeding tube once a day for 6 days. The final dose
for each group was administered to 36 h fasted animals 1 h prior
to a pyloric ligation procedure (Mabrouk et al. 2009) under
ketamine (i.p.) anesthesia. Keeping in view the relationship
between an empty stomach, increased acidity, and the propen-
sity for ulcerogenicity, we opted for a 36-h fasting period
(extended duration). The majority of NSAIDs are readily
absorbed from the gastrointestinal tract (GIT) within 60 min
of administration to produce systemic effects. However, there is
a relatively prolonged gut transit time after oral administration
increasing the possibility of localized effects on the gastric
mucosa after 60 min. Consequently, we have selected a 1-h
pretreatment period for the test compounds prior to the ligation
procedure. During the surgical procedure, the stomach was
carefully exposed and the pyloric sphincter was immediately
ligated with surgical silk avoiding any injury to blood vessels
and the abdominal wall was subsequently reclosed with sutures
prior to recovery. Food and water were withheld for 4 h to
permit gastric juice to accumulate, and after killing, the
stomachs were re-exposed. A knot was applied at the esopha-
geal end to avoid drainage of gastric juice, and samples of the
stomach tissue were dissected for histological studies.
Ethical approval
The study was approved under the project entitled “Evaluation
of the anti-inflammatory, antinociceptive and antipyretic ac-
tivities of selected synthetic and natural compounds in animal
models” by the “Ethical Committee of Department of Phar-
macy, University of Peshawar, Peshawar, Khyber
Pakhtunkhwa (KP), Pakistan” in its third meeting held on
June 15, 2011 and an approval certificate bearing number
15/EC/Pharm was obtained.
Evaluation of ulcerogenicity of aspirin, compound I
(4-(2-hydroxybenzyl) morpholin-4-ium chloride),
and compound II (1, 4-bis (2-hydroxybenzyl)
piperazine-1,4-diium chloride)
Evaluation of the ulcerogenic effects of aspirin and com-
pounds I and II was carried out according to a method reported
previously (Virupaksha and Shivkumar 2008). Briefly, the
animals were randomly divided into four treatment groups
(n =6 each). Treatments, doses, and the route of administration
are summarized in Table 2.
Macroscopic assessment of stomach mucosa
Table 2 Distribution of animals in the treatment groups
After ligation for 4 h, each stomach was removed and opened
along the greater curvature to assess the degree of ulceration in
terms of ulcer score under a magnifying glass (×10). Ulcer
scoring was performed according to a previously reported
method (Patidar 2011) as depicted in Table 3.
Group no. Treatment group
Dose
Route
(mg/kg) (per os)
I
Saline control (untreated)
–
p.o.
p.o.
p.o.
II
III
Standard treatment group (aspirin)
Test treatment group (compound I)
150
100
150
Biochemical gastric juice analysis
IV
Test treatment group (compound II) 100
150
p.o.
The volume of collected gastric juice was measured; then, it
was centrifuged at 1,000 rpm for 10 min at room temperature

Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:281–290
Table 3 Determination
285
Histological evaluation of ulcerogenicity of compound I
Score
Observation
of ulcer score
[4-(2-hydroxybenzyl) morpholin-4-ium chloride], compound
II [1,4-bis(2-hydroxybenzyl) piperazine-1,4-diium chloride],
and aspirin
0
Normal stomach
Red coloration
0.5
1.0
1.5
2.0
3.0
Spot ulcers
Gastric tissues were collected at the end of the experimental
procedure and subjected to histological examination using a
rotary microtome (SLEE Mainz CUT 5062, Germany) to
produce 3 μm tissue sections and a light microscope
(Labomed Lx 400 USA with digital camera Labomed iVu
3100). Practice procedures were employed for tissue process-
ing as well as hematoxylin and eosin (H&E) staining with
slight modifications (Falkeholm et al. 2001).
Hemorrhagic streaks
Ulcer number ≥3 but ≤5
Ulcer number >5
Mean ulcer score for
each animal group was
calculated
and the supernatant separated for pH, free acidity, total acidity,
and pepsin determination. Gastric parietal cells produce hy-
drochloric acid (HCl), which can exist in the gastric juice
either as free HCl representing free acidity and HCl bound
to other acidic compounds including acidic proteins in the
gastric juice (bound acidity). The term total acidity is used to
designate the sum of the free acidity, bound acidity, and
acidity due to other weak acids (organic acids and proteins).
Two to three drops of Topfer’s reagent (dimethylamino-
azobenzene) were added to 1 mL of supernatant and titrat-
ed against 0.01 N NaOH the end point being a color change
from light red to yellowish orange. The volume (milliliters)
of alkali required represents free acidity and was deter-
mined by the following formula (Patidar 2011; Reddy
et al. 2012):
Results
Effects of compound I, compound II, and aspirin
on biochemical parameters
One-way ANOVA followed by Dunnett’s post hoc analysis
revealed significant biochemical changes in gastric juice vol-
ume, pH, free acidity, and total acidity but not pepsin concen-
tration in the aspirin-treated group. No biochemical changes
were observed for the compound I- and compound II-treated
groups (Fig. 2a–e).
Effects of compound I, compound II, and aspirin on the gross
photomicrographic appearance of stomach mucosae
Acidity = volume of NaOH × N × 100 mEq/L/0.1,
where N =normality of NaOH. Total acidity was determined
by adding phenolphthalein indicator and titration with 0.01 N
NaOH continued until a red color reappeared. The volume of
alkali consumed was substituted in the acidity equation above
(Anoop and Jegadeesan 2003; Kore Kakasaheb et al., 2011;
Muniappan and Sundararaj 2003).
Examination of photomicrographs revealed no gross morpho-
logical changes such as spot ulcers or hemorrhagic streaks in
the stomach mucosae of animal groups treated with com-
pounds I and II and saline. However, visually detectable
changes were observed in the mucosae of the aspirin-treated
group (Fig. 3a–f).
To determine pepsin activity, gastric juice (0.1 mL) was
mixed with 1 mL of 0.5 % of bovine serum albumin (BSA)
in 0.01 N HCl. The mixture was then incubated at 37 °C for
20 min. A gastric juice blank containing no albumin was
prepared by adding 1 mL of 0.01 N HCl. After incubation,
the hydrolysis reaction was halted using 2 mL of 10 %
trichloroacetic acid (TCA). All tubes were heated in a boil-
ing water bath for 5 min which caused protein denaturation,
it was then cooled, and the precipitate was removed by
centrifugation (9,000×g, 10 min). To 1 mL of supernatant
sample of each tube, 0.4 mL NaOH (2.5 N) plus 0.1 mL
Folin–Ciocalteau phenol reagent was added. Lastly, the
volume was adjusted with distilled water up to 10 mL, and
protein absorbency was measured with ultraviolet visible
(UV/VIS) spectrophotometer at 700 nm wavelength. Activ-
ity was expressed in terms of micromoles of tyrosine per
milliliter or milligram per deciliter bovine serum albumin
(Kalra et al. 2011).
Effects of compound I, compound II, and aspirin on ulcer
score and body weight
One-way ANOVA followed by Dunnett’s post hoc analysis
revealed a significant increase in ulcer score in the aspirin-
treated but not in the compound I- and II-treated groups
(Fig. 4a). No significant change in body weight was observed
in all treated groups (Fig. 4b).
Effects of compound I, compound II, and aspirin on gastric
histology
Results of histological examination of control, aspirin, com-
pound I, and compound II are depicted in Fig. 5a–f [H&E
stain; ×10]. Histological examination of gastric mucosae
showed that oral administration of compound I and compound

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Fig. 2 Effect of aspirin and compounds I and II on gastric juice volume,
pH, free acidity, total acidity, and pepsin concentration. Saline (control),
aspirin (150 mg/kg), and compounds I and II (100 and 150 mg/kg) were
administered once a day orally for 6 days. The final dose was adminis-
tered to 36 h fasted rats in each experimental group 1 h before pyloric
ligation. Four hours after surgery, gastric juice was collected and analyzed
for volume (a), pH (b), free acidity (c), total acidity (d) and pepsin
concentration (e). Data presented as mean SEM (n =6) compared to the
saline vehicle-treated group (**P <0.01, ***P <0.001; one-way ANOVA
followed by Dunnett’s post hoc analysis)
II resulted in no significant morphological changes. On the
other hand, changes like disruption of epithelial lining and
edema were clearly observed in the case of the aspirin-treated
group (Fig. 5b).
show that these compounds are devoid of ulcerogenic proper-
ties as there was no change in both biochemical and histolog-
ical parameters at doses that have shown significant
antinociceptive, anti-inflammatory, and antipyretic activities
(Ali et al. 2013).
There is an explicit need to substitute the classical nonste-
roidal anti-inflammatory drugs such as aspirin by synthesizing
compounds sharing the anti-inflammatory, antinociceptive,
and antipyretic activities of the latter but lacking its serious
adverse effects like GIT ulceration, hemorrhage, and perfora-
tion (Singh and Rosen 1998).
Discussion
The synthesized salicyl alcohol nitrogen containing deriva-
tives, i.e., compound I and compound II, was evaluated for
ulcerogenic properties in comparison with aspirin. Our results

Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:281–290
287
Fig. 3 Gross photomicrographic appearance of the gastric mucosae of saline- (control) (a), compound I- (100 mg/kg, b; 150 mg/kg, d), compound II-
(100 mg/kg, c; 150 mg/kg, e), and aspirin-treated (150 mg/kg, f) groups. For each condition (repeated six times), a representative photomicrograph is shown
With respect to the synthesis of new pharmaceutical com-
pounds, bioisosterism has unique relevance and is performed
to curtail side effects or to alter the activity of a target molecule
(Wang et al. 2003). In the biomedical field, the aim of
exchanging one bioisostere for another is to boost the pre-
ferred pharmacological, biological, or physical qualities of a
substance without making substantial changes in the chemical
skeleton (Lima and Barreiro 2005; Patani and LaVoie 1996).
Fig. 4 Effect of aspirin and
compounds I and II on ulcer score
and body weight. A significant
increase in ulcer score was
observed for the aspirin- but not
for the compound I- and II-treated
groups (a). There was no
significant change in body weight
of any treated group (b) (n =6
each). ***P <0.001 (one-way
ANOVA followed by Dunnett’s
post hoc analysis)

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Fig. 5 Histological ulcerogenic
effects of control, aspirin,
compound I, or compound II
(H&E staining; ×10 original
magnification) (n =6 each).
Representative photomicrographs
of the glandular portion of the
stomach: a the saline-treated
group (control group) showing
normal appearance of mucosa
(bar) and submucosa (asterisk)
and b aspirin-treated group
(150 mg/kg) showing erosion
with disruption of mucosa
(large arrow) and inflammatory
exudates (small arrow). Normal
histological appearance of
mucosa (bar) and submucosa
(asterisk) were found in groups of
animals treated with c compound
I (100 mg/kg), d compound I
(150 mg/kg), e compound II
(100 mg/kg), and f compound II
(150 mg/kg)
In our previous study (Ali et al. 2013), utilizing FRED 2.1
and MOE software, molecular docking, and dynamics analy-
ses, compounds I and II show a higher binding energy with the
cyclooxygenase-2, predicting these compounds as potential
COX-2 inhibitors. The compounds have exhibited dose-
dependent antinociceptive, anti-inflammatory, and antipyretic
activities in animal models. Nonetheless, these compounds
did not show antinociceptive properties in the hot plate test
when compared with centrally acting standard analgesic
(morphine), thus suggesting a peripheral mechanism of action
in the mediation of antinociception (Ali et al. 2013).
In the present study, compounds I and II were investigated
for their ulcerogenic potential in comparison to aspirin. The
doses of the compounds selected for this study (100 and
150 mg/kg) were based on our previous observations that
these doses possessed significant antinociceptive, anti-
inflammatory and antipyretic activities (Ali et al. 2013).
The results of the current study clearly demonstrated that
treatment with I and II, compared to aspirin, lacked ulcero-
genic potential as there was no significant change in gastric
juice volume, pH, free or total acidity, pepsin concentration, or
ulcer score. Since these compounds possess neutral character-
istics, it may be concluded that their presence in the stomach
may not have influenced either the acidity or other biochem-
ical parameters of gastric juice to any significant extent.
The gross appearance of gastric mucosa revealed the ab-
sence of morphological changes like ulcer spots or hemor-
rhagic streaks in the control and in the compound I- and II-
treated groups in contrast to the aspirin group where such
changes were clearly evident. Moreover, histological exami-
nation of the stomach tissues revealed no surface epithelial
disruption, deep mucosal damage, edema, necrotic lesion, or
leucocyte infiltration following treatment with saline, com-
pound I, or compound II, while changes like mucosal

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aspirin. Furthermore, during the entire period of the study, no
significant changes in body weight or any other sign of
toxicity were observed in the compound I- and compound
II-treated animals.
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group (–COOH) which is generally considered an important
contributor of the ulcerative properties of the majority of
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Conclusions
In summary, the current study presents the bioisosteric syn-
thesis and evaluates the ulcerogenic potential of the synthetic
compounds (I and II) versus aspirin. Our findings clearly
indicate that unlike aspirin, these compounds do not show
significant ulcerogenic properties. The lack of the latter effects
might be ascribed to selective inhibition of COX-2 enzyme,
the absence of a free carboxylic acid moiety, and consequent-
ly, the neutral characteristics of these compounds.
Acknowledgments We sincerely thank and acknowledge the Higher
Education Commission (HEC) of Pakistan for support of the synthetic work.
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Conflict of interest We declare that we have no conflict of interest.
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