Anti-inflammatory effects of two new methyl and morpholine derivatives of diphenhydramine on rats

Article abstract of DOI:10.1007/s00044-011-9891-y

Diphenhydramines are one of the first-generation histamine H1-receptor antagonists of the ethanolamine class that demonstrate many pharmacological properties including anti-inflammatory effects. In this research, bromo (II) and two new tolyl derivatives of I, (Di [p-tolyl] [dimethylaminoethoxy] methane, III) and (Di [p-tolyl] [2-morpholinoethoxy] methane, IV) were synthesized. Their acute and chronic anti-inflammatory activities were evaluated with the formalin and histamine-induced rat paw edema. The vascular permeability in formalin and histamine-induced paw edema, in xylene-induced ear edema, and in peritonitis after acetic acid application into peritoneal cavity were also measured and compared to II. Cotton pellet-induced granuloma model was selected for inducing chronic inflammations in rats. The newly synthesized analogs of diphenhydramine seemed effective to decrease acute inflammations. It was concluded that the prominent anti-phlogistic effects of the new drugs could be related to its reduction vascular permeability mechanism(s) or to its antagonistic effects on H1 histamine receptors. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9891-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3532–3540
DOI 10.1007/s00044-011-9891-y
ORIGINAL RESEARCH
Anti-inflammatory effects of two new methyl and morpholine
derivatives of diphenhydramine on rats
•
Abbas Ahmadi Mohsen Khalili Ramin Hajikhani
•
•
•
Narjes Safari Babak Nahri-Niknafs
Received: 1 June 2011 / Accepted: 8 November 2011 / Published online: 22 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Diphenhydramines are one of the first-genera-
tion histamine H1-receptor antagonists of the ethanolamine
class that demonstrate many pharmacological properties
including anti-inflammatory effects. In this research,
bromo (II) and two new tolyl derivatives of I, (Di [p-tolyl]
[dimethylaminoethoxy] methane, III) and (Di [p-tolyl]
[2-morpholinoethoxy] methane, IV) were synthesized.
Their acute and chronic anti-inflammatory activities were
evaluated with the formalin and histamine-induced rat paw
edema. The vascular permeability in formalin and hista-
mine-induced paw edema, in xylene-induced ear edema,
and in peritonitis after acetic acid application into perito-
neal cavity were also measured and compared to II. Cotton
pellet-induced granuloma model was selected for inducing
chronic inflammations in rats. The newly synthesized
analogs of diphenhydramine seemed effective to decrease
acute inflammations. It was concluded that the prominent
anti-phlogistic effects of the new drugs could be related to
its reduction vascular permeability mechanism(s) or to its
antagonistic effects on H1 histamine receptors.
Keywords Diphenhydramine Á
Anti-inflammatory activities Á
Histamine H1-receptor antagonist
Introduction
Diphenhydramine (2-(diphenylmethoxy)-N,N-dimethyle-
thanamine), DPH, [CAS 58-73-1, I] is one of the first-
generation histamine H1-receptor antagonist of the etha-
nolamine class. It is a competitive inhibitor to interaction
between histamine and H1-receptor antagonist (Santiago-
Palma et al., 2001). In addition to their antihistamine
effects, H1-receptor antagonists possess such pharmaco-
logical properties as anti-inflammatory, anti-allergic, anti-
platelet effects, and suppressive impact to the respiratory
burst of professional phagocytes which are not uniformly
´
´
distributed in this class of drugs (Kralova et al., 2008;
Leurs et al., 2002). Histamine is an intercellular chemical
messenger which plays a critical role in several diverse
physiological processes. Four human G-proteins coupled
with histamine receptor subtypes (H1–4) are currently
recognized to mediate various actions by monoamine
histamine, including smooth muscle contraction, inflam-
matory responses, gastric acid secretion, and mediation
of neurotransmitter release in the central nervous system
(Saxena et al., 2006; Triggiani et al., 2001). Recently,
our knowledge about the histamine roles in specific
activation or blockade of the receptor subtypes, both in
physiology and pathology, has dramatically increased.
Among the four subtypes, histamine H1-receptor has
been an attractive target to drug discovery for several
A. Ahmadi (&) Á N. Safari
Department of Chemistry, Faculty of Science, Islamic Azad
University, Karaj Branch, P.O. Box 31485-313, Karaj, Iran
e-mail: a-ahmadi@kiau.ac.ir; ahmadi3957@yahoo.com
M. Khalili
Department of Physiology, Neuroscience and Herbal Medicine
Research Center, Shahed University, Tehran, Iran
R. Hajikhani
Department of Physiology, Faculty of Science,
Islamic Azad University, Karaj Branch, Karaj, Iran
B. Nahri-Niknafs
Islamic Azad University, Karaj Branch, Karaj, Iran
123

Med Chem Res (2012) 21:3532–3540
3533
years. H1-receptor antagonists have been proved to be
effective therapeutic agents in many diseases; hence they
incorporated into an important class of available drugs
(Saxena et al., 2006). Histamine is also a potent mediator
in inflammation and tissue remodeling and a modulator
in various autoimmune diseases such as rheumatoid
arthritis osteoarthritis and allergic diseases, respectively
were recorded with a Bruker 300 MHz (model AMX,
Karlsruhe, Germany) spectrometer (internal reference:
TMS). IR spectra were recorded with a Thermo Nicolet
FT-IR (Nexus-870, Nicolet Instrument Corp, and Madison,
WI, USA) spectrometer. Mass spectra were also recorded
with an Agilent Technologies 5973, Mass Selective
Detector (MSD) spectrometer (Wilmigton, USA). Column
chromatographic separations were performed over Acros
silica gel (No. 7631-86-9 particle size 35–70 lm, Geel,
Belgium). Adult female wistar rats (Pasteur Institute,
Tehran, Iran), weighing 250–300 g, were selected as the
subjects in pharmacological testing.
´
´
(Kralova et al., 2009; Estelle and Simons, 2003). In this
study, bromo (II, a well-known drug in this family) and
two new (III and IV) derivatives of I and intermediates
(1–4) were synthesized and their acute and chronic anti-
inflammatory activities were evaluated by the published
procedures (Matos Gomes et al., 2010; Gupta et al.,
2006; Winter et al., 1962; D’Arcy et al., 1960; Miles and
Miles, 1952).
Preparations (Schemes 1, 2, 3, 4)
Phenyl (p-bromophenyl) methanol (p-bromobenzhydrol)
(1)
Materials and methods
This compound was prepared following a published
method (Rieveschi and Woods, 1950) with some imparting
modification. To the solution of p-bromobenzaldehyde
(1.4 g, 0.1 mol) in diethyl ether (50 ml), phenyl magne-
sium bromide (prepared from 15.7 g bromobenzene and
2.43 g of Mg in 50 ml of dry ether) was added dropwise
and refluxed for 5 h. Poured into ice-NH4Cl, the organic
layer was separated, water-washed, re-extracted with die-
thyl ether, dried over MgSO4, and evaporated under vac-
uum. Eventually, the solid compound (m.p.: 60–62°C) was
obtained (Scheme 2).
General
4-Bromobenzaldehyde, 4-methylbenzaldehyde, acetyl bro-
mide, dimethylaminoethanol, 2-morpholino ethanol, mag-
nesium turning, diethyl ether, xylene, benzene, and all used
chemicals in this study were purchased from Merck
Chemical Co. (Darmstadt, Germany). Melting points
(uncorrected) were determined after with a digital Elec-
trothermal melting point apparatus (9100, Electrothermal
Engineering Ltd., Essex, UK). H and 13C NMR spectra
1
Scheme 1 Structure formulas
of Diphenhydramine
(2-(diphenylmethoxy)-N,
N-dimethylethanamine, I),
Bromodiphenhydramine
(Phenyl [p-bromophenyl]
[dimethylaminoethoxy]
methane, Bromodiphen, II), and
two new analogs of I (Di
[p-tolyl] [dimethylami-
noethoxy] methane,
Bromodiphen-1, III) and
(Di [p-tolyl]
[2-morpholinoethoxy] methane,
Bromodiphen-2, IV)
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Med Chem Res (2012) 21:3532–3540
Scheme 2 Synthesis of
intermediates 1 and 3
Scheme 3 Synthesis of
intermediates 2 and 4
Phenyl (p-bromophenyl) methyl bromide
(p-bromobenzhydryl bromide) (2)
This compound was used in the next step without further
purification (Scheme 3).
This compound was prepared following a published
method (Rieveschi and Woods, 1950) with some imparting
modification. A benzene (20 ml) solution of alcohol
(1,16 g, 0.06 mol) was added into acetyl bromide (11.4 g,
0.09 mol) and refluxed for 11 h. The reaction mixture was
concentrated to yield the desired compound as brown oil.
Phenyl (p-bromophenyl) (dimethylaminoethoxy) methane
(Bromodiphenhydramine, Bromodiphen) II
This compound was prepared following a published method
(Rieveschi and Woods, 1950) with some imparting modifi-
cation. A xylene (20 ml) solution of bromobenzhydryl
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Med Chem Res (2012) 21:3532–3540
3535
Scheme 4 Synthesis of target
compounds (II–IV)
bromide (2, 13 g, 0.4 mol) was slowly added into a xylene
(10 ml) solution of dimethylaminoethanol (5, 7.1 g,
0.08 mol) and refluxed for 24 h. The reaction mixture was
cooled, treated with water before the aqueous layer was
extracted with ether, re-extracted with 10% HCl, neutralized
with 10% NaOH, dried over MgSO4, and evaporated under
vacuum to obtain the desired oily compound. The hydro-
chloride salt of II (m.p.: 143–145°C) was prepared using
diethyl ether and HCl and was recrystallized from 2-propa-
nol (Scheme 4).
was obtained that was directly used in the next step without
further purification (Scheme 2).
IR (KBr): 3459, 2923, 2734, 1606, 1517, 1455, 1385,
1272, 1169, 1019, 809, 757 cm-1
.
1H-NMR. (CDCl3) (ppm): 2.19 (6H, s), 2.02 (OH, m),
5.78 (1H, s), 7.15 (4H, d. JHH = 16 Hz), 7.24 (4H, d.
JHH = 16 Hz).
13C{1H}-NMR. (CDCl3) (ppm): 25.9, 77.8, 128.5, 129.8,
134.2, 140.7.
MS: m/z (regulatory intensity): 212 (22).
Di (p-tolyl) methanol (Di [p-methylbenzhydrol]) (3)
Di (p-tolyl) methyl bromide (Di [p-methylbenzhydryl]
bromide) (4)
Phenyl magnesium bromide (prepared from 1.7 g p-bromo
toluene and 2.43 g of Mg in 25 ml of dry ether) was added
dropwise to a THF (50 ml) solution of p-methyl benzal-
dehyde (12 g, 0.1 mol) and refluxed for 6 days. It was
poured into ice-NH4Cl, washed with brine, re-extracted
with diethyl ether, dried over MgSO4, and evaporated
under vacuum. An oily compound (8.2 g, 38.67% yield)
A benzene (20 ml) solution of alcohol (3, 8 g, 0.038 mol)
was added into acetyl bromide (11.4 g, 0.09 mol) and
refluxed for 9 days. The reaction mixture was concentrated
to yield the desired compound as brown oil (5.4 g, 51%
yield). This compound was used in the next step without
further purification (Scheme 3).
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Med Chem Res (2012) 21:3532–3540
IR (KBr): 2923, 1610, 1514, 1448, 1253, 1176, 1116,
973, 872, 751 cm-1
13C{1H}-NMR. (CDCl3) (ppm): 26, 52.2, 56.7, 64, 73.1,
78.1, 128.5, 129.8, 134.3, 140.8.
.
1H-NMR. (CDCl3) (ppm): 2.19 (6H, s), 6.2 (1H, s),
7.15(4H, d. JHH = 17 Hz), 7.24(4H, d. JHH = 17 Hz).
13C{1H}-NMR. (CDCl3) (ppm): 25.7, 63.5, 128.2, 129.7,
134.4, 139.7.
MS: m/z (regulatory intensity): 325 (11).
Pharmacological methods
MS: m/z (regulatory intensity): 275 (23).
Animals
Di (p-tolyl) (dimethylaminoethoxy) methane
(Bromodiphen-1) III
Some adult female Wistar rats (Pasteur Institute, Tehran,
Iran), weighing 250–300 g, at the beginning of the exper-
iment, were randomly housed with three to four per cage,
in a temperature-controlled colony room under 12 h light/
dark cycle. Rats were given free access to water and
standard laboratory rat chow (Pars Company, Tehran,
Iran). All the experiments were conducted between 11 a.m.
and 4 p.m., under a normal room light and at 25°C. This
study was carried out in line with policies mentioned in the
Guidelines for the Care and Use of Laboratory Animals
(NIH) and the Research Council of Shahed University of
Medical Sciences (Tehran, Iran).
A xylene (20 ml) solution of Di [p-methylbenzhydryl]
bromide (4, 5 g, 0.018 mol) was added slowly to a xylene
(10 ml) solution of dimethylaminoethanol (5, 7.1 g,
0.08 mol) and refluxed for 90 h. The reaction mixture was
cooled, treated with water before the aqueous layer was
extracted with ether, re-extracted with 10% HCl, neutral-
ized with 10% NaOH, dried over MgSO4, and evaporated
under vacuum to obtain the desired oily compound (3.3 g,
57% yield). The hydrochloride salt of III was prepared
using diethyl ether and HCl and was recrystallized from
2-propanol (Scheme 4).
Anti-inflammatory activities
IR (KBr): 2919, 2645, 1615, 1510, 1469, 1357, 1177,
1020, 804, 763 cm-1
.
Acute inflammation Formalin-induced rat paw edema.
Thirty-two rats were divided into four groups (n = 8).
Control (normal saline) and treatment groups received II–
IV (17 mg/kg, i.p.), respectively. The administration of
drugs was 30 min before the injection of 50 ll of 3%
formalin into the right hind-paw subplantar in individual
rats. The paw volume was initially measured (zero time),
followed by formalin injection at 0.5, 1, 2, and 3 h using
caliper (Gupta et al., 2006). The observed differences in
paw diameters between control and treatment groups were
statistically analyzed.
1H-NMR. (CDCl3) (ppm): 2.19 (6H, s. 2CH3Ph–), 2.33
(6H, s. –N(CH3)), 2.86 (2H, t. –CH2–N), 3.65 (2H, t.
–CH2–O), 5.78 (1H, s), 7.19(4H, d. JHH = 17 Hz),
7.59(4H, d. JHH = 17 Hz).
13C{1H}-NMR. (CDCl3) (ppm): 26, 53.2, 60.8, 66.2,
77.8, 128.5, 129.8, 134.2, 140.7.
MS: m/z (regulatory intensity): 284 (16).
Di (p-tolyl) (2-morpholinoethoxy) methane
(Bromodiphen-2) IV
Histamine-induced rat paw edema. In this study, rats
were treated similarly to formalin-induced paw edema
models. Only histamine (300 lg, 100 ll) was applied in
hind-paw subplantar surface (Matos Gomes et al., 2010).
Vascular permeability in formalin and histamine-
induced paw edema. All treatments and induction of edema
in this experiment were identical to formalin or histamine
models, following the procedure in Miles and Miles (1952).
Thirty minutes after formalin or histamine injection, the
animals received intravenous injection of Evans Blue dye
(30 mg/kg). At the next 30 min, the rats were anesthetized
with CO2 inhalation and sacrificed. The inflamed formalin
or histamine paws were cut from their wrist region and
sectioned to pieces. The paw pieces were stored in a
mixture of acetone and sodium sulfate (1%) at ratio 3/1 at
room temperature applying 24 h shaking (IKA-Vibrax,
Germany). The mixed solution plus paw sections were
centrifuged before their supernatants were collected and
A xylene (20 ml) solution of Di [p-methylbenzhydryl]
bromide (4, 5 g, 0.018 mol) was added slowly to a xylene
(10 ml) solution of 2-morpholinoethanol (6, 10.48 g,
0.08 mol) and refluxed for 96 h. The reaction mixture was
cooled, treated with water before the aqueous layer was
extracted with ether, re-extracted with 10%, neutralized
with 10% NaOH, dried over MgSO4, and evaporated under
vacuum to obtain the desired oily compound (3.8 g, 58%
yield). The hydrochloride salt of IV was prepared using
Diethyl ether and HCl was recrystallized from 2-propanol
(Scheme 4).
IR (KBr): 2921, 2850, 1608, 1514, 1461, 1377, 1276,
1113, 1015, 795 cm-1
.
1H-NMR. (CDCl3) (ppm): 2.17 (6H, s. 2CH3Ph–),
2.29–2.37 (6H, m), 3.2–3.9 (6H, m), 5.78 (1H, s),
7.16(4H, d. JHH = 16 Hz), 7.53(4H, d. JHH = 16 Hz).
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Med Chem Res (2012) 21:3532–3540
3537
absorbance at 590 nm were measured as the scores of
inflammation (Spectronic 20, Germany).
Results
Vascular permeability in acetic acid-induced to perito-
neal cavity. Test drugs or vehicles in this study were
administered to only rats. Thirty minutes after every rat
was given an intravenous injection of Evans Blue solution
(30 mg/kg), they received an intra peritoneal injection of
0.7% acetic acid at 10 ml/kg (Winter et al., 1962). Rats
were sacrificed by cervical dislocation in the next 30 min
following the acetic acid injection, whereas their peritoneal
cavity was washed three times with 10 ml of total normal
saline. Saline washes were combined and centrifuged for
10 min at 581 rpm in a table top centrifuge (Sigma-4-10,
Germany). Supernatants were collected and their absor-
bance at 590 nm was measured with a spectrophotometer
(Spectron 20 Genesys, USA). The amount of Evans Blue
extruded into the peritoneal cavity was estimated based on
the standard curve.
Chemistry
Bromodiphenhydramine (Phenyl [p-bromophenyl] [dimethyl-
aminoethoxy] methan, Bromodiphen, II, as a well-known
drug in this family) and two new analogs of I (Di [p-tolyl]
[dimethylaminoethoxy] methane, Bromodiphen-1, III) and
(Di [p-tolyl] [2-morpholinoethoxy] methane, Bromodiphen-2,
IV) were synthesized by the reaction of substituted benzhy-
dryl bromide (2 and 4) re-agents and substituted for amino-
ethanol compounds (5, 6). Methyl group on the aromatic
rings bears a high electron donating character that produces
an electron density and dipole moments on the molecules
(Ahmadi et al., 2009, 2010, 2011; Johnson et al., 1981).
Replacing dimethylamine group with a morpholine with
several pharmacological properties (Chen et al., 2003;
Kaneyoshi et al., 1994; Regnie et al., 1991; Rekka et al.,
1990) induced more anti-inflammatory activities on our
newly synthesized drugs (III and IV, Table 1). The known
procedures were applied in synthesizing the compounds II,
1, and 2 after appropriate modifications (Rieveschi and
Vascular permeability in xylene-induced ear edema.
This experiment followed the previously described proce-
dure (Winter et al., 1962), except for inducing ear edema as
xylene (0.03 ml) was applied topically on both surfaces of
the right ear. Ear disks of 8.0 mm in diameter were pun-
ched out, sectioned to pieces and, subjected to Evans blue
extraction and measurement methods as previously men-
tioned in acetic acid peritonitis.
Woods, 1950). Spectroscopic data (IR, H and 13C-NMR,
1
Mass) confirmed the structure of the compounds 3, 4, III,
and IV. The melting points of the known compounds also
confirmed their identity. The purity of individual com-
pounds was checked by TLC using ethyl acetate–hexane as
the eluent.
Chronic inflammation: cotton pellet-induced granuloma
formation The control and treatment rats were anaesthe-
tized (ketamine 100 mg/kg) before sterile cotton pellets,
weighing 30 1 mg, were subcutaneously implanted into
both sides of the groin region of individual rats (D’Arcy
et al., 1960). The animals in both groups received the
vehicle (saline) or drugs for 7 consecutive days after cotton
pellet implantation. On the 8th day, the animals were
anaesthetized and the pellets together with the granuloma
tissues were carefully removed and cleaned from extrane-
ous tissues. The wet pellets were weighed, dried in an oven
at 60°C for 24 h to a constant weight before the dried
pellets were re-weighed. Increment in the dry weight of the
pellets was taken as a measure of granuloma formation.
Pharmacology
General consideration
In this study, mortality (number of death), morbidity
(defined as any abnormal condition or behavior due to a
disorder), irritability (a condition of aggressiveness or
increased response on handling), and other related abnor-
mal states were observed in experimental animals. How-
ever, the motor coordination index (measured by Rota-rod
apparatus, Harvard, UK) did not indicate any significant
differences between treated rats.
Table 1 Effects of II–IV on formalin and histamine paw inflammations, xylene-induced ear edema, and acetic acid-induced peritonitis
Pontamine sky blue dye extracted solution light absorbance (inflammation score)
Inflammation-induced
methods
Histamine injection
to hind paw
Formalin injection
to hind paw
Xylene injection
to ear
Acetic acid injection
to peritoneal
Animal groups tested
Control
0.35 0.08
0.31 0.08
0.15 0.02 *$
0.14 0.03 *$
$
0.30 0.06
0.21 0.02
0.19 0.04
0.19 0.02
0.18 0.01
0.49 0.07
Brom (II)
0.28 0.05
0.10 0.08 **$$
0.12 0.04 **$$
0.37 0.06
0.17 0.04 **$
0.18 0.05 **$
Brom1 (III)
Brom2 (IV)
$$
* P \ 0.05, ** P \ 0.01 (Versus control group); P \ 0.05, P \ 0.01 (Versus Brom (II) treatment animals)
123

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Med Chem Res (2012) 21:3532–3540
Acute inflammation
Vascular permeability in formalin and histamine-induced
paw edema As indicated in Table 1, compounds III and
IV could reduce dye extrusion (edema) from plantar sur-
face of paws at 57.14 and 60.00%, respectively in the
histamine-induced inflammation model (P \ 0.05). Also,
III and IV revealed more anti-inflammatory effects than II
(P \ 0.05) (Table 1). In the formalin-induced edema
method, a prominent anti-phlogistic effect for III and IV
were obtained at about 66.66 and 60.00%, respectively
(P \ 0.001).
Formalin-induced rat paw edema The anti-inflammatory
activity of II–IV measured at the dose of 25 mg/kg b.w.
against acute paw edema induced by formalin is summarized
in Fig. 1. As illustrated, 1 h after formalin injection, II and
III could produce significant (P \ 0.05) anti-inflammatory
activities as 14.28 and 34.69%, respectively. However,
comparing the effects of II and III showed a marked potent
anti-inflammatory effect more for III than II.
Histamine-induced rat paw edema The anti-inflammatory
effects of II–IV against acute paw edema induced by
phlogistic agent histamine are shown in Fig. 2. An hour
after histamine injection, compounds II and III generated
more significant anti-inflammatory effects as 22.44 and
40.81%, respectively (P \ 0.05) than the control groups.
Vascular permeability in xylene-induced ear edema Neither
original nor new drugs showed any significant inhibitory
effects on the xylene-induced ear edema compared to
control groups (Table 1).
Vascular permeability in acetic acid-induced to peritoneal
cavity Compounds III and IV were able to inhibit acetic
acid-induced dye extrusion into the peritoneal cavity by
65.30 and 63.26%, respectively (Table 1). Statistical
analysis showed the significant effects for new drugs
compared to control group (P \ 0.01) as well as compound
II to experimental groups (P \ 0.05).
Control
0.6
Brom
Brom1
Brom2
0.5
*
0.4
*$
0.3
0.2
0.1
0
Chronic inflammation: cotton pellet-induced granuloma
formation in rats
0
0.5
1
2
3
The effects of compounds II–IV on cotton pellet-induced
granuloma formation are shown in Fig. 3. Any significant
increment in the weight of cotton pellet from 67.10
11.58 g in control group rats to 76.20 18.24 g for
compound I, 73.17 14.47 g for compound III and
70.95 9.26 g for compound IV was not observed.
Time(hr)
Fig. 1 Anti-inflammatory effects of II–IV in formalin-induced rat
paw edema. Edema was measured in 0, 0.5, 1, 2, and 3 h after
formalin injection. Bars show mean SEM of paw diameter. * and $
show P \ 0.05 compared with control and compound II, respectively
(n = 12)
Control
0.6
100
80
60
40
20
0
Brom
Brom1
Brom2
0.5
*
0.4
*$
0.3
0.2
0.1
0
0
0.5
1
2
3
Cont
Brom
brom1
brom2
Time(hr)
Group test
Fig. 2 Anti-inflammatory effects of II–IV in histamine-induced rat
paw edema. Edema was measured in 0, 0.5, 1, 2, and 3 h after
histamine injection. Bars show mean SEM of paw diameter. * and
$ show P \ 0.05 compared with control and compound II, respec-
tively (n = 12)
Fig. 3 Chronic anti-inflammatory effects of II–IV in cotton plate
implantation model. Significant effects between control and treatment
animals are not observed. Bars show mean SEM cotton dry weight
(n = 12)
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Med Chem Res (2012) 21:3532–3540
3539
Discussion
opiate component) which act through l receptors (Zhang et al.,
2005), and because of agonistic actions of those drugs via these
receptors (Seo et al., 2011), some remarkable effects occur.
However, the non-efficiency effects of these drugs on allevi-
ating chronic inflammation bring us the probability of their
short half-lives or complexity of chronic inflammation mech-
anism(s) which are unaffected by (only) histamine release.
Nevertheless, it must be reminded that anti-inflammatory
actions by these new drugs in both formalin and histamine
injection tests show that they must act through other unknown
mechanism(s) than preventing histamine release. Regarding to
higher vessel permeability, as a main factor for developing
inflammation (McDougall et al., 2004), it seems the antago-
nizing action by these new drugs against high vessel perme-
ability may cause their more anti-inflammatory effects.
Compounds with the potential of H1-antihistaminic activ-
ities might be considered for more useful therapeutic
qualities in the treatment of various allergic and inflam-
matory conditions due to histamine release. Although,
antihistamines belong to different chemical classes, they
mostly render remarkable chemical similarities (Settimo
et al., 1992). Since, their discovery and early developments
in the 1940s, histamine H1-receptor antagonists (antihista-
mines) have been known as one of the most widely used
classes of medications for allergic disorders. Earlier ‘‘first-
generation’’ antihistamines exhibited a high binding
affinity for H1-receptors, although they mostly exhibited
binding affinities for other classes of cellular receptors,
such as muscarinic cholinergic subtypes (M1–M5) (Orze-
chowski et al., 2005). It has been reported that the first-
generation of the H1-receptor antagonists is among the
most widely used medications around the world. These
compounds induce the inhibition effects of histamine
mediated by H1–receptors (Simons and Simons, 1994), as
they are the receptor-independent G-protein activators in
HL-60 cells, basophils, and mast cells (Burde et al., 1996).
Diphenhydramine (I) has a relatively low molecular weight
with high lipid solubility which allows easy blood–brain
barrier and placental passages (Moraes et al., 2004). So in
this study, some of its new derivatives (III and IV) were
synthesized and their anti-inflammatory effects were tested
by the known procedures. The results indicate that inclu-
sion of the methyl group with high electron distribution and
dipole moments (Ahmadi et al., 2009, 2010, 2011; Johnson
et al., 1981) on phenyl rings beside the morpholine group
with many pharmacological properties (Chen et al. 2003;
Kaneyoshi et al. 1994; Regnie et al., 1991; Rekka et al.,
1990) cause more anti-inflammatory activities on the newly
synthesized drugs (III and IV) than II in control groups
(Table 1). However, these effects were remarkable only
when methyl groups were replaced for bromine in phenyl
rings (III), so the new drug could diminish inflammatory
effects in all acute inflammation tests (Figs. 1, 2; Table 1).
Also, it seems replacing dimethylamine by morpholine is
not effective by itself; only with methyl group in phenyl
rings (IV), it could effectively diminish the inflammatory
effects. This finding could be explained with methyl high
electron distribution and dipole moments which makes it
also effective in dimethylamine for activating basicity
potent of nitrogen atom (in amine group) in this moiety.
From pharmacokinetic point of view, more antagonizing
actions done by these new drugs (especially III) on H1 hista-
mine receptors and their inhibitory effects of histamine release
from must cells might cause their more anti-inflammatory
activities. Meanwhile, because of anti-inflammatory actions
(Schiene et al., 2011) by some analgesic drugs (especially
Conclusions
Compound I new derivatives, including III and IV could
markedly diminish acute anti-inflammatory effects.
However, the prominent anti-phlogistic effects of these
newly synthesized drugs might be related to their reduc-
ing potential for vascular permeability mechanism(s) or to
their more potent antagonistic effects on H1 histamine
receptors.
Acknowledgments This article is a report made out a research
project that enjoyed the financial supports of Iran National Founda-
tion Support (INFS), and conducted at Islamic Azad University, Karaj
Branch, Iran. The authors would like to express their gratitude to
Mojdeh Javadi for her assistance in chemical experiments, Fariba
Ansari for her assistance in the pharmacological tests, Dr. Natasha Q.
Pourdana, the CamTESOL International editor, and Dr. Ahmad Jahan
Latibari for their patience with proofreading the initial drafts of this
article.
Conflict of interest This research is not a part of the researchers’
normal lectures, employment, consultation, and involvement; no
institution would require any rights from this work, either. In addition,
no patent has been applied, nor any commercial rights have been
given to any company and/or institution, as it will not be done later
either.
References
Ahmadi A, Khalili M, Abbassi S, Javadi M, Mahmoudi A, Hajikhani
R (2009) Synthesis and study on analgesic effects of 1-[1-[4-
methylphenyl][cyclohexyl]-4-piperidinol and 1-[1-[4-methoxy-
phenyl][cyclohexyl]]-4-piperidinol as two new phencyclidine
derivatives. Arzneimittelforschung 59(4):202–206
Ahmadi A, Khalili M, Mihandoust F, Barghi L (2010) Synthesis and
determination of acute and chronic pain activities of 1-[1-[3-
methyphenyl][tetralyl]] piperidine as a new derivative of phen-
cyclidine via tail immersion and formalin tests. Arzneimittelf-
orschung 60(1):30–35
123

3540
Med Chem Res (2012) 21:3532–3540
Ahmadi A, Khalili M, Hajikhani R, Naserbakht M (2011) Synthesis
and determination of acute and chronic pain activities of 1-[1-[4-
methylphenyl][cyclohexyl] morpholine as a new phencyclidine
derivative in rats. Arzneimittelforschung 61:1–11
Burde R, Dippel E, Seifert R (1996) Receptor-independent G-protein
activation may account for the stimulatory effects of first
generation H-receptor antagonists in HL-60 cells, basophils, and
mast cells. Biochem Pharmacol 51:125–131
Chen DM, Chen K, Wang H (2003) Antithrombotic effects of
morpholine and piperazine ring derivatives and their molecular
mechanism. Yao Xue Xue Bao 38(9):641–645
D’Arcy EM, Howard PW, Muggletone M, Townsend SB (1960) The
anti-inflammatory action of griseofulvin in experimental ani-
mals. J Pharm Pharmacol 12:659–665
Estelle R, Simons MD (2003) H1-antihistamines: more relevant than
ever in the treatment of allergic disorders. J Allergy Clin
Immunol 112(4):S42–S52
Gupta M, Mazumder UK, Gomathi P, Thamil SV (2006) Antiinflam-
matory evaluation of leaves of Plumeria acuminate. BMC
Complet Altern Med 6:36–42
Johnson PY, Pan R, Quan Wen J, Halfman CJ (1981) Synthesis of
amine derivatives of phencyclidine. J Org Chem 46:2049–2054
Kaneyoshi K, Takayuki D, Mitsuo Y (1994) Assignee: Takeda
Chemical Industries LTD., Japan; Tetracyclic morpholine
derivatives and their use as analgesics. European Patent;
EP0610793
rats: effects on physical and neurobehavioral development and
on neurochemical parameters. Neurotoxicol Teratol 26:681–692
Orzechowski RF, Currie DS, Valancius CA (2005) Comparative
anticholinergic activities of 10 histamine H1-receptor antagonists
in two functional models. Eur J Pharmacol 506:257–264
Rekka E, Retsas S, Demopoulos VJ, Kourounakis PN (1990)
Lipophilicity of some substituted morpholine derivatives syn-
thesized as potential antinociceptive agents. Arch Pharm
(Weinheim) 323(1):53–56
Regnie G, Guillonneau C, Lepagnol J, Lestage P (1991) Assignee:
Adir et Compagnie, Morpholine compounds, compositions and
use. United State Patent; US 5026701
Rieveschi G, Woods GP (1950) Assignee: Park Davis Company,
Detroit, MI. b-Dimethylamino-ethyl p-halobenzhydryl ethers
and their salts. United State Patent; US 2527963
Santiago-Palma J, Fischberg D, Kornick C, Khjainova N, Gonzales G
(2001) Diphenhydramine as an analgesic adjuvant in refractory
cancer pain. J Pain Symptom Manag 22(2):699–703
Saxena M, Gaur S, Prathipati P, Saxena K (2006) Synthesis of some
substituted pyrazinopyridoindoles and 3D QSAR studies along
with related compounds: piperazines, piperidines, pyrazinoiso-
quinolines, and diphenhydramine, and its semi-rigid analogs as
antihistamines (H1). Bioorg Med Chem 14:8249–8258
Schiene K, De Vry J, Tzschentke TM (2011) Antinociceptive and
antihyperalgesic effects of tapentadol in animal models of
inflammatory pain. J Pharmacol Exp Ther 339(2):537–544
Seo YJ, Kwon MS, Choi SM, Lee JK, Park SH, Jung JS (2011)
Differential cross-tolerance development between single and
repeated immobilization stress on the antinociceptive effect
induced by b-endorphin, 5-hydroxytryptamine, morphine, and
WIN55,212-2 in the inflammatory mouse pain mode. Arch
Pharm Res 34(2):269–280
Settimo AD, Primofiore G, Settimo FD, Calzolari L, Cazzulani P,
Passoni A (1992) 1-Substituted 2-benzylaminobenzimidazole
derivatives: compounds with H1-antihistaminic activity. Eur J
Med Chem 27:395–400
Simons FER, Simons KJ (1994) The pharmacology and use of H1
receptor-antagonist drug. N Engl J Med 330:1663–1670
Triggiani M, Gentile M, Secondo A, Granata F, Oriente A,
Taglialatela M (2001) Histamine induced exocytosis and IL-6
production from human lung macrophages through interaction
with H1-receptors. J Immunol 166:4083–4091
´
´
´
´
´
´
´
Kralova J, Nosal R, Drabikova K, Jancvinova V, Denev P, Moravcova
A (2008) Comparative investigations of the influence of H1-
antihistamines on the generation of reactive oxygen species by
phagocytes. Inflamm Res 57(1):S49–S50
´
´
´
´
´
Kralova J, Rackova L, Pekarova M, Kubala L, Nosal R (2009) The
effects of H1-antihistamines on the nitric oxide production by
RAW 264.7 cells with respect to their lipophilicity. Int
Immunopharmacol 9:990–995
Leurs R, Church MK, Taglialatela M (2002) H1-antihistamines:
inverse agonist, anti-inflammatory actions and cardiac effects.
Clin Exp Allergy 32:489–498
Matos Gomes ND, Moraes de Rezende C, Paredes Fontes S, Eline
Matheus M (2010) Characterization of the antinociceptive and
anti-inflammatory activities of fractions obtained from Copaifera
multijuga Hayne. J Ethnopharmacol 128(1):177–183
McDougall JJ, Baker CL, Hermann PM (2004) Attenuation of knee
joint inflammation by peripherally administered endomorphin-1.
J Mol Neurosci 22(1–2):125–137
Miles AA, Miles EM (1952) Vascular reaction to histamine,
histamine liberator and leukotaxine in the skin of guinea-pigs.
J Physiol 118:228–257
Moraes AP, Schwarz A, Spinosa HS, Florio JC, Bernardi MM (2004)
Maternal exposure to diphenhydramine during the fetal period in
Winter CA, Risley EA, Nuss GW (1962) Carrageenin-induced edema
in hind paws of the rat as an assay for anti-inflammatory drug.
Proc Soc Exp Biol Med 111:544–547
Zhang RX, Wang L, Liu B, Qiao JT, Ren K, Berman BM (2005) Mu
opioid receptor-containing neurons mediate electroacupuncture-
produced anti-hyperalgesia in rats with hind paw inflammation.
Brain Res 1048(1–2):235–240
123