Design, synthesis, and docking studies of novel benzopyrone derivatives as H1-antihistaminic agents

Article abstract of DOI:10.1016/j.bmc.2008.08.039

Two new series of 2H-1-benzopyran-2-one derivatives substituted at C-6 and/or C-7 with propanolamines, and/or piperazine propanol derivatives have been synthesized and assayed for the H₁-histamine antagonist. Twelve of the 20 newly synthesized

Full text of DOI:10.1016/j.bmc.2008.08.039

Bioorganic & Medicinal Chemistry 16 (2008) 9009–9017
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and docking studies of novel benzopyrone derivatives
as H1-antihistaminic agents
a
b
Nahla. A. Farag a, , Shadia. R. Mohamed , Gamal A. H. Soliman
*
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, El-Kasr El-Aini Street, Cairo 11562, Egypt
b Pharmacology Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new series of 2H-1-benzopyran-2-one derivatives substituted at C-6 and/or C-7 with propanolam-
ines, and/or piperazine propanol derivatives have been synthesized and assayed for the H1-histamine
antagonist. Twelve of the 20 newly synthesized 4- substituted benzopyrones have shown potent antihis-
taminic H1 activity. In addition, molecular modeling and docking of the tested compounds into high affin-
ity histamine binding protein (HBP) and histamine N-methyltranseferase (HNMT) active site in complex
with its bound inhibitor (diphenhydramine) was performed in order to predict the affinity and orienta-
tion of these compounds at the active sites. The ICM score values show good agreement with predicted
binding affinities obtained by molecular docking studies as verified by pharmacological screening. The
results showed similar orientation of the target compounds at HBP, and HNMT active sites compared
with reported histamine H1 antagonist. Also, it was concluded that in order for the compounds to be
active, they must bind with both active sites of HNMT enzyme (two pockets) to inhibit it. Compounds
8c, 8i, 11g, 11i, and 11k; observe the maximum activities.
Received 23 April 2008
Revised 4 August 2008
Accepted 20 August 2008
Available online 26 August 2008
Keywords:
2H-1-benzopyran-2-one
Histamine binding protein
Histamine N-methyltransferase
Histamine H1 antagonists
Docking
Internal coordinate mechanics (ICM)
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tion of the inhibitor with HNMT, they all occupy the histamine-
binding site, thus blocking access to the enzyme’s active site.5 Near
Histamine is an intercellular chemical messenger and plays a crit-
ical role in several diverse physiological processes. Four human
G-protein coupled histamine receptor subtypes (H1–4) are currently
recognized to mediate various actions of monoamine histamine.
Among the four subtypes, the histamine H1 receptor has been an
attractive target for drug discovery for several years and H1 receptor
antagonist have proved to be effective therapeutic agents for respira-
tory distress, thus contributing to an important class of drugs today.1
In mammals, histamine action is terminated mainly through
metabolic inactivation by histamine N-methyltransferase (HNMT)
the N terminus of HNMT; several aromatic residues (Phe9, Tyr15,
and Phe19) adopt different rotamer conformations or become dis-
ordered in the enzyme–inhibitor complexes, accommodating the
diverse, rigid hydrophobic groups of the inhibitors. The maximized
shape complementarily between the protein aromatic side chains
and aromatic rings of the inhibitors are responsible for the tight
binding of the inhibitor.6 Also, visualization of diphenhydramine
bound to HNMT will assist in the development of new generation
H1-receptor antagonists with ‘dual’ actions.6
Quinacrine antimalarial drug is a potent inhibitor of HNMT, and
by transferring a methyl group from S-adenosyl-
(AdoMet) to the N-2 atom of the imidazole ring, yielding methyl
histamine and S-adenosyl-
-homocysteine (AdoHcy),2 HNMT has
L-methionine
a competitive inhibitor with respect to histamine.3
A common feature of first generation H1 antihistaminic com-
pounds includes two aryl or two heteroaryl rings linked to an ali-
phatic tertiary amine via a side chain (diphenhydramine and
pheniramine). Also the second generation compounds (terfenadine
and cetirizine) also contain many of the structural features of first
generation compounds.7
L
two-domain structure including a histamine-binding domain and
the methyl donor (AdoMet) binding domain.3 Histamine receptors
and histamine binding sites in HNMT may be similar as both are
inhibited by many H1-antagonists.4
It was reported that histamine receptor H1 antagonist diphen-
hydramine, the antimalarial drug amodiaquine, the antifolate drug
metoprine, and the anticholinesterase drug tacrine are surprisingly
all potent HNMT inhibitors, having inhibition constants in the
range of 10–100 nM. Determining the structural mode of interac-
The key pharmacophoric moieties consist of the same common
feature functions of two ring aromatic features which are essential
for significant H1-receptor affinity which must be capable of adopt-
ing a noncoplanar conformation relative to each other for optimal
interaction and a basic amine function which represent the posi-
tive ionizable feature.1
The piperazines or cyclizines can also be considered to be cyclic
ethylenediamines (cyclizines), where terminal amine functionality
* Corresponding author. Tel.: +202 24534418; fax: +202 3628426.
E-mail address: anahla_69@yahoo.com (Nahla. A. Farag).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.039

9010
Nahla. A. Farag et al. / Bioorg. Med. Chem. 16 (2008) 9009–9017
as well as the nitrogen atom of the connecting group are all part of
a piperazine moiety. Both nitrogen atoms in these compounds are
aliphatic and thus display comparable basicities. The piperazines
are moderately potent antihistaminics with a lower incidence of
drowsiness and exhibit antimuscarinic activity and may be respon-
sible for antiemetic and antivertigo effects. The activity of the
piperazine-type antihistaminics is characterized by a slow onset
and long duration.7
CH3
O
HO
O
6
epichlorohydrin/ KOH
Furthermore, the works of Wieland et al.8 on the active antago-
nistic site region of histamine H1 receptor prove that, one of the
CH3
O
aromatic rings of the antagonists forms favorable aromatic
staking interactions with Phe 433 and Phe 436, the other ring
establishes aromatic staking with trp 167, additionally, the
p–p
O
p–p
O
O
nitrogen establishes a salt bridge interaction with ASP 116.
A literature survey reveals excellent antihistaminic activity in
quinazolines and condensed quinazolines.9,10 Cromolyn sodium
and nedocromil sodium which are chromone derivatives have been
shown to inhibit the release of the mediators of allergic reaction.11–
7
Amines, piperazines/ absolute ethanol
13
Furthermore, it has been observed that certain benzopyranone
derivatives possess significant antihistaminic activity.11–20
Encouraged by these findings, we thought of preparing new
derivatives 8a–i, 11a–k of benzopyran-2-ones substituted at C-6
and/or C-7 with different propanolamines and/or piperazine pro-
panol derivatives in order to screen them for H1 antihistaminic
activity and to study the systematic evaluation of the positional
isomers in the benzopyranone nucleus, also docking the prepared
and tested compounds into histamine binding protein (HBP) and
histamine N-methyltransferase (HNMT) active site in order to pre-
dict the affinity and orientation of these compounds at the active
sites in both enzymes.
CH3
OH
R
O
O
O
8 a-i
R= NH(n-C3H7), , N(C2H5)2
,
,
N
O
N
,
N
,
N
N
N
N
N
,H
C N
HN
,
N
N
3
,
Cl
OMe
2. Results and discussion
2.1. Chemistry
Scheme 1.
In this study we synthesized two new series of 4-methyl-6-
substituted-2H-1-benzopyran-2-one 8, and 4-phenyl-7-substi-
tuted-2H-1-benzopyran-2-one 11 substituted with 2-hydroxy pro-
panolamines, 2-hydroxy propanol piperazines to be tested against
H1-antihistaminic activity.
of isolated guinea pig ileum decreased when pretreated with phenir-
amine maleate and the test compounds.
Cumulative concentration–response curves of histamine
diphosphate showed that the test compounds 8b, 8c, 8f, 8g, 8i,
11a, 11d, 11e, 11f, 11g, 11i, and 11k inhibited the histamine-
induced contraction of isolated guinea pig ileum in a concentra-
tion-dependent fashion. The standard antihistaminic pheniramine
maleate and three different concentrations of compounds 8b, 8c,
8f, 8g, 8i, 11a, 11d, 11e, 11f, 11g, 11i, and 11k showed clear right-
ward shifts compared to that produced with saline. As shown in
Figures. 1 and 2, the test compounds as 11g and 11k produced
competitive inhibition at low concentrations and non-competitive
inhibition at high concentrations of histamine-induced contrac-
tions of isolated guinea pig ileum.
The synthesis of our target compounds is outlined in Schemes 1
and 2. The starting compounds, 6-hydroxy-4-methyl-2H-1-benzo-
pyran-2-one 6, and 7-hydroxy-4-phenyl-2H-1-benzopyran-2-one
9 were first condensed with epichlorohydrin in alcoholic potassium
hydroxide to give the corresponding epoxides 6-(2,3-epoxyprop-
oxy)-4-methyl-2H-1-benzopyran-2-one 7, 7-(2,3-epoxypropoxy)-
4-phenyl-2H-1-benzopyran-2-one 10, then 7, 10 were reacted
smoothly with appropriate amine and/or piperazine derivatives to
give 1-(4-methyl-2H-1-benzopyran-2-one-6-yloxy)-3-(substituted
amino) propan-2-ol 8a–d, 1-(4-methyl-2H-1-benzopyran-2-one-
6-yloxy)-3-(substituted piperazino) propan-2-ol 8e–i. Scheme 1,
and 1-(4-phenyl-2H-1-benzopyran-2-one-7-yloxy)-3-(substituted
amino) propan-2-ol 11a–f, 1-(4-phenyl-2H-1-benzopyran-2-one-
7-yloxy)-3-(substituted piperazino) propan-2-ol 11g–k Scheme 2.
The EC50 of histamine obtained in the presence of pheniramine
maleate (5 l )
g mLꢁ1) and two concentrations (0.5 and 1.0 mg mLꢁ1
of compounds 8b, 8c, 8f, 8g, 8i, 11a, 11d, 11e, 11f, 11g, 11i, and
11k were significantly higher than those for saline (Table 2). How-
ever, there were no significant differences among the EC50s ob-
tained for 0.1 mg mLꢁ1 of the same compounds.
2.2. Pharmacology
The maximum response to histamine diphosphate obtained in
the presence of 1.0 mg mL-1 of compounds 8b, 8c, 8f, 8g, 8i, 11a,
11d, 11e, 11f, 11g, 11i, and 11k were significantly lower than
those of saline. The maximum responses to histamine obtained
The compounds 8b, 8c, 8f, 8g, 8i, 11a, 11d, 11e, 11f, 11g, 11i,
and 11k showed promising activity among the newly synthesized
compounds.
in the presence of pheniramine maleate (5 l ) and
g mLꢁ1
1.0 mg mLꢁ1 of compounds: 8b, 8c, 8f, 8g, 8i, 11a, 11d, 11e, 11f,
11g, 11i, and 11k were significantly lower than that of saline
(Table 2). However, the maximum response to histamine obtained
in the presence of 0.1 and 0.5 mg mLꢁ1 of the test compounds were
not significantly different from that of saline.
Histamine diphosphate elicited contractile responses from the
ileum. The threshold value for the contractile effect of histamine
on the ileum was obtained at a concentration of 2 ꢀ 10ꢁ3
l
g mLꢁ1
,
while the maximal response was attained at 9 ꢀ 10ꢁ3
l
g mLꢁ1 of
histamine (Table 1). The potency of histamine-induced contraction

Nahla. A. Farag et al. / Bioorg. Med. Chem. 16 (2008) 9009–9017
9011
120
100
80
60
40
20
0
C6H5
Saline
HO
O
O
Pheniramine
Comp. 11g (0.1)
Comp. 11g (0.5)
Comp. 11g (1.0)
9
epichlorohydrin/ KOH
C6H5
O
O
O
O
1
2
3
4
5
6
7
8
9
10
10
Histamine conc. (X 10-3 ug mL-1)
Amines, piperazines/ absolute ethanol
Figure 1. Cumulative concentration–response curves of histamine-induced con-
traction of guinea pig ileum in the presence of saline, pheniramine maleate
(5 l
g mLꢁ1) and three increment concentrations (0.1, 0.5, and 1.0 mg mLꢁ1) of
compound 11g. Number of observation = 5.
C6H5
120
R
O
O
O
Saline
100
80
60
40
20
0
OH
Pheniramine
Comp. 11k (0.1)
Comp. 11k (0.5)
Comp. 11k (1.0)
11a-k
,
,
R= NH(n-C3H7), NH(n-C4H9), N(CH3)2, N(C2H5)2
O
N
N
,
N
N
N
N
,
N
N
, H
C N
HN
,
N
N
3
Cl
OMe
Scheme 2.
1
2
3
4
5
6
7
8
9
10
Table 1
A measure of sensitivity of isolated guinea pig ileum to histamine diphosphate
Histamine conc. (X 10-3 ug mL-1)
Concentration
Response of
isolated ileum
Average height of
contraction (cm)
Figure 2. Cumulative concentration–response curves of histamine-induced con-
(ꢀ10ꢁ3
l
g mLꢁ1
)
traction of guinea pig ileum in the presence of saline, pheniramine maleate
(5 l
g mLꢁ1) and three increment concentrations (0.1, 0.5, and 1.0 mg mLꢁ1) of
<1
2*
3
4
5
No response
Contraction
Contraction
Contraction
Contraction
Contraction
Contraction
Contraction
Contraction
Contraction
0.00
0.28
0.65
0.96
1.25
1.42
1.48
1.54
1.62
1.62
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
compound 11k. Number of observation = 5.
n,n-dimethylamine) available through the RCSB Protein Data Bank
(PDB entry 2AOT).6
6
7
The ICM score values obtained by docking our titled compounds
on histamine N-methyltransferase (HNMT) shows more accurate
result than that on histamine binding protein. It also shows good
agreement with the predicted binding affinities obtained by molec-
ular docking studies on (2AOT), and as verified by pharmacological
screening (Table 3 and Fig. 10).
8
9**
10
() Number of observations.
*
Threshold value (T.R.).
Maximal response (M.R.).
**
Figure 3 shows the docking solutions of 8b, 8c, 8f, 8g, and 8i
with histamine binding protein (1QFT), Figure 4 shows the docking
solutions of 11a, 11d, 11e, 11f, 11g, 11h, 11i, and 11k with hista-
mine binding protein (1QFT), Figures 5–8 shows the docking solu-
tions with the highest predicted binding affinity for histamine
N-methyltransferase (2AOT) and different benzopyrone derivatives
8a, 8i, 11c, and 11k, respectively, while Figure 9 shows orientations
of diphenhydramine, amodiaquine, tacrine and metoprine at
HNMT active site at (2AOT).
With respect to ICM scores on histamine N-methyltransferase
(2AOT), it reveals that compound 8i is pharmacologically active
and has the least score in this series ꢁ95.75, while 8a is inactive
pharmacologically and has the highest score in this series
ꢁ79.96, additionally, it shows that compound 11k is the most ac-
2.3. Docking studies
To understand the pharmacological data on structural basis, we
evaluate the titled compounds (two different classes of benzopyro-
nes) through docking techniques using Molsoft ICM 3.4-8C pro-
gram. We docked our titled compounds on one of the crystal
structures of histamine-binding protein (HBPs) available through
the RCSB Protein Data Bank (PDB entry 1QFT),22 which reveals a
lipocalin fold novel in containing two binding sites for the same
ligand (histamine), and on another crystal structures of histamine
N
-methyltransferase (HNMT) in complex with its bound
inhibitor diphenhydramine (1H n-[(2-bezhydroxy) ethyl]

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Nahla. A. Farag et al. / Bioorg. Med. Chem. 16 (2008) 9009–9017
Table 2
Table 3
EC50 and Emax of histamine diphosphate in the presence of saline, pheniramine
maleate and the test compounds (n = 5)
ICM Scores of the tested compounds
Compound
ICM docking
scores of
2AOT
No. of
hydrogen
bonds
ICM docking
scores of
1QFT
No. of
hydrogen
bonds
Solutions
Concentration
—
EC50 (ꢀ10-3
5.1 0.28
6.8 0.30***
l
g mLꢁ1
)
Emax (%)
Saline
100 0.00
78 3.82***
Diphenhydramine
Pheniramine
Cyclizine
Amodiaquine
Metoprine
Tacrine
8a
ꢁ79.47
ꢁ79.48
ꢁ78.04
ꢁ91.00
ꢁ76.94
ꢁ6.76
0
0
0
0
5
0
5
7
1
6
3
1
2
2
1
3
1
5
4
2
5
6
3
4
2
2
ꢁ61.98
ꢁ60.49
ꢁ60.08
—
—
—
1
1
0
—
—
—
6
6
6
4
5
6
4
4
7
4
4
5
4
5
5
5
3
6
7
6
Pheniramine maleate
5
l
g mLꢁ1
8b
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
5.2 0.34
6.0 0.31*
8.5 0.32***
100 0.00
96 4.46
58 3.28***
8c
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
5.5 0.14
100 0.00
98 4.46
6.5 0.31*
8.3 0.28***
ꢁ79.96
ꢁ84.67
ꢁ87.26
ꢁ80.98
ꢁ79.55
ꢁ86.24
ꢁ94.16
ꢁ91.22
ꢁ95.75
ꢁ85.09
ꢁ93.24
ꢁ81.46
ꢁ83.99
ꢁ91.09
ꢁ86.00
ꢁ84.72
ꢁ87.22
ꢁ100.03
ꢁ96.48
ꢁ99.68
ꢁ75.42
ꢁ66.78
ꢁ71.42
ꢁ64.29
ꢁ67.39
ꢁ70.23
ꢁ70.15
ꢁ70.83
ꢁ74.51
ꢁ74.76
ꢁ78.38
ꢁ64.83
ꢁ73.25
ꢁ75.94
ꢁ69.20
ꢁ69.30
ꢁ68.52
ꢁ72.41
ꢁ65.88
ꢁ71.37
55 3.85***
8b
8c
8d
8e
8f
8g
8h
8i
11a
11b
11c
11d
11e
11f
11g
11h
11i
8f
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
5.8 0.30
6.0 0.31*
8.1 0.31***
100 0.00
98 5.06
54 3.11***
8g
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
5.2 0.34
6.3 0.31*
8.5 0.28***
100 0.00
95 5.72
60 3.82***
8i
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
5.8 0.29
6.2 0.30*
8.5 0.28***
100 0.00
96 5.40
58 3.82***
11a
11d
11e
11f
11g
11i
11k
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
0.1 mg mLꢁ1
0.5 mg mLꢁ1
1.0 mg mLꢁ1
5.6 0.27
6.3 0.30*
8.5 0.32***
100 0.00
100 0.00
53 2.86***
5.9 0.29
100 0.00
95 5.72
6.4 0.33**
8.9 0.32***
11j
11k
60 4.18***
5.5 0.29
6.2 0.30*
8.3 0.32***
100 0.00
95 5.00
58 3.85***
antagonist. In our studies, we focused on substitution of benzopy-
ran-2-ones at C-6 and/or C-7 with different propanol amines and/
or propanol piperazines derivatives 8a–i, 11a–k in order to screen
them for H1 antihistaminic activity.
Molecular docking studies on the two crystal structures of his-
tamine binding protein (1QFT), and histamine N-methyltransferase
(2AOT) reveals that the results of docking is more accurate with
histamine N-methyl transferase enzyme, and we conclude that:
5.6 0.32
6.0 0.31*
8.9 0.30***
100 0.00
94 4.96
60 3.74***
5.8 0.29
100 0.00
100 0.00
53 3.22***
6.3 0.29**
8.6 0.27***
5.4 0.13
100 0.00
94 4.95
6.3 0.29**
8.3 0.4.3***
54 2.18***
5.7 0.29
6.2 0.30*
8.9 0.31***
100 0.00
96 5.27
(1) The only active amine derivatives in which its molecule is
bound with both pockets in the enzyme are 8b, 8c, 11a,
11d, and 11f, while the inactive amine derivatives occupy
52 3.18***
Significant at: *P 6 0.05; **P 6 0.01; ***P 6 0.001.
the histamine binding site only not S-adenosine-L-homocys-
teine binding site.
(2) Molecular docking and test results showed that, all the
piperazine derivatives bind with both pockets of the
enzyme, N-phenyl substitution of piperazine moiety retain
activity, replacement of ortho proton of phenyl piperazine
by chloro group abolish the activity, while its substitution
with methoxy group significantly increase the activity in
both series.
tive compound and has nearly the least score in this series ꢁ99.68,
while 11c is inactive pharmacologically and has the highest score
in this series ꢁ81.46. Also orientation of compounds 8a, 8i, 11c,
11k, and diphenhydramine at the active site reveals that the mol-
ecule of the active compounds must bind with both active sites
diphenhydramine (pocket 2, red), and S-adenosyl-L-homocysteine
(AdoHcy) (pocket 1, blue) in the enzyme to inhibit it (Figs. 5–8).
It was mentioned earlier that histamine action is terminated
through metabolic inactivation by histamine N-methyltransferase
(HNMT), and diphenhydramine, amodiaquine, metoprine, and ta-
crine and they are surprisingly all potent HNMT inhibitors.6 From
our Docking studies, it was revealed that they all occupy the hista-
mine-binding site and give moderate ICM scores (Table 3), and all
of them occupy only the histamine binding site pocket (red pocket)
The experimental findings are in good agreement with pre-
dicted binding affinities obtained by molecular docking studies
on histamine N-methyltransferase. The present work may be al-
lowed an at least ranking of the new synthesized and tested H1-
antihistaminic agents.
not S-adenosyl-L-homocystein pocket (blue one) Figure 9.
4. Experimental
4.1. General
Also, structural comparisons reveal that benzopyrane-2-one
moiety may share common interaction pattern with the protein
binding pockets, despite their different substituents.
Remarks: All melting points are uncorrected and determined by
the open capillary method using Gallen Kamp melting point appa-
ratus (MFB-595-010M). Microanalysis was carried out at the
microanalytical unit, Faculty of Science, Cairo University. Infrared
spectra were determined (KBr) using Schimadzu Infrared Spec-
3. Conclusion
From the literature, we knew about the importance of the two
aromatic rings feature and the basic amine for the H1-histamine

Nahla. A. Farag et al. / Bioorg. Med. Chem. 16 (2008) 9009–9017
9013
Figure 6. Orientation of 8i, diphenhydramine (red pocket), and S-adenosyl-L-
homocysteine (blue pocket) at HNMT active site.
Figure 3. Orientation of active compounds of 8 at HBP active site.
Figure 7. Orientation 11c, diphenhydramine (red pocket), and S-adenosyl-L-
homocysteine (blue pocket) at HNMT active site.
Figure 4. Orientation of active compounds of 11 at HBP active site.
Figure 8. Orientation of 11k, diphenhydramine (red pocket), and S-adenosyl-
L-
homocysteine (blue pocket) at HNMT active site.
Figure 5. Orientation of 8a, diphenhydramine (red pocket), and S-adenosyl-L-
homocysteine (blue pocket) at HNMT active site.
spectra were carried out using Finnigan SSQ 7000 Gas Chromato-
graph–Mass spectrometer. TLC was carried out using Art. 5735,
DC-Plastikfolien, Kieselgel 60 F254 sheets (Merck), the developing
solvents were CCl4/CH3OH (9:1) and the spots were visualized by
UV 366 and 254 nm.
trometer (IR-435) and FT-IR 1650 (Perkin Elmer). 1H NMR Spectra
were carried out using Joel, FX 90Q, NMR Spectrometer at 200 MHz
and Fourier transform EM-390, 300 MHz NMR Spectrometer. Mass

9014
Nahla. A. Farag et al. / Bioorg. Med. Chem. 16 (2008) 9009–9017
(s, 1H, C3-H), 7.70–6.75 (m, 8H, ArH). Anal. Calcd for C18H14O4
(294): C, 73.47; H, 4.76. Found: C, 73.38; H, 4.59.
4.2.5. General method for preparation of 6-[2-hydroxy-3-
(substituted) propoxy]-4-methyl-2H-1-benzopyran-2-one (8a–
i)
A solution of 6-(2,3-epoxypropoxy)-4-methyl-2H-1-benzopy-
ran-2-one (7, 2.32 g, 0.01 mol) and the appropriate amine or piper-
azine (0.01 mol) in ethanol 50 mL was stirred at reflux for 90 min
and allowed to cool, The precipitate obtained was collected, dried
and crystallized from ethanol.
4.2.5.1. 6-[2-hydroxy-3-(propylamino)propoxy]-4-methyl-2H-
1-benzopyran-2-one (8a). Yield 63%; mp: 130 °C. 1H NMR
(DMSO-d6, D2O) d ppm: 1.00 (t, 3H, –N–CH2– CH2–CH3), 2.20 (m,
2H, –N–CH2– CH2–CH3), 2.43 (s, 3H, C4–CH3), 2.64–3.04 (m, 4H,
–CH2–N–CH2– CH2–CH3), 3.89 (m, 1H, –CH–CH2–N–pr), 4.19 (m,
2H, OCH2), 6.38 (s, 1H, C3–H), 7.22–7.31 (m, 3H, ar-H), 9.57 (s,
1H, OH, or NH, exch.).
Figure 9. Orientation of diphenhydramine, amodiaquine, tacrine, and metoprine
(red pocket), and S-adenosyl-L-homocysteine (blue pocket) at HNMT active site.
Anal. Calcd for C16H21NO4: C, 65.98; H, 7.22; N, 4.81. Found: C,
66.47; H, 7.35; N, 4.94.
4.2. Chemistry
4.2.5.2. 6-[2-Hydroxy-3-(diethylamino)propoxy]-4-methyl-2H-
1-benzopyran-2-one (8b). Yield 74%; mp: 125 °C. 1H NMR
(DMSO-d6, D2O) d ppm: 1.00 (t, 6H, –N–(CH2–CH3)2), 2.43 (s, 3H,
C4–CH3), 2.48 (q, 4H, –N–(CH2–CH3)2), 2.64–3.03 (m, 2H, –CH2–
N–(CH2–CH3)2), 3.98 (m, 1H, –CH–CH2–N–(CH2–CH3)2), 4.19 (m,
2H, OCH2), 6.38 (s, 1H, C3–H), 7.21–7.50 (m, 3H, ar-H), 9.60 (s,
1H, OH, or NH, exch.). Anal. Calcd for C17H23NO4: C, 67.11; H,
7.54; N, 4.59. Found: C, 67.33; H, 7.57; N, 4.79.
4.2.1. Synthesis of 6-hydroxy-4-methyl-2H-1-benzopyran-2-
one (6).
Was previously reported.23-25
4.2.2. Synthesis of 7-hydroxy-4-phenyl-2H-1-benzopyran-2-
one (9).
Was previously reported.23-25
4.2.5.3. 6-[2-Hydroxy-3-(piperidino-1-yl)propoxy)]-4-methyl-
2H-1-benzopyran-2-one (8c). Yield 67%; mp: 128 °C. 1H NMR
4.2.3. Synthesis of 6-(2,3-epoxypropoxy)-4-methyl-2H-1-
benzopyran-2-one (7).
*
(DMSO-d6, D2O) d ppm: 1.93–2.23 (m, 6H, 3 CH2), 2.43 (s, 3H,
Was previously reported.23-25
C4–CH3), 2.54–2.61 (m, 6H, CH2–N–(CH2)2-), 3.77 (m, 1H, –CH–
CH2–N–), 4.21 (m, 2H, OCH2), 6.38 (s, 1H, C3–H), 7.10–7.48 (m,
3H, ar-H), 9.60 (s, 1H, OH, or NH, exch.). Anal. Calcd for
4.2.4. 7-(2,3-epoxypropoxy)-4-phenyl-2H-1-benzopyran-2-one
(10)
C18H23NO4: C, 68.14; H, 7.56; N, 4.42. Found: C, 67.83; H, 7.65;
Epichlorohydrin (2.8 mL, 0.03 mol) was added carefully while
stirring to a solution of 7-hydroxy-4-phenyl-2H-1-benzopyran-2-
one (9, 2.38 g, 0.01 mol) in ethanol 50 mL and potassium hydroxide
(0.56 g, 0.01 mol). The mixture was refluxed for 2 h, filtered while
hot, the solvent was removed under reduced pressure and the res-
idue partitioned between water and ethyl acetate. Evaporation of
the dried magnesium sulfate organic phase gave the product.
Recrystallization from ethanol afforded 2.4 g, yield 81%, mp: 126 °C.
1H NMR (CDCl3-d6) d ppm: 2.76–2.73 (d, 2H, CH2, J = 2.25 Hz),
4.05–2.79 (m, 1H, CH), 4.30–4.27 (d, 2H, OCH2, J = 2.52 Hz), 6.208
N, 4.78.
4.2.5.4. 6-[2-Hydroxy-3-(morpholin-1-yl)propoxy)]-4-methyl-
2H-1-benzopyran-2-one (8d). Yield 65%; mp: 144 °C. 1H NMR
(DMSO-d6, D2O) d ppm: 2.43 (s, 3H, C4–CH3), 2.76–2.79 (m, 6H,
CH2-N-(CH2)2), 3.42–3.66 (m, 4H, O–(CH2)2), 3.81–3.94 (m, 1H,
–CH–CH2–N–), 4.39 (m, 2H, OCH2), 6.38 (s, 1H, C3–H), 7.10–7.48
(m, 3H, ar-H), 9.70 (s, 1H, OH, or NH, exch.). Anal. Calcd for
C17H21NO5: C, 63.95; H, 6.58; N, 4.39. Found: C, 63.68; H, 6.26; N
4.60.
0
-20
-40
ICM docking
scores of 2AOT
-60
ICM docking
scores of 1QFT
-80
-100
-120
Compounds
Figure 10. Chart representation of Table 3.

Nahla. A. Farag et al. / Bioorg. Med. Chem. 16 (2008) 9009–9017
9015
4.2.5.5. 6-(2-Hydroxy-3-(piperazin-1-yl)propoxy)-4-methyl-2H-
1-benzopyran-2-one (8e). Yield 95%; mp: 159 °C. 1H NMR
(CDCl3-d6, D2O) d ppm: 2.44 (s, 3H, C4–CH3), 2.73–3.07 (m, 10H,
N–(CH2)5), 3.45, 3.99 (m, 1H, –CH–CH2–N–), 4.20 (s, 2H, OCH2),
6.37 (s, 1H, C3–H), 7.25–7.28 (m, 3H, ar-H), 9.58 (s, 1H, OH and/
or NH). Anal. Calcd for C17H22N2O4: C, 64.15; H, 6.92; N, 8.81.
Found: C, 64.22; H, 6.71; N, 8.85.
4.2.6.3. 7-(3-(Dimethylamino)-2-hydroxypropoxy)-4-phenyl-
2H-1-benzopyran-2-one (11c). Yield 65%; mp: 150 °C. 1H NMR
(CDCl3, D2O) d ppm: 2.33 (s, H, CH2–N(CH3)2), 3.44–3.52 (m, 1H,
–CH–CH2–N–), 4.03 (m, 2H, OCH2), 6.21 (s, 1H, C3–H), 6.90–7.29
(m, 8H, ar-H), 9.29 (s, 1H, OH, or NH, exch.). Anal. Calcd for
C20H21NO4: C, 70.79; H, 6.19; N, 4.13. Found: C, 70.57; H, 6.24; N,
4.23.
4.2.5.6. 6-(2-Hydroxy-3-(methylpiperazin-1-yl)propoxy)-4-met-
hyl-2H-1-benzopyran-2-one (8f). Yield 80%; mp: 207 °C. 1H NMR
(CDCl3-d6, D2O) d ppm: 2.41 (s, 3H, C4–CH3), 2.49 (s, 3H, –N–CH3),
2.51–3.11 (m, 10H, N–(CH2)5), 3.39, 3.67 (m, 1H, –CH–CH2–N–),
4.20 (s, 2H, OCH2), 6.37 (s, 1H, C3–H), 7.25–7.28 (s, 3H, ar-H),
9.55 (s, 1H, OH and/or NH). Anal. Calcd for C18H24N2O4: C, 65.06;
H, 7.23; N, 8.43. Found: C, 65.35; H, 7.62; N 8.80.
4.2.6.4. 7-(3-(Diethylamino)-2-hydroxypropoxy)-4-phenyl-2H-
1-benzopyran-2-one (11d). Yield 80%; mp: 229 °C. 1H NMR
(DMSO-d6, D2O) d ppm: 1.00 (t, 6H, –N–(CH2–CH3)2), 2.33 (q, 4H,
–N–(CH2–CH3)2), 2.64–3.30 (m, 2H, –CH2–N–(CH2–CH3)2), 4.03
(m, 1H, –CH–CH2–N–(CH2–CH3)2), 4.19 (m, 2H, OCH2), 6.21 (s,
1H, C3–H), 7.52–7.85 (m, 8H, ar-H), 9.29 (s, 1H, OH, or NH, exch.).
Anal. Calcd for C22H25NO4: C, 71.93; H, 6.81; N, 3.81. Found: C,
71.66; H; 6.31; N, 3.85.
4.2.5.7. 6-(2-Hydroxy-3-(phenylpiperazin-1-yl)propoxy)-4-met-
hyl-2H-1-benzopyran-2-one (8g). Yield 93%; mp: 137 °C. 1H
NMR (DMSO-d6, D2O) d ppm: 2.43 (s, 3H, C4–CH3), 2.62–3.65 (m,
10H, N–(CH2)5), 3.73, 3.93 (m, 1H, –CH–CH2–N–), 4.05 (s, 2H,
OCH2), 6.38 (s, 1H, C3–H), 6.56–7.29 (m, 8H, ar-H), 15.68 (s, 1H,
OH and/or NH). Anal. Calcd for C23H26N2O4: C, 70.05; H, 6.60; N,
7.11. Found: C, 70.11; H, 6.62; N, 7.15.
4.2.6.5. 7-(2-Hydroxy-3-(morpholin-1-yl)propoxy)-4-phenyl-
2H-1-benzopyran-2-one (11e). Yield 85%; mp: 226 °C. 1H NMR
*
(DMSO-d6, D2O) d ppm: 1.93–2.23 (m, 6H, 3 CH2), 3.31–3.10 (m,
6H, –CH2–N–(CH2)2–), 3.73 (m, 1H, –CH–CH2–N-), 4.23 (m, 2H,
OCH2), 6.21 (s, 1H, C3–H), 6.89–7.74 (m, 8H, ar-H), 9.60 (s, 1H,
OH, or NH, exch.). Anal. Calcd for C23H25NO4: C, 72.81; H, 6.59;
N, 3.69. Found: C, 73.11; H, 5.92; N, 3.66.
4.2.5.8.
6-(2-Hydroxy-3-(20-chloropiperazin-1-yl)propoxy)-
4-methyl-2H-1-benzopyran-2-one (8h). Yield 75%; mp: 100 °C.
MS: m/z (%) = 428.15 (M+, 1.59), 429.65 (0.78), 430 (0.78), 395.4
(0.63), 209 (100), 176 (50.16).1 H NMR (DMSO-d6, D2O) d ppm:
2.44 (s, 3H, C4–CH3), 3.33 (m, 10H, N–(CH2)5), 3.56–3.78 (m, 1H, –
CH–CH2–N–), 4.06 (s, 2H, OCH2), 6.38 (s, 1H, C3–H), 7.24–7.30 (m,
7H, ar-H), 9.59 (s, 1H, OH and/or NH). Anal. Calcd for C23H25N2O4Cl:
C, 64.41; H, 5.83; N, 6.53. Found: C, 64.58, H 5.36, N 6.37.
4.2.6.6. 7-(2-Hydroxy-3-(piperidin-1-yl)propoxy)-4-phenyl-2H-
1-benzopyran-2-one (11f). Yield 75%; mp: 218 °C. 1H NMR
(DMSO-d6, D2O) d ppm: 2.71–2.77 (m, 6H, CH2–N–(CH2)2), 2.85–
3.45 (m, 4H, O–(CH2)2), 3.85–3.98 (m, 1H, –CH–CH2–N–), 4.43
(m, 2H, OCH2), 6.24 (s, 1H, C3–H), 6.88–7.56 (m, 8H, ar-H), 9.70
(s, 1H, OH, or NH, exch.). Anal. Calcd for C22H23NO5: C, 69.29; H,
6.04; N, 3.67. Found: C, 69.30; H, 6.17; N, 3.69.
4.2.5.9. 6-(2-hydroxy-3-(20-methoxypiperazin-1-yl)propoxy)-4-
methyl-2H-1-benzopyran-2-one (8i). Yield 95%; mp: 125 °C. 1H
NMR (DMSO-d6, D2O) d ppm: 2.26 (s, 3H, C4–CH3), 2.45–3.29 (m,
10H, N–(CH2)5), 3.44 (m, 1H, –CH–CH2–N–), 3.80 (s, 3H, O CH3),
4.08 (s, 2H, OCH2), 6.41 (s, 1H, C3–H), 6.77–7.34 (m, 7H, ar-H),
9.57 (s, 1H, OH and/or NH). Anal. Calcd for C24H28N2O5: C, 67.92;
H, 6.60; N, 6.60. Found: C, 68.14; H, 6.46; N, 6.63.
4.2.6.7. 7-(2-Hydroxy-3-(piperazin-1-yl)propoxy)-4-phenyl-2H-
1-benzopyran-2-one (11g). Yield 75%; mp: 210 °C. 1H NMR
(CDCl3-d6, D2O) d ppm: 2.46–2.84 (m, 10H, N–(CH2)5), 3.37, 3.63
(m, 1H, –CH–CH2–N–), 4.10 (s, 2H, OCH2), 6.21 (s, 1H, C3–H),
7.29–7.55 (m, 8H, ar-H), 9.55 (s, 1H, NH, exch.), 9.57 (s, 1H, OH,
exch.). Anal. Calcd for C22H24N2O4: C, 69.47; H, 6.32; N, 7.37.
Found: C, 69.70; H, 6.33; N, 7.32.
4.2.6. General method for preparation of 7-2-hydroxy-(3-substi-
tuted) propoxy)-4-phenyl-2H-1-benzopyran-2-one (11a–k)
A solution of 7-(2,3-epoxypropoxy)-4-phenyl-2H-1-benzopy-
ran-2-one (10, 2.94 g, 0.01 mol) and the appropriate amine or
piperazine (0.01 mol) in ethanol 50 mL was stirred at reflux for
90 min and allowed to cool, The residue obtained was collected,
dried and crystallized from ethanol.
4.2.6.8. 7-(2-Hydroxy-3-(4-methylpiperazin-1-yl)propoxy)-4-
phenyl-2H-1-benzopyran-2-one (11h). Yield 85%; mp: 233 °C.
1H NMR (DMSO-d6, D2O) d ppm: 2.49 (s, 3H, –N–CH3), 2.51–
3.11 (m, 10H, N–(CH2)5), 3.39, 3.67 (m, 1H, –CH–CH2–N–), 4.20
(s, 2H, OCH2), 6.37 (s, 1H, C3–H), 7.25–7.28 (s, 3H, aromatic pro-
tons), 9.55 (s, 1H, OH and/or NH, exch.). Anal. Calcd for
C23H26N2O4: C, 70.05; H, 6.59; N, 7.11. Found: C, 69.79; H,
6.27; N, 7.35.
4.2.6.1. 7-(2-Hydroxy-(3-propylamino)propoxy)-4-phenyl-2H-
1-benzopyran-2-one (11a). Yield 65%; mp: 230 °C. 1H NMR
(DMSO-d6, D2O) d ppm: 1.00 (t, 3H, –N–CH2–CH2–CH3), 2.64 (m,
2H, –N–CH2–CH2–CH3), 2.85–3.12 (m, 4H, –CH2–N–CH2–CH2–
CH3), 3.64 (m, 1H, –CH–CH2–N–pr), 4.21 (m, 2H, OCH2), 6.22 (s,
1H, C3–H), 7.09–7.54 (m, 8H, ar-H), 9.57 (s, 1H, OH, or NH, exch.).
Anal. Calcd for C21H23NO4: C, 71.39; H, 6.52; N, 3.97. Found: C,
71.66; H, 6.31; N, 3.98.
4.2.6.9. 7-(2-Hydroxy-3-(4-phenylpiperazin-1-yl)propoxy)-4-
phenyl-2H-1-benzopyran-2-one (11i). Yield 90%; mp: 91 °C. 1H
NMR (DMSO-d6, D2O) d ppm: 2.50–3.28 (m, 10H, N–(CH2)5),
3.69–3.89 (m, 1H, –CH–CH2–N–), 4.22 (s, 2H, OCH2), 6.22 (s, 1H,
C3–H), 6.89–7.75 (m, 13H, ar-H), 15.69 (s, 1H, OH and/or NH,
exch.). Anal. Calcd for C28H28N2O4: C, 73.68; H, 6.14; N, 6.14.
Found: C, 73.59; H, 5.91; N 6.11.
4.2.6.2. 7-(3-Butylamino)-2-hydroxypropoxy)-4-phenyl-2H-1-
benzopyran-2-one (11b). Yield 55%; mp: 228 °C. 1H NMR
(DMSO-d6, D2O) d ppm: 1.00 (t, 3H, –N–CH2–CH2–CH2–CH3), 1.70–
1.92 (m, 4H, –N–CH2–CH2–CH2–CH3), 2.43 (s, 3H, C4–CH3), 2.85–
3.12 (m, 4H, –CH2–N–CH2–CH2– CH2–CH3), 3.74 (m, 1H, –CH–CH2–
N–bu), 4.23 (m, 2H, OCH2), 6.22 (s, 1H, C3–H), 7.012–7.56 (m, 8H,
ar-H), 9.55 (s, 1H, OH, or NH, exch.). Anal. Calcd for C22H25NO4: C,
71.93; H, 6.81; N 3.81. Found: C, 71.72; H, 6.47; N, 3.83.
4.2.6.10. 7-(2-Hydroxy-3-(20-chlorophenylpiperazin-1-yl)pro-
poxy)-4-phenyl-2H-1-benzopyran-2-one (11j). Yield 95%; mp:
139 °C. MS: m/z (%) = 490.25 (M+, 1.05), 294.15 (2.44), 238 (6.64),
209 (100). 1H NMR (CDCl3-d6, D2O) d ppm: 2.62–2.90 (m, 10H,
N–(CH2)5), 3.07–3.12 (m, 1H, –CH–CH2–N–), 4.10 (s, 2H, OCH2),
6.23 (s, 1H, C3–H), 6.77–7.49 (m, 12H, ar-H), 9.21 (s, 1H, OH and/
or NH, exch.). Anal. Calcd for C28H27N2O4Cl: C, 68.50; H, 5.50; N,
5.71. Found: C, 68.36; H, 5.74; N 5.84.

9016
Nahla. A. Farag et al. / Bioorg. Med. Chem. 16 (2008) 9009–9017
4.2.6.11. 7-(2-Hydroxy-3-(20-methoxyphenylpiperazin-1-yl)pro-
poxy)-4-phenyl-2H-1-benzopyran-2-one (11k). Yield 85%; mp:
129 °C. 1H NMR (CDCl3-d6, D2O) d ppm: 2.62–2.87 (m, 10H, N–
(CH2)5), 3.31–3.60 (m, 1H, –CH–CH2–N–), 3.87 (s, 3H, O CH3),
4.11 (s, 2H, OCH2), 6.22 (s, 1H, C3–H), 6.951 (s, 4H, 2-methoxy-
phenyl-H), 6.77–7.34 (m, 8H, ar-H), 9.24 (s, 1H, OH and/or NH,
exch.). Anal. Calcd for C29H30N2O5: C, 71.60; H, 6.17; N, 5.76.
Found: C, 71.46; H, 5.86; N, 5.66.
4.3.2. Statistical analysis
The heights of the contractions were determined and the cumu-
lative concentration–response curves were plotted for all the
recordings. EC50 and maximum response to histamine data were
expressed as means SEM. The EC50 and maximum response to
histamine in the presence of pheniramine maleate and different
concentrations of test compounds were compared with those ob-
tained in the presence of saline using Student’s t-test.29
4.3. Pharmacology
4.4. Docking studies
4.3.1. Materials and methods
All docking studies were performed using ‘‘Internal Coordinate
Mechanics (Molsoft ICM 3.4-8C)”.
To understand the obtained pharmacological data on a struc-
tural basis, we evaluate compounds (two different classes of benz-
opyrone derivatives) through molecular modeling and docking
techniques using program.
Twenty representative new compounds, 8a, 8b, 8c, 8d, 8e, 8f,
8g, 8h, 8i, 11a, 11b, 11c, 11d, 11e, 11f, 11g, 11h, 11i, 11j and
11k were tested for their antihistaminic activity.
4.3.1.1. Animals. Male guinea pigs (250–400 g) were used for all
experiments.
ICM docking is probably the most accurate predictive tool of
binding geometry today.30–33
4.3.1.2. Tissue preparation. Male guinea pigs were sacrificed by a
blow to the base of the skull and cervical dislocation. Clean ileal seg-
ments of 2–3 cm long were prepared from the guinea pigs. One
end was fixed to a glass aerating tube and the other to isotonic trans-
ducer (Bioscience, England). The wholepreparation was set up in 10-
mL organ bath containing aerated Tyrode solution (NaCl: 1.0; CaCl2:
0.2; KCl: 0.2; MgCl2ꢂ6H2O: 0.2; NaH2PO4: 0.05; NaHCO3: 1, and glu-
cose: 2.0 g/dL). The Tyrode solution was maintained at 37 °C.
The aim of the flexible docking calculations is prediction of cor-
rect binding geometry for each binder. The goal is to have an ade-
quate 3D-model of the receptor pocket we are planning to dock
compounds to. Usually dock the tested compounds to determine
ICM scores.
In order to compare the binding affinity of the newly synthe-
sized benzopyrone analogs, we docked the compounds 8a–i,
11a–k into the empty binding site of the experimentally known
Histamine-binding protein (HBPs) which reveals two binding sites
for the same ligand (histamine) (1QFT) and histamine N-methyl-
transferase (HNMT) complexes with Diphenhydramine (2AOT),6
(Table 3) which offer a new strategy for controlling histamine-
based diseases.21
25
4.3.1.3. Protocols.
(1) The possible in vitro antihistaminic activity of the 20 test
compounds was examined by producing a cumulative con-
centration–response curve of histamine-induced contraction
of guinea pig ileum after exposing tissue to each experimen-
tal solution.26 In addition to histamine diphosphate solution,
sixty two experimental solutions were used:
4.4.1. Generation of ligand and enzyme structures
The crystal structure of histamine-binding protein (HBPs)
(1QFT),21 and the crystal structure of histamine N-methyltransfer-
ase (HNMT) complexed with diphenhydramine (2AOT)6 were
downloaded through the Protein Data Bank PDB/RCSB site and
(a) Three increment concentrations of each of the 20 test
compounds (0.1, 0.5, and 1.0 mg mLꢁ1).
(b) Pheniramine maleate (5 l
g mLꢁ1).
(c) 0.01 mL normal saline. The threshold value (T.R) is the
*
saved as .pdb file.
concentration of histamine (ꢀ10-3
l
g mLꢁ1) at which
A set of benzopyrone analogs synthesized to inhibit (HBP) and
(HNMT) was compiled by us earlier.
the strip of ileum recorded the first contraction, whereas
the maximal response (M.R) is the concentration at
which further increases in concentration does not pro-
duce higher height of contractions. Consecutive concen-
trations of histamine were added every 2 min (between
the threshold value and the maximal response). The per-
centage of maximum contraction obtained with each
concentration in proportion to the maximum contrac-
tion obtained with saline was plotted against the con-
centration of histamine.27 The effective concentration
Molecular modeling of the target compounds were built using
ChemDraw Ultra version 8.0.3 and minimized their energy through
Chem3D Ultra version 8.0.3/MOPAC, Jop Type: Minimum RMS Gra-
dient of 0.010 kcal/mol and RMS distance of 0.1 Å , and saved as
*
MDL MolFile ( .mol).
4.4.2. Docking using Molsoft ICM 3.4-8C program
(1) Convert our PDB file (1QFT) and (2AOT) into an ICM object:
This conversion involves addition of hydrogen bonds, assignment
of atoms types, and charges from the residue templates.
Click on MolMechanics/Convert/Protein, and then delete water
molecules.
of histamine causing 50% of maximum response (EC50
)
in each experiment was extrapolated from the concen-
tration–response curve of the corresponding experi-
ment.28 The shift of the cumulative concentration–
response curves obtained in the presence of different
concentrations of the test compounds and pheniramine
maleate was examined by comparing the EC50 of each
solution with that of saline.
(2) To perform ICM small molecule docking:
(a) Setup docking project:
(1) Set project name: Click on docking/set project name,
press OK.
(2) In addition, the maximum response (Emax) to histamine
obtained in the presence of all test compound concentra-
tions and pheniramine maleate was compared with that of
saline. The effect of different concentrations of the test com-
pounds and pheniramine maleate was tested on different
ileum strips (n = 5).
(2) Setup the receptor: Click on docking/receptor setup,
enter the receptor molecule in the receptor mole-
*
cule data entry box (a_ ) will do, then click on iden-
tify the binding sites button to identify the potential
ligand binding pockets, press OK. After the receptor

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setup is complete, the program normally displays
the receptor with selected binding site residues
highlighted in yellow xstick presentation.
(3) Review and adjust binding site: ICM makes a box
around the ligand binding site based on the infor-
mation entered in the receptor setup section. The
position of the box encompasses the residues
expected to be involved in ligand binding. Click on
the menu docking/review/adjust ligand/box.
(4) Make receptor maps: the step now is to construct
energy maps of the environment within the docking
box. Click on menu docking/make receptor maps,
select the resolution of the map by entering a value
into the grid cell size data entry box which is 0.5,
this step takes few minutes.
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(b) Start docking simulation: Use interactive docking to dock
one ligand at a time. Click on menu docking/interactive docking/
mol table ligand, use the drop down arrow to find the table of li-
gand and/or Compounds we wish to dock, and then enter the thor-
oughness which represent the length of simulation. Generally 1 is
reasonable value, select Calc ICM Score, then select Display run
which display the ligand sampling the energy in the ligand binding
project.
(3) Display the result: Click docking/browse/stack conforma-
tions. ICM stochastic global optimization algorithm attempts to
find the global minimum of the energy function that include five
grid potentials describing interaction of the flexible ligand with
the receptor and internal conformational energy of the ligand, dur-
ing this process a stack of alternative low energy conformations is
saved (Table 3 and Fig. 10).
28. Ortiz De Urbina, A. V.; Martin, M. L.; Montero, M. J.; Carron, R.; Servilla, M. A.;
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References and notes
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Functional Aspects. In Neuromethods 5. Neurotransmitter Enzymes; Boulton, A.
A., Baker, G. B., Tu, P. H., Eds.; Humana Press: Clifton, NJ, 1986; p 147.
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32. Halperin, I.; Ma, B.; Wolfson, H.; Nussinov, R. Proteins: Struct. Funct. Genet.
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