Novel 4-substituted-2(1H)-phthalazinone derivatives: Synthesis, molecular modeling study and their effects on α-receptors

Article abstract of DOI:10.1007/s12272-013-0095-5

Novel 4-(4-bromophenyl)phthalazine derivatives connected via an alkyl spacer to amine or N-substituted piperazine were designed and synthesized as promising α-adrenoceptor antagonists. The structures of the phthalazine derivatives were established using e

Full text of DOI:10.1007/s12272-013-0095-5

Arch. Pharm. Res. (2013) 36:671–683
DOI 10.1007/s12272-013-0095-5
RESEARCH ARTICLE
Novel 4-substituted-2(1H)-phthalazinone derivatives: synthesis,
molecular modeling study and their effects on a-receptors
•
Nadia A. Khalil Eman M. Ahmed
•
•
Hosam A. Elshihawy Sawsan A. Zaitone
Published online: 1 April 2013
Ó The Pharmaceutical Society of Korea 2013
Abstract Novel 4-(4-bromophenyl)phthalazine deriva-
tives connected via an alkyl spacer to amine or N-substituted
piperazine were designed and synthesized as promising a-
adrenoceptor antagonists. The structures of the phthalazine
derivatives were established using elemental and spectral
analyses. Twelve of the tested compounds displayed sig-
nificant a-blocking activity. Molecular modeling studies
were performed to rationalize the biological results. Among
the tested compounds, 7j displayed the best-fitting score and
the highest in vitro activity.
a-Adrenergic receptors (a-ARs) play an important role in the
regulation of a variety of physiological processes, particularly
within the cardiovascular system and are divided into two main
subtypes a1-anda2-ARs (Jain et al. 2008). The a1-ARs mediate
actions in the sympathetic nervous system through the binding
ofthe catecholamines, epinephrine andnorepinephrine (Romeo
et al. 2011). Compounds acting as antagonists at various post-
junctional a1-ARs are frequently used in the therapy of
hypertension, benign prostatic hyperplasia, lower urinary tract
symptoms and cardiac arrhythmia (Frishman and Kotob 1999;
Kojima et al. 2009). Prazosin I, a common antihypertensive
drug, is considered the prototype of this class which selectively
blocks postsynaptic a1-ARs (Jain et al. 2008), (Fig. 1).
Keywords Phthalazinones ꢀ a-Adrenoceptor antagonists ꢀ
Molecular modeling ꢀ Arylpiperazine
In this context, the search for selective a1-adrenoceptor
antagonists has been, and still is, an important goal in the
medicinal chemistry. In recent decades, various new a1-AR
antagonists have been synthesized, many of them contain
arylpiperazine moiety (Handzlik et al. 2008; Handzlik et al.
2010; Sharma et al. 2010). It has been reported that aryl-
piperazine moiety linked to different heterocyclic systems
through polymethylene spacer is a key element for a1-AR
affinity (Barbaro et al. 2001; Kulig et al. 2009; Abou-Seri
et al. 2011). Some known arylpiperazine derivatives such
as SGB 1534 II, 5-methylurapidil III, RA36 IV, WAY-
100635 V and the imidazopyridazinone derivative VI can
be considered as classical examples of potent a1-adreno-
ceptor antagonists which is useful in various pharmaco-
logical assays including binding studies, as well as in vivo
tests (Betti et al. 2002) (Fig. 1). Besides, there are many
reports in the literature regarding the effect of compounds
containing a pyridazin-3(2H)-one fragment and their benzo
analogue, phthalazinone, on the cardiovascular system
(Demirayak et al. 2004a; Del Olmo et al. 2006; Strappa-
ghetti et al. 2006). A phthalazine derivative, hydralazine,
one of the first antihypertensive agents, is considered as a
Introduction
The adrenergic system is an essential regulator of neuronal,
endocrine, cardiovascular, andmetabolicfunctions. Adrenergic
receptors (ARs) are endogenously activated by adrenaline and
noradrenaline and are members of the G-protein coupled
receptors (GPCRs) super-family that transmit their signal
across the plasma membrane (Calzada and de Artin˜ano 2001).
N. A. Khalil ꢀ E. M. Ahmed (&)
Department of Pharmaceutical Organic Chemistry, Faculty
of Pharmacy, Cairo University, Cairo, Egypt
e-mail: dr_eman2001@hotmail.com
H. A. Elshihawy
Organic Chemistry Division, Faculty of Pharmacy, Suez Canal
University, Ismailia 41522, Egypt
S. A. Zaitone
Department of Pharmacology & Toxicology, Faculty
of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
123

672
N. A. Khalil et al.
Fig. 1 Structures of potent
selective a1-adrenoceptor
antagonists
NH2
H
N
O
H3CO
H3CO
N
N
N
OCH3
N
N
O
O
N
N
O
Prazosin I
SGB 1534 II
H3CO
N
H3CO
N
CH3
N
CH3
N
H
N
N
O
N
N
N
O
N
H3C
CH3
H
O
IV
RA36
5-Methylurapidil
III
H3CO
O
N
N
O
N
N
N
N
N
OCH3
N
N
N
VI
WAY-100635
V
lead for developing new drugs, due to its direct vasodilator
action (Bang et al. 1998). Structural modification of
hydralazine led to the discovery of phthalazine derivatives
with broad spectra on the cardiovascular system including
antihypertensive effects (Demirayak et al. 2004b), inhibi-
tion of platelet aggregation (Sotelo et al. 2002), and inhi-
bition of phosphodiesterases (Kagayama et al. 2009).
A specially useful model considering the structural
requirements for a1-AR affinity concerning phenylpiper-
azine derivatives, has been postulated by Barbaro et al.
(2001). Barbaro’s model, has postulated five pharmacophore
features: A positively ionizable group, corresponding to the
more basic nitrogen atom of the arylpiperazine ring (A). An
ortho- or para substituted phenyl ring, both of which con-
stitute the arylpiperazine system (B) A polar group (corre-
sponding to the phthalazinone ring) that provides a hydrogen
bond acceptor (HBA) feature, filling one of the portions of
the pharmacophore that is required at the edge of the mole-
cule opposite the arylpiperazine moiety (C). Additionally,
they possess an alkyl spacer between piperazine nitrogen and
nitrogen placed close to carbonyl oxygen (HBA), which may
consist of 2–7 carbons (D). Finally, a hydrophobic moiety,
corresponding to the terminal molecular portions directly
linked to the phthalazinone ring, was hypothesized to fit one
of the five features of the model (E).
Taking in consideration the structural requirements for
a1-AR affinity, the final phthalazinone targets, 4a–f, 5a–c, 6
and 7a–j were designed and synthesized. This study was
directed at the modification of (i) a pharmacophoric portion
constituted by the introduction of 2- or 4-methoxy; 2-ethoxy;
4-chloro into the phenyl ring or replacement the phenylpip-
erazine moiety by a tertiary amino function (ii) to probe the
influence of the terminal cyclic substituent on a-AR blocking
activity, a 4-bromophenyl moiety was placed at the 4-posi-
tion of the phthalazin-1(2H)-one system (iii) an alkyl chain
linker of one, two or three carbon atoms between these two
substructures for investigation as a-AR antagonists, (Fig. 2).
Materials and methods
Chemistry
Melting points were determined on Griffin apparatus and the
values given are uncorrected. IR spectra were determined on
Shimadzu IR 435 spectrophotometer (KBr, cm-1). 1H-NMR
123

Phthalazinone derivatives, effects on a-receptors
673
Fig. 2 General structures of the
newly designed compounds
C
D
O
O
A
R1
N
R
N
N
N
n
N
N
B
n
N
R
E
Br
Br
4a-e, 5a-c, 7g-j
4f, 6, 7a-f
spectra were carried out using Varian Mercury-300
(300 MHz) Spectrophotometer using TMS as internal stan-
dard. Chemical shift values are recorded in ppm on d scale,
Microanalytical Center, Cairo University, Egypt. Mass
spectra were recorded on a GCMP-QP1000 EX Mass spec-
trometer, Microanalytical Center, Cairo University, Egypt.
Elemental analyses were carried out at the Microanalytical
Center, CairoUniversity,Egypt. Progressofthereactionswas
monitored using TLC sheets precoated with UV fluorescent
silica gel Merck 60F 254 using acetone/benzene (1:9) and
were visualized using UV lamp.
phthalazin-1(2H)-one (1) (3.01 g, 0.01 mol) and 1-bromo-3-
chloropropane (4.72 g, 2.96 mL, 0.03 mol). The reaction
mixturewaspouredontocoldwater, extractedwithmethylene
chloride (3 9 10 mL) and dried (Na2SO4). The solvent was
evaporated under reduced pressure, the residue was triturated
with benzene, and the formed white solid product was filtered
then crystallized from benzene-petroleum ether (60–80 °C).
Yield 47 %; mp 161–162 °C; IR (KBr) cm-1: 3,070
(C–H aromatic), 2,978, 2,885 (C–H aliphatic), 1,654
1
(C=O), 1,604 (C=N); HNMR (DMSO–d6); 1.85–2.05 (m,
2H, –CH2–), 2.68 (t, 2H, J = 6.3 Hz, CH2–N), 3.98 (t, 2H,
J=6.3 Hz, CH2–Cl), 6.95–7.67 (m, 6H, ArH), 7.90–8.03
(m, 2H, ArH); MS (EI) m/z (% rel. Int.): 300 (C14H9BrN2O,
100), 376 (M?, 0.08), 378 (M ? 2, 0.07), 380 (M ? 4,
0.07). Anal. Calcd for C17H14BrClN2O: C, 54.06; H, 3.74;
N, 7.42. Found: C, 53.95; H, 3.88; N, 7.35.
All chemicals were obtained from Aldrich, Fluka, or
Merck chemicals.
4-(Bromophenyl)phthalazin-1(2H)-one was prepared as
reported (Colotta et al. 1994).
4-(4-Bromophenyl)-2-(2-chloroethyl)phthalazin-1(2H)-one
(2)
General procedure for the synthesis of compounds 4a–
f and 5a–c
A mixture of 4-(bromophenyl)phthalazin-1(2H)-one (1) (3.01 g,
0.01 mol), 1-bromo-2-chloroethane (7.17 g, 4.16 mL, 0.05 mol)
and anhydrous K2CO3 (6.90 g, 0.05 mol) in anhydrous DMF
(30 mL) was stirred at room temperature 2 h. The mixture was
poured onto water, the resulting white solid was filtered, dried and
crystallized from benzene-petroleum ether (60–80 °C).
A mixture of an appropriate 2 or 3 (0.003 mol), anhydrous
K2CO3 (2.07 g, 0.015 mol) and few specks of KI in dry
acetonitrile (20 mL) was heated under reflux for 30 min.
An appropriate secondary amine or N-arylpiperazine
(0.009 mol) was added to the hot reaction mixture and the
heating was continued for 10 h. After cooling, the reaction
mixture was poured with continuous stirring onto ice-cold
water (25 mL). The separated solid was filtered, and
crystallized from isopropanol.
Yield 62 %; mp 119–120 °C; IR (KBr) cm-1: 3,097 (C–H
aromatic), 2,950, 2,897 (C–H aliphatic), 1,658 (C=O), 1,589
(C=N); 1HNMR (DMSO–d6): 4.04 (t, 2H, J = 6.3 Hz, CH2–
N), 4.53 (t, 2H, J = 6.3 Hz, CH2–Cl), 7.54–7.95 (m, 6H,
ArH), 8.32–8.40 (m, 2H, ArH); MS (EI) m/z (% rel. Int.): 300
(C14H9BrN2O, 69.82), 362 (M?, 36.71), 364 (M ? 2, 46.65),
366 (M ? 4, 14.62). Anal. Calcd for C16H12BrClN2O: C,
52.85; H, 3.33; N, 7.70. Found: C, 52.55; H, 3.15; N, 7.85.
4-(4-Bromophenyl)-2-[2-(4-phenylpiperazin-1-yl)ethyl]
phthalazin-1(2H)-one (4a) Yield: 58 %; mp 240–241
°C; IR (KBr) cm-1: 3,074 (C–H aromatic), 2,947, 2,850
(C–H aliphatic), 1,651 (C=O), 1,600 (C=N); 1HNMR
(DMSO–d6): 2.60–2.64 (m, 4H, piperazine), 2.80 (t, 2H,
CH2–N), 2.82–2.95 (m, 4H, piperazine), 4.37 (t, 2H, CH2–
N), 6.90–7.86 (m, 11H, ArH), 8.21–8.30 (m, 2H, ArH);
EIMS (% rel. abundance): 175 (C11H15N2, 78.67), 312
(C15H9BrN2O, 27.06), 488 (M?, 6.63), 490 (M ? 2, 4.84).
4-(4-Bromophenyl)-2-(3-chloropropyl)phthalazin-1(2H)-
one (3)
The compound 3 was prepared following the procedure
described for the compound 2 by using 4-(bromophenyl)
123

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N. A. Khalil et al.
Anal. Calcd for C26H25BrN4O, C, 63.81; H, 5.15; N, 11.45;
Found C, 63.55; H, 5.00; N, 11.54.
100), 313 (C15H10BrN2O, 4.10), 328 (C16H13BrN2O, 27),
(M?, 10.94), 520 (M ? 2, 10.76). Anal. Calcd for
C27H27BrN4O2, C, 62.43; H, 5.24; N, 10.79; Found C, 62.15;
H, 5.00; N, 10.98.
4-(4-Bromophenyl)-2-{2-[4-(4-chlorophenyl)piperazin-1-yl]
ethyl}phthalazin-1(2H)-one (4b) Yield: 64 %; mp 141–
142 °C; IR (KBr) cm-1: 3,074 (C–H aromatic), 2,950,
2,850 (C–H aliphatic), 1,651 (C=O), 1,602 (C=N); 1HNMR
(DMSO–d6): 2.58–2.62 (m, 4H, piperazine), 2.79 (t, 2H,
CH2–N), 2.98–3.15 (m, 4H, piperazine), 4.37 (t, 2H, CH2–
N), 6.91–6.95 (m, 2H, ArH), 7.19–7.22 (m, 2H, ArH),
7.56–7.92 (m, 6H, ArH), 8.31–8.39 (m, 2H, ArH); EIMS
(% rel. abundance): 209 (C11H14ClN2, 100), 522 (M?,
3.97), 524 (M ? 2, 3.77), 526 (M ? 4, 1.51). Anal. Calcd
for C26H24BrClN4O, C, 59.61; H, 4.62; N, 10.70; Found C,
59.55; H, 4.82; N, 10.95.
4-(4-Bromophenyl)-2-[2-(piperidin-1-yl)ethyl]phthalazin-1
(2H)-one (4f) Yield: 62 %; mp 99–100 °C; IR (KBr)
cm-1: 3,081 (C–H aromatic), 2,934, 2,863 (C–H aliphatic),
1
1,651 (C=O), 1,600 (C=N); HNMR (CDCl3): 1.21–1.26
(m, 2H, CH2 piperidine), 1.50–1.75 (m, 4H, 2CH2 piperi-
dine), 1.85–2.10 (m, 4H, 2CH2 piperidine), CH2–N), 3.98
(t, 2H, J = 6.9 Hz, CH2–N), 4.61 (t, 2H, J=6.3 Hz, CH2–
N), 7.27–8.52 (m, 6H, ArH), 8.49 (d, 2H, ArH); Anal.
Calcd for C21H22BrN3O, C, 61.17; H, 5.38; N, 10.19;
Found C, 61.45; H, 5.15; N, 10.22.
4-(4-Bromophenyl)-2-{2-[4-(2-ethoxyphenyl)piperazin-1-yl]
ethyl}phthalazin-1(2H)-one (4c) Yield: 65 %; mp
167–168 °C; IR (KBr) cm-1: 3,043 (C–H aromatic), 2,935,
2,897 (C–H aliphatic), 1,654 (C=O), 1,604 (C=N); 1HNMR
(DMSO–d6): 1.31 (t, 3H, J = 6.9 Hz, CH3CH2), 2.58–2.64
(m, 4H, piperazine), 2.79 (t, 2H, J = 6.9 Hz, CH2–N),
2.85–3.01 (m, 4H, piperazine), 3.99 (q, 2H, J = 6.9 Hz,
CH3CH2), 4.35 (t, 2H, J = 6.3 Hz, CH2–N), 6.82–6.96 (m,
4H, ArH), 7.44–7.90 (m, 6H, ArH), 8.27–8.36 (m, 2H,
ArH); EIMS (% rel. abundance): 219 (C13H19N2O, 100),
532 (M?, 5.39), 534 (M ? 2, 5.44). Anal. Calcd for
C28H29BrN4O2, C, 63.04; H, 5.48; N, 10.50; Found C,
62.90; H, 5.35; N, 10.68.
4-(4-Bromophenyl)-2-{3-[4-(4-chlorophenyl)piperazin-1-yl]
propyl}phthalazin-1(2H)-one (5a) Yield: 55 %; mp 143–
144 °C; IR (KBr) cm-1: 3,074 (C–H aromatic), 2,947, 2,816
(C–H aliphatic), 1,662 (C=O), 1,593 (C=N); 1HNMR
(DMSO–d6) d: 2.35–2.45 (m, 2H, CH2), 2.60–2.70 (m, 4H,
piperazine), 2.80 (t, 2H, J = 6.3 Hz, CH2–N), 3.18–3.20 (m,
4H, piperazine), 4.20 (t, 2H, J = 6.3 Hz, CH2–N), 6.89–7.97
(m, 10H, ArH), 8.32–8.35 (m, 2H, ArH); EIMS (% rel.
abundance):
312
(C15H799BrN2O,
16.97),
314
(C15H891BrN2O,17.44), 536 (M?, 0.06), 538 (M ? 2, 0.02).
Anal. Calcd for C27H26BrClN4O, C, 60.29; H, 4.87; N,
10.42; Found C, 60.10; H, 4.55; N, 10.58.
4-(4-Bromophenyl)-2-{3-[4-(2-ethoxyphenyl)piperazin-1-yl]
propyl}phthalazin-1(2H)-one (5b) Yield: 55 %; mp
198–199 °C; IR (KBr) cm-1: 3,090 (C–H aromatic), 2,927,
2,855 (C–H aliphatic), 1,651 (C=O), 1,605 (C=N); 1HNMR
(DMSO–d6) d: 1.32 (t, 3H, CH3CH2), 2.40–2.45 (m, 2H,
CH2), 2.55–2.60 (m, 4H, piperazine), 2.85 (t, 2H,
J = 6.3 Hz, CH2–N), 3.22–3.28 (m, 4H, piperazine), 4.05
(q, 2H, CH3CH2), 4.32 (t, 2H, J = 6.3 Hz, CH2–N),
7.48–7.83 (m, 10H, ArH), 8.24–8.26 (m, 2H, ArH); EIMS
4-(4-Bromophenyl)-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]
ethyl}phthalazin-1(2H)-one (4d) Yield: 60 %; mp
234–235 °C; IR (KBr) cm-1: 3,105 (C–H aromatic), 2,931,
2,893 (C–H aliphatic), 1,654 (C=O), 1,589 (C=N); 1HNMR
(DMSO–d6): 2.56–2.59 (m, 4H, piperazine), 2.85 (t, 2H,
CH2–N), 3.00–3.09 (m, 4H, piperazine), 3.77 (s, 3H,
OCH3), 4.38 (t, 2H, CH2–N), 6.85–6.94 (m, 2H, ArH),
7.54–7.91 (m, 8H, ArH), 8.32–8.35 (m, 2H, ArH); EIMS
(% rel. Abundance): 205 (C12H17N2O, 2.53), 313
(C15H10BrN2O, 0.09), 518 (M?, 0.13), 520 (M ? 2, 0.12).
Anal. Calcd for C27H27BrN4O2, C, 62.43; H, 5.24; N,
10.79; Found C, 62.11; H, 5.35; N, 10.66.
(% rel. abundance): 219 (C13H19N2O, 8.17), 233
79
(C14H21N2O, 36.74), 313 (C15H BrN2O, 3.19),
81
10
315(C15H BrN2O, 3.55), 546 (M?, 3.05), 548 (M ? 2,
10
2.58). Anal. Calcd for C29H31BrN4O2, C, 63.62; H, 5.71;
N, 10.23; Found C, 63.55; H, 5.82; N, 10.35.
4-(4-Bromophenyl)-2-{2-[4-(4-methoxyphenyl)piperazin-1-yl]
ethyl}phthalazin-1(2H)-one (4e) Yield: 70 %; mp
225–226 °C; IR (KBr) cm-1: 3,090 (C–H aromatic), 2,943,
2,835 (C–H aliphatic), 1,654 (C=O), 1,596 (C=N); 1HNMR
(DMSO–d6): 2.59–2.62 (m, 4H, piperazine), 2.80 (t, 2H,
CH2–N), 2.92–3.05 (m, 4H, piperazine), 3.66 (s, 3H, OCH3),
4.35 (t, 2H, CH2–N), 6.80–6.84 (m, 2H, ArH), 6.94 (d, 2H,
ArH), 7.44 (d, 2H, ArH), 7.57–7.91 (m, 4H, ArH), 8.30–8.45
(m, 2H, ArH); EIMS (% rel. Abundance): 205 (C12H17N2O,
4-(4-Bromophenyl)-2-{3-[4-(4-methoxyphenyl)piperazin-1-
yl]propyl}phthalazin-1(2H)-one (5c) Yield: 67 %; mp 128
–129 °C; IR (KBr) cm-1: 3,100 (C–H aromatic), 2,927, 2,855
(C–H aliphatic), 1,657 (C=O), 1,620 (C=N); 1HNMR
(DMSO–d6) d: 2.32–2.40 (m, 2H, CH2), 2.53–2.60 (m, 4H,
piperazine), 2.80 (t, 2H, CH2–N), 3.20–3.40 (m, 4H,
piperazine), 3.66 (s, 3H, OCH3), 4.40 (t, 2H, CH2–N),
6.81–7.60 (m, 10H, ArH), 7.90–8.20 (m, 2H, ArH); EIMS
123

Phthalazinone derivatives, effects on a-receptors
675
(% rel. abundance): 315 (C15H BrN2O, 32.15), 532 (M?,
81
60.31; H, 5.06; N, 10.55. Found: C, 60.59; H, 4.80; N,
10.85.
10
11.96), 534 (M ? 2, 13.05). Anal. Calcd for C28H29
BrN4O2, C, 63.04; H, 5.48; N, 10.50; Found C, 62.85; H,
5.25; N, 10.45.
4-(4-Bromophenyl)-2-(pyrrolidin-1-ylmethyl)phthalazin-1
(2H)-one (7c) Yield 65 %; mp 164–165 °C; IR (KBr)
cm-1: 3,078 (C–H aromatic), 2,947, 2,873 (C–H aliphatic),
1,651 (C=O), 1,604 (C=N); 1HNMR (DMSO–d6): 1.62
(t, 4H, 2CH2 pyrrolidine), 2.75 (t, 4H, 2CH2–N pyrroli-
dine), 5.14 (s, 2H, N–CH2), 7.54–7.93 (m, 6H, ArH),
8.36–8.39 (m, 2H, ArH); Anal. Calcd for C19H18BrN3O: C,
59.39; H, 4.72; N, 10.94. Found: C, 59.50; H, 4.78; N,
11.22.
2-{[4-(4-Bromophenyl)-1-oxophthalazin-2(1H)-yl]methyl}
isoindoline-1,3-dione (6) A mixture of 1 (0.301 g, 0.001
mol), N-bromomethylphthalimide (0.24 g, 0.001 mol) and
anhydrous K2CO3 (0.41 g, 0.003 mol) in dry DMF
(10 mL) was stirred at room temperature for 24 h. The
reaction mixture was poured onto ice-cold water, the
resulting precipitate was filtered, washed with water and
crystallized from ethanol.
Yield 75 %; mp 221–222 °C; IR (KBr) cm-1: 3,097 (C–H
aromatic), 2,927, 2,897 (C–H aliphatic), 1,662 (C=O), 1,589
(C=N); 1HNMR (DMSO–d6): 5.65 (s, 2H, N–CH2),
7.54–7.91 (m, 10H, ArH), 8.32–8.35 (m, 2H, ArH); (EI)
m/z (% rel. Int.): 459 (M?, 0.35), 461 (M ? 2, 0.32). Anal.
Calcd for C23H14BrN3O3: C, 60.02; H, 3.07; N, 9.13. Found:
C, 59.65; H, 3.16; N, 9.32.
4-(4-Bromophenyl)-2-[(diethylamino)methyl]phthalazin-1
(2H)-one (7d) Yield 75 %; mp 139–140 °C; IR (KBr)
cm-1: 3,030 (C–H aromatic), 2,966, 2,855 (C–H aliphatic),
1,651 (C=O), 1,605 (C=N); 1HNMR (DMSO–d6): 1.06
(t, 6H, 2CH3), 2.70 (q, 4H, 2CH2), 5.13 (s, 2H, N–CH2),
7.76–7.89 (m, 6H, ArH), 8.30–8.37 (m, 2H, ArH); Anal.
Calcd for C19H20BrN3O: C, 59.08; H, 5.22, N, 10.88.
Found: C, 59.25; H, 4.93; N, 10.94.
General procedure for the compounds 7a–j
4-(4-Bromophenyl)-2-[(dibenzylamino)methyl]phthalazin-1
(2H)-one (7e) Yield 60 %; mp 261–262 °C; IR (KBr)
cm-1: 3,060 (C–H aromatic), 2,950, 2,847 (C–H aliphatic),
1,647 (C=O), 1,600 (C=N); 1HNMR (DMSO–d6): 5.46
(s, 4H, 2N–CH2), 5.49 (s, 2H, N–CH2), 6.67–7.95 (m, 16H,
ArH), 8.36–8.42 (m, 2H, ArH); MS (EI) m/z (% rel. Int.):
510 (M?, 6.40), 512 (M ? 2, 7.26). Anal. Calcd for
C29H24BrN3O: C, 68.24; H, 4.74; N, 8.23. Found: C, 68.43;
H, 4.92; N, 8.00.
To a solution of 1 (0.301 g, 0.001 mol) in absolute ethanol
(10 mL), a mixture of formalin (0.048 g, 0.001 mol) and
the appropriate secondary amine or substituted piperazine
(0.001 mol) in ethanol (5 mL) was added and the reaction
mixture was heated under reflux for 4 h. The hot solution
was filtered, the filtrate was concentrated to half its volume
and cooled. The separated solid was filtered and crystal-
lized from methanol.
4-(4-Bromophenyl)-2-(morpholinomethyl)phthalazin-1(2H)
-one (7a) Yield 75 %; mp 184–185 °C; IR (KBr) cm-1
:
4-(4-Bromophenyl)-2-[(diphenylamino)methyl]phthalazin-
1(2H)-one (7f) Yield 65 %; mp 244–245 °C; IR (KBr)
cm-1: 3,050 (C–H aromatic), 2,968, 2,846 (C–H aliphatic),
1,643 (C=O), 1,605 (C=N); 1HNMR (DMSO–d6): 5.46
(s, 2H, N–CH2), 6.67–7.94 (m, 16H, ArH), 8.32–8.41
(m, 2H, ArH); MS (EI) m/z (% rel. Int.): 301
(C14H10BrN2O, 75.53), 314 (C15H11BrN2O, 0.13),
481(M?, 0.06), 483 (M ? 2, 0.05). Anal. Calcd for
C27H20BrN3O: C, 67.23; H, 4.18; N, 8.71. Found: C, 67.30;
H, 4.33; N, 8.50.
3,050 (C–H aromatic), 2,950, 2,855 (C–H aliphatic), 1,651
(C=O), 1,604 (C=N); 1HNMR (DMSO–d6): 2.67
(t, 4H, J = 4.5 Hz, 2CH2–N), 3.54 (t, 4H, J = 4.5 Hz,
2CH2–O), 5.04 (s, 2H, N–CH2), 7.53–7.93 (m, 6H, ArH),
8.37–8.40 (m, 2H, ArH); Anal. Calcd for C19H18BrN3O2: C,
57.01; H, 4.53; N, 10.50. Found: C, 56.59; H, 4.64; N, 10.80.
4-(4-Bromophenyl)-2-(piperidin-1-ylmethyl)phthalazin-1
(2H)-one (7b) Yield 60 %; mp 219–220 °C; IR (KBr)
cm-1: 3,050 (C–H aromatic), 2,935, 2,893 (C–H aliphatic),
1,651 (C=O), 1,604 (C=N); 1HNMR (DMSO–d6):
1.31–1.47 (m, 2H, CH2 piperidine), 2.48–2.50 (m, 4H,
2CH2 piperidine), 2.63–2.67 (m, 4H, 2CH2 piperidine),
5.03 (s, 2H, N–CH2), 7.53–7.92 (m, 6H, ArH), 8.36–8.39
(m, 2H, ArH); MS (EI) m/z (% rel. Int.): 301 (C14H10
BrN2O, 16.59), 314 (C15H11BrN2O, 1.77), 397 (M?, 0.77),
399 (M ? 2, 0.72). Anal. Calcd for C20H20BrN3O: C,
4-(4-Bromophenyl)-2-[(4-methylpiperazin-1-yl)methyl]
phthalazin-1(2H)-one (7g) Yield 80 %; mp 252–
253 °C; IR (KBr) cm-1: 3,074 (C–H aromatic), 2,955,
2,865 (C–H aliphatic), 1,647 (C=O), 1,605 (C=N); 1HNMR
(DMSO–d6): 2.09 (s, 3H, N–CH3), 2.27–2.32 (m, 4H,
piperazine), 2.68–2.72 (m, 4H, piperazine), 5.48 (s, 2H,
N–CH2), 7.54–7.93 (m, 6H, ArH), 8.32–8.41 (m, 2H, ArH).
123

676
N. A. Khalil et al.
Anal. Calcd for C20H21BrN4O: C, 58.12; H, 5.12; N, 13.56.
Found: C, 58.30; H, 5.20; N, 13.75
asparagine and glutamine were deprotonated. Prime 1.5 was
used to check the energy content of the protein segments and
loops. The docking process involved the standard precision
docking (SP) in which ligand poses that were expected to have
unfavourable energies would be rejected. The presumption is
that only active compounds will have available poses that avoid
these penalties. SP docking is appropriate for screening ligands
of unknown quality in large numbers. SP is a soft docking
programme that was adept at identifying ligands that have a
reasonable propensity to bind and 20 % of the final poses pro-
duced from the SP docking were subjected to the Extra Precision
mode of Glide docking (XP) to perform the more expensive
docking simulation on worthwhile poses. XP docking mode is
harder than SP docking mode in that it penalizes the poses that
violate established charges. Flexible docking was selected to
generate conformations of all possible ligand poses, which is
more realistic as this occurs in reality because the protein
undergoes side chain and back bone movement or both, upon
ligand binding. Five and six-membered rings were allowed to
flip and amide bonds which were not cis or trans configuration
were penalised. 5,000 Poses per ligand for the initial phase of
docking with a scoring window for keeping poses of 100 kJ/mol
were set up. The best poses which fulfil those conditions were
subjected to energy minimization on the OPLS-AA non-bonded
interaction grid with a distance dielectric constant of 2 and
maximum number of conjugate gradient steps of 500 iterations.
The ligands of the poses selected by the initial screening were
subsequently minimized in the field of the receptor using a
standard molecular mechanics energy function (OPLS-AA
force field) in conjunction with a distance-dependant dielectric
model. Finally, the lowest energy poses obtained in this fashion
were subjected to a Monte Carlo procedure that examines
nearby torsion minima. The complex was minimized using the
conjugate gradients algorithm until an energy convergence
criterion of 0.1 kJ/mol was reached with iteration cycle of
10,000. Molecular dynamics (MD) at 300 K were then per-
formed on the solvated system for a 10 ps equilibration and
100 ps of production employing a 1 fs time step using OPLS-
2005 force field, from which 100 structures were sampled at
1 ps intervals and averaged. The final averaged structure was
then finally minimized (Cairns et al. 2001). The free energy of
binding was calculated from the following equation.
4-(4-Bromophenyl)-2-[(4-phenylpiperazin-1-yl)methyl]
phthalazin-1(2H)-one (7h) Yield 60 %; mp 187–188
°C; IR (KBr) cm-1: 3,051 (C–H aromatic), 2,944, 2,850
(C–H aliphatic), 1,647 (C=O), 1,597 (C=N); 1HNMR
(DMSO–d6): 2.80–2.83 (m, 4H, piperazine), 3.10–3.13 (m,
4H, piperazine), 5.13 (s, 2H, N–CH2), 6.85–7.92 (m, 11H,
ArH), 8.30–8.40 (m, 2H, ArH); MS (EI) m/z (% rel. Int.):
301 (C14H10BrN2O, 13.14), 314 (C15H11BrN2O, 3.71), 474
(M?, 24.84), 476 (M ? 2, 25.23). Anal. Calcd for
C25H23BrN4O: C, 63.16; H, 4.88; N, 11.79. Found: C,
63.50; H, 4.80; N, 12.05.
4-(4-Bromophenyl)-2-{[4-(2-methoxyphenyl)piperazin-1-
yl]methyl}phthalazin-1(2H)-one (7i) Yield 65 %; mp
199–200 °C; IR (KBr) cm-1: 3,051 (C–H aromatic), 2,950,
2,855 (C–H aliphatic), 1,654 (C=O), 1,597 (C=N); 1HNMR
(DMSO–d6): 2.83–2.84 (m, 4H, piperazine), 2.92–2.94
(m, 4H, piperazine), 3.76 (s, 3H, OCH3), 5.12 (s, 2H,
N–CH2), 6.85–7.94 (m, 10H, ArH), 8.31–8.40 (m, 2H,
ArH); MS (EI) m/z (% rel. Int.): 301 (C14H10BrN2O,
30.35), 314 (C15H11BrN2O, 0.84), 504 (M?, 6.05), 506
(M ? 2, 6.13). Anal. Calcd for C26H25BrN4O2: C, 61.79;
H, 4.99; N, 11.09. Found: C, 61.50; H, 4.75; N, 11.00.
4-(4-Bromophenyl)-2-{[4-(4-methoxyphenyl)piperazin-1-yl]
methyl}phthalazin-1(2H)-one (7j) Yield 72 %; mp 219–
220 °C; IR (KBr) cm-1: 3,051 (C–H aromatic), 2,943, 2,831
(C–H aliphatic), 1,651 (C=O), 1,604 (C=N); 1HNMR
(DMSO–d6): 2.65–2.70 (m, 4H, piperazine), 2.90–2.95 (m,
4H, piperazine), 3.91 (s, 3H, OCH3), 5.07 (s, 2H, N–CH2),
6.75–7.89 (m, 10H, ArH), 8.33–8.41 (m, 2H, Ar–H); Anal.
Calcd for C26H25BrN4O2: C, 61.79; H, 4.99; N, 11.09.
Found: C, 61.66; H, 4.85; N, 11.23.
Molecular modeling procedure
Molecular modeling was carried out on Schrodinger computa-
tional software workstation using Maestro 7.5 graphic user
interface (GUI) and Red Hat Linux nash Enterprise version V
4.1.18 on Batchmin V 9.1 modeling engine. The atomic coor-
dinates for the polypeptide segments of MTHF and Hcy binding
domains of MetS enzyme extracted from human liver were
obtained from the protein bank database [PDB (Brookhaven
protein database)]. Explicit calcium ion counter ions were
DEBind ¼ DEComplex ꢁ ðDEReceptor þ DELigand
Þ
Pharmacological assessment of a-adrenergic blocking
activity
˚
included instead of cadmium and positioned at a 4 A distance
Drugs
from the homocysteine binding site. The basic and acidic amino
acids were neutralized at pH 7 2 by protonation of the ter-
minal amino groups such as histidine, lysine and arginine.
Moreover, the terminal carboxylic acid groups of amino acids
and acidic amino acids such as glutamic acid, aspartic acid,
Acetylcholine hydrochloride, noradrenaline and prazosin
hydrochloride were purchased from Sigma-Aldrich (MO,
USA). All other chemicals were of analytical grades and
obtained from ADWIC Company (Cairo, Egypt).
123

Phthalazinone derivatives, effects on a-receptors
677
Isolated rabbit intestine preparation
results were statistically analyzed using one-way ANOVA
followed by Bonferroni’s multiple comparisons test.
P B 0.05 was considered as a significant difference. All
statistical tests were done employing SPSS program ver-
sion 17 (SPSS Software, SPSS Inc., Chicago, USA).
The IC50 values (a concentration necessary for 50 %
inhibition of maximal inhibitory effect of norepinephrine
hydrochloride upon Ach contraction) was determined by
plotting the % inhibition for NE effect versus the concen-
tration of the test compounds or prazosin. Dose–response
curve was plotted and IC50 values were calculated using
Microsoft EXCEL, version 2007.
It has been reported that a-receptors mediate the inhibitory
effects in the isolated small intestine of the rabbit (Cutler
et al. 1985). The novel final targets 4a–f, 5a–c, 6 and 7a–
j were tested for their blocking effect on the a-receptors.
The a-blocking activity was determined by measuring the
degree of blocking of the inhibitory action of norepineph-
rine (NE), which is mediated via a-ARs by a method
described by Cutler et al. (1980). A segment of small
intestine (2–3 cm) was suspended in oxygenated Ringer–
Locke solution of the following composition (g/L): 9.0 g
NaCl, 0.42 g KC1, 0.17 g CaCl2, 0.5 g NaHCO3 and 1.0 g
glucose at 37 °C.
Muscle contractions were recorded by a transducer and
displayed on a calibrated oscillograph. The preparation
was allowed to equilibrate for 30–40 min, then 0.4 mL of
0.01 % acetylcholine (Ach) was added and the contraction
amplitude was measured over a 1 min period. The bath
was then washed out three times with Ringer–Locke
solution. The response of the same dose of Ach was re-
evaluated after 3 min of pre-treatment with 0.8 mL of
0.01 % norepinephrine solution. The bath was then
washed out three times with Ringer–Locke solution and
the effect of test compounds (antagonists) to be assayed
was measured by their ability to attenuate the NE-induced
inhibition on the amplitude of contraction induced by
Ach.
Results and discussion
Chemistry
The synthetic pathways to compounds 4a–f and 5a–c are
shown in Scheme 1. The new intermediates 4-(4-bromo-
phenyl)-2-(2-chloroethyl)phthalazin-1(2H)-one (2) and 4-(4-
bromophenyl)-2-(3-chloropropyl)phthalazin-1(2H)-one (3)
were obtained by alkylation of 4-(bromophenyl)phthalazin-
1(2H)-one (1) (Colotta et al. 1994) with the respective
bromochloroalkane in dry DMF in presence of anhydrous
potassium carbonate. Nucleophilic displacement of chlorine
in 2 or 3 with various N-(substituted)arylpiperazines or
piperidine gave the relevant 4a–f or 5a–c respectively in
55–70 % yields. The structures of the new compounds were
confirmed by elemental analyses and spectral data. 1HNMR
spectra of compounds 4a–e and 5a–c displayed two broad
signals of piperazine ring in the range of d 2.55–2.70 and
2.82–3.40 ppm. All the other signals in 1HNMR were
observed in the expected regions.
The test compounds as well as prazosin hydrochloride (a
reference standard) were dissolved in dimethylsulfoxide
(DMSO) as stock solution (20 mL, 1 %). DMSO was used in
control experiments. The % decrease in the amplitude of
contraction induced by Ach was recorded in case of pre-
treatment with NE alone as well as in case of pre-treatment
with NE and the test compounds (or prazosin) (0.01 %
solution, 1 mL) (Table 2).
The synthesis of compounds 6 and 7a–j is illustrated in
Scheme 2. The synthesis of compound 6 was achieved by
alkylation of 1 with N-bromomethylphthalimide in dry
DMF at room temperature. On the other hand, the
phthalazinone Mannich bases 7a–j were obtained through
Mannich reaction of 1 with formaline and a various sec-
ondary amines or arylpiperazines following standard
reaction conditions. IR spectral data showed the disap-
pearance of the NH absorption band at 3,250 cm-1 of the
starting phthalazinone 1. Moreover, the formation of 6 and
the Mannich bases 7a–j was confirmed through appear-
ance of a singlet peak corresponding to methylene group
flanked by two nitrogen atoms in the range of d
5.03–5.65 ppm.
Compared to Ach ? NE treatment, compounds that
exhibited significant change in % decrease in the amplitude
of contraction relative to Ach treatment were considered to
have promising a-blocking activity. Different concentra-
tions of prazosin (standard compound) or the test com-
pounds (antagonists) to be assayed (0.01 % solution) were
added to the bathing solution 3 min before addition of
norepinephrine. Ach was added to the bathing solution and
the response was recorded.
For all of the suggested a-adrenergic blocking agents,
the percentage decrease in the amplitude of contraction was
calculated as follows:
In addition, 1HNMR spectra of compounds 7g–j showed
the appearance of two characteristic broad multiplets of
piperazine ring in the range d 2.27–2.84 and 2.68–3.13 ppm.
All the other aromatic and aliphatic protons were observed in
the expected regions.
% decrease in amplitude of contraction = (amplitude of
contraction due to Ach-amplitude of contraction due to
Ach after treatment with the test compound and NE)/
amplitude of contraction due to Ach. The mean of six
measures was taken and expressed as mean SEM. The
123

678
N. A. Khalil et al.
Scheme 1 Reagents and
conditions: a 1-bromo-2-
chloroethane, anhydrous
K2CO3, DMF, rt; b 1-bromo-3-
chloropropane, anhydrous
K2CO3, DMF, rt; c HNRR1,
K2CO3, CH3CN, reflux
R
O
O
O
Cl
N
a
c
R1
N
N
NH
N
N
N
Br
Br
Br
1
2
4a-f
NRR1 4a =
4 b =
N
N
N
b
N
Cl
O
O
C2H5O
N
R
N
N
N
N
Cl
4c=
N
R1
c
N
H3CO
N
N
4d=
Br
Br
N
N
OCH3
4e=
4f=
5a-c
3
NRR1 5a =
5 b =
N
N
Cl
N
N
N
C2H5O
N
OCH3
N
5c =
Molecular modeling studies
prazosin was used with subsequent synthesized compounds
in this article. Compound 7j showed low free energy of
binding -223.79 kJ/mol and a high reduction percentage
in the contraction of the intestine (3.3 0.3 %). The root
mean square deviation (RMSD) for the compound was
Molecular modeling investigations were done on a1-
adrenergic receptor and the designed inhibitors including
the known drug prazosin were docked into the receptor.
There is a direct proportionality between the free energy of
binding of the inhibitors (vasodilators) and the percentage
decrease in the degree of contraction induced by them. The
smaller the free energy of binding, the more plausible and
tight is the binding of the inhibitors to their binding domain
with the suppression of the vasoconstriction action (Cairns
et al. 2001). The active site map analysis clearly showed
the presence of a large hydrophobic region deep inside the
binding domain. Prazosin was docked inside the
a1-adrenergic receptor as illustrated in Figs. 3 and 4. Pra-
zosin docking poses showed a maximum fitting to the
receptor cavity where the hydrophobic side chain at N-2
docked deeply inside the binding domain and the quinaz-
oline moiety at the surface of the receptor as shown in
Fig. 4. The free energy of binding and the percentage
decrease in the degree of contraction collected were quiet
small (-257.81 kJ/mol, 5.2 0.6). The thermodynamic
enthalpy parameterization set up with reference drug
˚
1.96 A. The docking poses of the compound 7j showed a
similar docking poses of prazosin, where the phthalazine
moiety docked at outside part of the receptor and the
hydrophobic side chain at N-2 of the phthalazine nucleus at
the hydrophobic pocket at deep part of the receptor
(Figs. 5, 6). Methoxy group at the side chain of the ligand
made a hydrogen bond with lysine 267. Therefore, the
ligand showed a higher docking score and a better elec-
trostatic and van der Waals energy interactions with the
receptor.
Ligand 4a showed a moderate free energy of binding
-125.51 kJ/mol and a moderate reduction percentage in the
contraction of the intestine (37.5 7.0). The RMSD for the
˚
ligand was 2.03 A (Table 1). The ligand formed a hydrogen
bond with threonine 1050 and the amino group of the side
chain at N-2 of the phthalazine nucleus (Figs. 7, 8). The
ligand produced a moderate type of electrostatic interactions
between lysine 1050 and threonine 1050 from one side and
123

Phthalazinone derivatives, effects on a-receptors
679
Scheme 2 Reagents and
conditions: a N-
bromomethylphthalimide,
K2CO3, DMF, rt; b HCHO,
HNRR1, ethanol, reflux
O
O
N
N
NRR1
NH
N
b
Br
Br
7a-j
1
NRR1
a
N
CH3
N
O
7g =
7h =
7i =
N
N
7a =
O
O
7b =
7c =
7d =
7e =
N
N
N
N
N
N
N
N
O
H3CO
N
CH2CH3
N
N
OCH3
CH2CH3
N
7j =
CH2C6H5
CH2C6H5
Br
6
C6H5
C6H5
7f =
N
Fig. 4 The docked prazosin in the a1-adrenergic receptor (receptor-
prazosin binding site surfaces
Fig. 3 The docked prazosin in the a1-adrenergic receptor
˚
ligand was 4.42 A therefore the docking poses of ligand 5a
the side chain at N-2 of the phthalazine nucleus. The
phthalazine moiety tended to point away from the receptor
due to high energy barrier present between the nucleus
threonine 1050 and this contributes unfavourably to the free
energy of binding of the ligand into its binding domain.
The ligand 5a showed the highest free energy of binding
6.54 kJ/mol and poor reduction percentage in the con-
traction of the intestine (62.5 7.0 %). The RMSD for the
and prazosin are different (Figs. 9, 10). The side chain of
the ligand started to fold up on itself to avoid the high
energy barrier between the ligand and receptor. This led to
the ligand docked at the surface of the domain. The
hydrophobic chain of the ligand resulted in the expulsion of
the ligand from the receptor to avoid the high steric clashes
with the hydrophobic residues inside the receptor pocket
123

680
N. A. Khalil et al.
Table 1 The calculated energy terms for the interaction of the syn-
thesized compounds and % decrease in amplitude of contraction
relative to acetylcholine their RMSD values
Ligand
Energy term (KJ/mol)
DEBind DEComplex DEReceptor DELigand
Prazosin -257.81 -360.51 120.20
RMSD
˚
(A)
-222.90
-186.09
-190.24
-385.62
-209.90
-289.94
-259.91
-388.22
-299.94
-298.90
-303.02
-279.90
-259.91
-280.94
-298.90
-269.04
-269.01
-326.11
-289.94
-185.62
-120.92
1.56
4a
4b
4c
4d
4e
4f
-125.51
222.72
88.880 2.03
86.090 1.90
80.400 4.42
-162.93 -267.08
4.07 -301.15
-100.00 -321.50
-105.89 -340.55
-84.80 -321.50
6.54 -301.22
114.20
109.20
5.09
5.13
23.210 4.11
80.460 4.42
5a
5b
5c
6
Fig. 5 The docked pose of compound 7j in the a1-adrenergic
receptor
-140.81 -345.55
-131.73 -341.55
-125.71 -341.55
-111.81 -321.50
-84.80 -321.50
-150.19 -331.05
-131.73 -341.55
-152.54 -340.50
-75.52 -321.52
-90.62 -390.03
-29.81 -210.55
-195.93 -301.15
-223.79 -240.51
114.48
3.12
89.080 6.11
87.180 6.11
70.210 5.09
23.210 4.11
7a
7b
7c
7d
7e
7f
100.08
1.9
89.080 6.11
81.080 6.01
23.010 4.11
26.700 1.6
7g
7h
7i
109.200 5.13
80.400 2.42
7j
104.20
1.96
Fig. 6 The docked compound 7j in the a1-adrenergic receptor
(receptor-ligand binding site surfaces)
and stacked to the surface of the binding domain. Therefore
the free binding energy is enormous due to unfavourable
ligand–receptor interactions.
In summary, the thermodynamic algorithms were oper-
ated on the different synthesized ligands revealed that the
hydrophobic side chain at position 2 increased the van der
Waals repulsive interactions. In addition, the presence of
polar groups attached to the hydrophobic side chain at
position 2 improved the free energy of docking.
Pharmacological evaluation
Fig. 7 The docked pose of compound 4a in the a1-adrenergic
receptor
Norepinephrine caused inhibition of the spontaneous con-
traction as well as contractions induced by Ach in the
isolated preparation of the small rabbit intestine, suspended
in Ringer–Locke solution. The amplitude of spontaneous
contraction was inhibited by norepinephrine by 65 7 %
in comparison to the amplitude of contraction due to Ach.
Pre-treatment with prazosin significantly reduced the per-
centage inhibition of contraction after norepinephrine by
123

Phthalazinone derivatives, effects on a-receptors
681
Table 2 IC50 values and % decrease in amplitude of contraction
relative to acetylcholine
Compounds
% Decrease in amplitude of IC50 (lg/mL)
contraction relative to Ach
Ach
0
–
Ach ? NE
65 7.0#
5.2 0.6*
37.5 7.0*
50 6.0
–
Ach ? NE ? Prazosin
Ach ? NE ? 4a
Ach ? NE ? 4b
Ach ? NE ? 4c
Ach ? NE ? 4d
Ach ? NE ? 4e
Ach ? NE ? 4f
Ach ? NE ? 5a
Ach ? NE ? 5b
Ach ? NE ? 5c
Ach ? NE ? 6
Ach ? NE ? 7a
Ach ? NE ? 7b
Ach ? NE ? 7c
Ach ? NE ? 7d
Ach ? NE ? 7e
Ach ? NE ? 7f
Ach ? NE ? 7g
Ach ? NE ? 7h
Ach ? NE ? 7i
Ach ? NE ? 7j
0.498
1.101
–
62.5 8.0
20 3.0*
21.9 2.5*
16 2.0*
62.5 7.0
13.3 2.0*
26.6 3.0*
26.6 3.0*
39 7.0
–
1.42
0.696
0.915
–
Fig. 8 The docked compound 4a in the a1-adrenergic receptor
(receptor-ligand binding site surfaces)
0.679
1.614
1.605
–
40 3.6
–
16.6 2*
23.3 3*
12 1.7*
59.4 6.0
53.2 4.0
80 6.0
0.692
0.70
0.81
–
–
–
0.093 0.01*
3.3 0.3*
0.621
0.51
Data are expressed as mean SEM and analyzed using one-way
ANOVA followed by Bonferroni’s multiple comparison’s test. Each
individual compound was compared with the first three groups sep-
arately. IC50 (concentration necessary for 50 % inhibition of maximal
inhibitory effect of norepinephrine hydrochloride upon Ach
contraction)
Ach acetylcholine, NE norepinephrine
#
Significantly different from Ach at P \ 0.05
Fig. 9 The docked pose of compound 5a in the a1-adrenergic
receptor
* Significantly different from Ach ? NE at P \ 0.05, n = 6
5.2 0.6 %. Therefore, prazosin blocked the action of
norepinephrine on the isolated rabbit intestine.
Contraction due to Ach was returned near the baseline
value in case of pre-treatment with various tested com-
pounds before NE addition. Those compounds that resulted
in significant changes in the amplitude of contraction rel-
ative to that recorded after treatment with Ach ? NE were
considered to possess a-antagonistic activity (Table 2).
Compound 7j produced a-antagonistic activity that is fairly
similar to that produced by prazosin. However, compounds
4b, 4c, 5a, 7a, 7b, 7f, 7g, 7h did not show any significant
activity.
For compounds that produced significant a-blocking
activity, IC50 values were determined, and the results
showed that compound 7j exhibited the lowest IC50 value
Fig. 10 The docked compound 5a in the a1-adrenergic receptor
(receptor-ligand binding site surfaces)
123

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Fig. 11 Dose-response curve
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in comparison to prazosin
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