Design and Synthesis of Substituted Pyridazinone-1-Acetylhydrazones as Novel Phosphodiesterase 4 Inhibitors

Article abstract of DOI:10.1002/ardp.201500363

A series of novel pyridazin-6-one-1-acetylhydrazone hybrids were rationally designed to inhibit phosphodiesterase 4 (PDE4B). The prepared compounds were evaluated for their in vitro ability to inhibit the PDE4B enzyme; several of these compounds showed mo

Full text of DOI:10.1002/ardp.201500363

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Full Paper
Design and Synthesis of Substituted Pyridazinone-1-
Acetylhydrazones as Novel Phosphodiesterase 4 Inhibitors
Hamdy M. Abdel-Rahman1, Mohamed Abdel-Aziz2, Heather N. Tinsley3, Bernard D. Gary4,
Joshua C. Canzoneri4, and Gary A. Piazza4
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt
Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia, Egypt
Department of Biology, Chemistry and Mathematics, University of Montevallo, Montevallo, AL, USA
Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
2
3
4
A series of novel pyridazin-6-one-1-acetylhydrazone hybrids were rationally designed to inhibit
phosphodiesterase 4 (PDE4B). The prepared compounds were evaluated for their in vitro ability to
inhibit the PDE4B enzyme; several of these compounds showed moderate activity compared to the
reference drug, rolipram. Compounds 6, 12, and 14 emerged as the most potent inhibitors in this
series. The [3-(4-methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]acetic acid [1-(3,4,5-trimethoxy-
phenyl)ethylidene]hydrazide (12) showed an IC50 value of 13 mM against PDE4B. Docking of 6, 12,
and 14 into the active site of PDE4B illustrates their possible binding mode and provides insights for
further optimization of this drug scaffold.
Keywords: Docking / N-Acylhydrazones / PDE4B inhibitors / Pyridazinones
Received: October 6, 2015; Revised: November 22, 2015; Accepted: November 26, 2015
DOI 10.1002/ardp.201500363
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
inflammatory effect with reduced side effects. Thus, selective
inhibition of PDE4B has received the most attention by
Introduction
Phosphodiesterase 4 (PDE4) is a member of the PDE enzyme
family that plays a key role in controlling the intracellular
concentration of cyclic adenosine monophosphate (cAMP) [1].
The critical role of cAMP in regulating inflammatory processes
makes PDE4 an attractive target for treating inflammatory
diseases such as asthma, chronic obstructive pulmonary
disease (COPD), adult respiratory distress syndrome (ARDS),
rheumatoid arthritis, and psoriasis. Also it may be effective in
treating central nervous system (CNS) disorders such as
depression and cancer [2, 3].
medicinal chemists for providing new drug candidates [4, 5].
Roflumilast (Fig. 1) is a selective PDE4 inhibitor that currently
has FDA approval as an anti-inflammatory drug for treating
COPD and its dose limiting side effects, namely emesis,
highlight the need for the design and discovery of novel PDE4
inhibitors [6, 7].
Moreover, the pyridazinone scaffold is one of the core
structures in the medicinal chemistry search for PDE4
inhibitors. Zardaverine [8], compound A [9], and PDE3/4
inhibitor hybrid [10] are examples of pyridazinone-containing
PDE4 inhibitors (Fig. 1). Furthermore, N-acylhydrazones
derivatives which are considered as simplified analogs of
the pyridazinone scaffold [11] demonstrate wide range of
biological activities such as PDE inhibition [11, 12], anti-
inflammatory [13], and vasodilatation effect [14].
PDE4 exists in four different isoforms PDE4A, B, C, and D.
Inhibitors of PDE4 with PDE4B selectivity may retain the anti-
Promoted with the above-mentioned studies, this work
gathers the two bioactive scaffolds, the pyridazinone (A) and
the N-acylhydrazones (B) in one compact structure aiming
for the purpose of synergistic effect (Fig. 2). Moreover, the
PDE4B inhibitory activities for the prepared compounds were
Correspondence: Prof. Hamdy M. Abdel-Rahman, Department of
Medicinal Chemistry, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt.
E-mail: hamdym01@yahoo.com
Fax: þ20 88 2332776
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Pyridazinone-1-Acetylhydrazones as PDE4B Inhibitors
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Figure 1. Chemical structures of selected PDE4
inhibitors.
evaluated. Docking studies were also carried out for the
designed compounds in the active site of PDE4B to illustrate a
likely binding mode and to give insight into strategies for
further optimization of this novel hybrid scaffold.
hydrochloride afforded the corresponding esters 1 and
2 [16–18]. Heating ester 1 or 2 at reflux with hydrazine
monohydrate afforded the corresponding hydrazide 3 and 4
in a good yield [18]. The hydrazides 3 or 4 on heating at reflux
with different aromatic aldehydes or ketones afforded the
corresponding Schiff bases 5–14 (Scheme 1). The synthesized
compounds were characterized by spectroscopic (1H NMR,
13C NMR, and mass spectra) as well as elemental analyses.
Results and discussion
Chemistry
Biological studies
All the synthesized compounds (1–14) were evaluated for
their in vitro inhibition of recombinant human PDE4B by using
PDE4B enzyme isolated from Sf9 cells. Rolipram (Fig. 1) was
3-Aroylpropionic acid derivatives were prepared by Friedel–
Crafts acylation of substituted benzene derivatives with
succinic anhydride [15]. Cyclocondensation of the prepared
3-aroylpropionic acid derivatives with ethyl hydrazinoacetate
Figure 2. Design of the pyridazinone/N-
acyhydrazone hybrid compounds as PDE4B
inhibitors.
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Scheme 1. Synthesis of the pyridazinone/
N-acetyl hydrazone hybrid compounds.
Reagents and conditions: (a) (CH2CO)2O,
AlCl3, heat; (b) H2NNHCH2COOC2H5.HCl,
Et3N, C2H5OH, heat; (c) H2NNH2, C2H5OH,
reflux; (d) different aldehydes or ketones,
C2H5OH, reflux.
used as a positive control for PDE4 inhibition. Initially, all
compounds were screened at a single dose of 50 mM in
to some of the synthesized Schiff bases namely 5, 8, 9, and 13.
The Schiff base 6 with the 3-(4-methoxyphenyl) moiety
showed the most potent PDE4B inhibitory activity among
this series (68% at 50 mM). Furthermore, Schiff bases 12, 14
with the 3-(4-methoxyphenyl) moiety and additional a-methyl
triplicate, followed by
a full dose response for active
compounds to calculate the exact IC50 values. The full dose–
response screening was performed for compounds displaying
initial PDE4B inhibition ꢀ60% and tested a range of 10
concentrations with at least two replicates per concentration.
The results summarized in Fig. 3 (see also Supporting
Information) show that compounds 6, 12, and 14 exhibit the
best PDE4B inhibitory activity within this series of compounds
at 50 mM.
These results revealed that the presence of the 4-
methoxyphenylpyridazinone moiety is critical for activity.
Thus, the starting 4-methoxyphenylpyridazinone-containing
esters and hydrazides 2 and 4, respectively, showed high
PDE4B inhibitory activity compared to their corresponding
4-fluorophenylpyridazinone-containing derivatives 1 and 3.
This trend was also observed in each pair of the final hybrids.
In general, the results shown in Fig. 3 indicate that the
formation of Schiff base hybrids 5–14 results in improvement
of PDE4B inhibitory activity. Yet, the starting 4-methoxy-
phenylpyridazinone-containing ester 2, although lacking the
N-acylhydrazone moiety, showed superior PDE4B inhibition
Figure 3. Percent PDE4B inhibition of compounds 1–14 at 50 mM
single-dose screening. The rolipram control was tested at its
IC50 of 150 nM.
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Pyridazinone-1-Acetylhydrazones as PDE4B Inhibitors
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group (R1 ¼ CH3) also showed the increased PDE4B inhibitory
activity (more than 60% at 50 mM).
The molecular modeling of the selected compounds showed
that the 4-methoxy moieties form a network of H-bonding
with water molecules that in turn form H-bonding with active
site amino acids as Gln443, His234, Met347, and Asn283.
Furthermore, these compounds are also oriented within the
PDE4B hydrophobic region comprised of Ile410, Phe414,
Phe446, and Phe506. A docking pose of compound 12 in the
PDE4B active site is shown in Fig. 4 (for docking poses of
compounds 6 and 14, see Supporting Information).
These observations show that these compounds are bound
to the PDE4B active site in a similar manner to other selective
inhibitors [21–23]. Additionally, this docking experiment
shows that compounds as 6 or 12 are existing as two 4-
methoxyphenylpyridazinone units that individually contrib-
ute to the enzyme binding. Most importantly, the docking
provides guidelines for the future design and synthesis of new
analogs with anticipated improved PDE4B inhibitory activities
such as (i) addition of the a-alkyl groups (R1 ¼ CH3) to Schiff
bases; (ii) using MeO isosteres at the 4-methoxyphenyl-
containing Schiff bases; and (iii) increasing bulkiness on the
Schiff base aromatic ring may also alter the activity.
Therefore, compounds 6, 12, and 14 that displayed initial
PDE4B inhibition >60% were subjected to the full dose–
response inhibition assay screening to determine their IC50
values against PDE4B enzyme compared to rolipram.
As shown in Table 1, compound 12 showed the most potent
activity of this series with an IC50 value of 13mM. On the other
hand, compounds 14 and 6 showed good PDE4B inhibitory
activity with IC50 values of 18 and 26.6 mM, respectively.
Although these three compounds were not as potent as the
control drug rolipram, all are still more potent than some
known inhibitors such as moexipril (Fig. 1) (PDE4B IC50 ¼ 38 mM)
[19]. From these results, several conclusions can be deduced
regarding SAR for this class of compounds; 4-methoxyphenyl-
pyridazinone moiety is critically significant for activity, forma-
tion of the hybrid Schiff bases generally improves the activity
and trimethoxyphenyl-containing Schiff base 12 showed
superior PDE4B inhibitory activity over other derivatives.
Molecular modeling study
Molecular docking studies of compounds 6, 12, and 14 were
performed in order to rationalize the obtained biological
results and understand their likely interactions with PDE4B
enzyme active site.
Conclusion
Docking studies were performed by MOE (molecular operat-
ing environment) software [20] using PDE4B enzyme co-
crystallized with 5-carbamoyl-2-phenylpyrimidine derivative
as a template (PDB ID: 3WD9) [21]. The protein was prepared
by removing other chain, nonreactive water molecules, and
ligand molecules other than crystal ligand. The synthesized
molecules were built using MOE builder. Approximately, 100
dockingswereperformedfortheinteractionoftheligandinthe
enzyme active site and the top scoring configuration of the
ligand–enzyme complexes was selected on energetic grounds.
Prior to docking the synthesized molecules into target,
crystal structure of ligand was used to perform control
docking to ensure whether the docking method was valid.
The amino acid residues that interact with the ligand
produced by the re-docking process were then compared
with amino acid residues that bind in the crystal structure; a
root-mean-square deviation (RMSD) value obtained was
We have reported here the design, synthesis, and characteriza-
tion of a novel class of pyridazinone/N-acyhydrazones hybrid
scaffold compounds that achieve the intended inhibitory
potential toward PDE4B. The trimethoxyphenyl Schiff base
12 was the most potent inhibitor in this series with an IC50 of
13 mM. Molecular modeling of compounds 6, 12, and 14 bound
to the active site of PDE4B illustrates the elements that
contribute to the enzyme binding and provides insight into
modifications for the further design and development of
additional novel PDE4B inhibitors with improved potency.
Experimental
Chemistry
Melting points were determined on an electrothermal
apparatus (Stuart Scientific, England) and were uncorrected.
1H NMR and 13C NMR spectra were run on a Bruker 400 MHz
(Varian, Palo Alto, CA, USA) using TMS as internal reference.
Chemical shifts (d) values are given in parts per million (ppm)
using CDCl3 (7.29 for proton and 76.98 for carbon) as solvent
and coupling constants (J) in Hz. Splitting patterns are
designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. Elemental analyses were performed on “Analy-
tischer Funktionstest vario EL Fab.-Nr. 11982027” (Germany).
All reactions were monitored by thin-layer chromatography
(TLC) using silica gel 60 GF245 precoated sheets 20 ꢁ 20 cm,
layer thickness 0.2 mm (E. Merck, Germany) and were
visualized by UV-lamp at wavelength (l) 254 nm. All chemicals
and solvents were of reagent grade, and the latter were
distilled and dried before use.
̊
1.13 A that was defined as reasonable criterion. This indicated
that the parameter set for docking was suitable for
reproducing the X-ray structure (for re-docking figures, see
the Supporting Information Figs S1–S3).
Table 1. PDE4B inhibition induced by compounds 6, 12,
and 14.
Compound
PDE4B inhibition IC50 (mM)
6
12
14
Rolipram
26.6
13.0
18.4
0.150
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Figure 4. 2D representation showing the dock-
ing pose interactions of compound 12 in the
PDE4B active site.
Generalprocedureforsynthesisof[3-(4-substitutedphenyl)-
6-oxo-5,6-dihydro-4H-pyridazin-1-yl]acetic acid ethyl esters
1 and 2
General procedure for synthesis of 3-(4-substitutedphenyl)-
6-oxo-5,6-dihydro-4H-pyridazin-1-yl]acetic acid hydrazides
3 and 4
Compounds 1 and 2 were prepared according to the reported
procedure [16–18]. An equimolar amount of the 3-(4-substi-
tuted)benzoyl propionic acid (prepared according to reported
procedures[15]),ethylhydrazinoacetateHCl,andtriethylamine
were refluxed for 4 h in ethanol. The reaction mixture was
cooled, evaporated under reduced pressure. The residue was
extracted by dichloromethane, washed with water and brine.
The organic layer was dried over anhydrous MgSO4, filtered,
evaporated where the solid product was obtained.
Compounds 3 and 4 were prepared according to the reported
procedure [15, 17]. A solution of the appropriate ester 1 or 2
(10 mmol) in ethanol (10 mL) was mixed with hydrazine
monohydrate (99%, 3 mL) and refluxed for 3 h then cooled to
room temperature. The bright solid material obtained was
filtered off and dried under vacuum.
[3-(4-Fluorophenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-
acetic acid hydrazide 3
White powder in 85% yield, mp 196–197°C; 1H NMR (400 MHz,
–
[3-(4-Fluorophenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-
DMSO) d (ppm): 2.56 (t, 2H, J ¼ 8.00 Hz, CH C N), 3.03 (t, 2H,
–
2
–
–
acetic acid ethyl ester 1
White powder in 75% yield, mp 86–87°C; H NMR (400 MHz,
J ¼ 8.00 Hz, CH C O), 4.24 (s, 2H, NH ), 4.32 (s, 2H, CH –N),
2
2
2
1
7.26 (m, 2H, Ar–H), 7.80 (m, 2H, Ar–H), 9.11 (s, 1H, NH);
13C NMR (100 MHz, DMSO) d (ppm): 22.42, 26.12, 50.10,
115.28, 115.50, 128.14, 131.99, 149.61, 164.10, 165.50, 166.48.
Anal. calcd. for C12H13FN4O2 (264.10): C, 54.54; H, 4.96; N,
21.20. Found: C, 54.24; H, 5.35; N, 20.85.
CDCl3) d (ppm): 1.21 (t, 3H, J ¼ 7.00 Hz, CH2CH3), 2.58 (t, 2H,
J ¼ 8.00 Hz, CH2), 2.93 (t, 2H, J ¼ 8.00 Hz, CH2), 4.15 (q, 2H,
J ¼ 7.00 Hz, CH2CH3), 4.51 (s, 2H, CH2–N), 7.00 (d, 2H, J ¼ 6.80
Hz, Ar–H), 7.62 (d, 2H, J ¼ 6.80 Hz, Ar–H); 13C NMR (100 MHz,
CDCl3) d (ppm): 14.17, 23.14, 26.67, 50.49, 61.33, 115.49,
115.71, 128.03, 131.59, 149.99, 162.53, 165.02, 165.82, 168.61.
Anal. calcd. for C14H15FN2O3 (278.11): C, 60.42; H, 5.43; N,
10.07. Found: C, 60.53; H, 5.61; N, 9.93.
[3-(4-Methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-
yl]acetic acid hydrazide 4
White powder in 82% yield, mp 193–195°C; 1H NMR (400 MHz,
–
CDCl ) d (ppm): 2.58 (t, 2H, J ¼ 8.00 Hz, CH C N), 2.93 (t, 2H,
–
3
2
–
–
[3-(4-Methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-
yl]acetic acid ethyl ester 2
White powder in 70% yield, mp 47–48°C (reported as
syrupy [18]). Anal. calcd. for C15H18N2O4 (290.13): C, 62.06;
H, 6.25; N, 9.65. Found: C, 61.66; H, 6.33; N, 9.61.
J ¼ 8.00 Hz, CH C O), 3.77 (s, 3H, OCH ), 3.96 (s, 2H, NH ), 4.47
2
3
2
(s, 2H, CH2–N), 6.86 (d, 2H, J ¼ 7.00 Hz, Ar–H), 7.28 (s, 1H, NH)
7.64 (d, 2H, J ¼ 7.00 Hz, Ar–H); 13C NMR (100 MHz, CDCl3) d
(ppm): 22.90, 26.80, 51.54, 55.43, 114.03, 127.33, 127.67,
152.04, 161.33, 166.31, 169.20. Anal. calcd. for C13H16N4O3
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Pyridazinone-1-Acetylhydrazones as PDE4B Inhibitors
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–
(276.12): C, 56.51; H, 5.84; N, 20.28. Found: C, 56.57; H, 6.23; N,
20.19.
J ¼ 8.10 Hz, CH –C O), 3.76 (s, 3H, OCH ), 3.78 (s, 3H, OCH ),
–
2
3
3
3.82 (s, 3H, OCH3), 5.02 (s, 2H, CH2–N), 6.82 (d, 2H, J ¼ 8.92 Hz,
Ar–H), 6.86–6.88 (m, 1H, Ar–H), 6.99 (t, 1H, J ¼ 8.00 Hz, Ar–H),
7.40–7.45 (m, 1H, Ar–H), 7.62 (d, 2H, J ¼ 8.92 Hz, Ar–H), 8.02 (s,
General procedure for the synthesis of Schiff bases 5–14
To a stirred solution of 3 or 4 (0.5 mmol) in ethanol (10 mL),
the appropriate aldehyde or ketone (0.5 mmol) was added
followed by few drops of glacial acetic acid. The mixture was
then refluxed for 30 min and cooled to room temperature.
The formed precipitate was filtered off and recrystallized
from aqueous ethanol.
1H, CH N), 9.01 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d (ppm):
–
–
23.08, 26.91, 50.82, 55.38, 55.84, 61.74, 113.84, 114.01, 117.73,
124.34, 127.28, 127.56, 128.16, 139.86, 148.49, 150.74, 152.82,
160.88, 166.39, 169.35; DART-MS: 425.20 [Mþ1]. Anal. calcd.
for C22H24N4O5 (424.17): C, 62.25; H, 5.70; N, 13.20. Found: C,
62.26; H, 6.03; N, 12.99.
[3-(4-Fluorophenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-
acetic acid (4-methoxy-benzylidene)hydrazide 5
[3-(4-Fluorophenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-
acetic acid (3,4,5-trimethoxybenzylidene)hydrazide 9
Off-white powder in 60% yield; mp 207–208°C; 1H NMR
Off-white powder in 73% yield; mp 181–182°C; 1H NMR
–
–
(400 MHz, CDCl ) d (ppm): 2.63 (t, 2H, J ¼ 8.00 Hz, CH –C N),
(400 MHz, CDCl ) d (ppm): 2.63 (t, 2H, J ¼ 8.20 Hz, CH –C N),
–
–
3
2
3
2
–
–
–
–
2.94 (t, 2H, J ¼ 8.00 Hz, CH –C O), 3.75 (s, 3H, OCH ), 5.00 (s, 2H,
2.95 (t, 2H, J ¼ 8.20 Hz, CH –C O), 3.81 (s, 6H, 2 OCH ), 3.86
2
3
2
3
CH2–N), 6.80 (d, 2H, J ¼ 7.40 Hz, Ar–H), 6.95–6.99 (m, 2H, Ar–H),
7.46(d, 2H, J¼ 7.40 Hz, Ar–H),7.63–7.71(m, 3H, Ar–H), 9.62(s, 1H,
NH);13CNMR(100 MHz,CDCl3)d(ppm):23.12,26.79,50.75,55.37,
114.20, 115.42, 115.64, 126.32, 127.95, 128.03, 128.75, 129.35,
131.75, 144.44, 149.69, 161.31, 162.45, 164.94, 166.19, 169.63;
DART-MS: 383.20[Mþ1]. Anal. calcd. forC20H19FN4O3 (382.14):C,
62.82; H, 5.01; N, 14.65. Found: C, 62.47; H, 5.22; N, 14.39.
(s, 3H, OCH3), 5.03 (s, 2H, CH2–N), 6.80 (s, 2H, Ar–H), 6.96–
–
–
7.03 (m, 2H, Ar–H), 7.60 (s, 1H, CH N), 7.63–7.68 (m, 2H,
Ar–H), 9.40 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d (ppm):
23.14, 26.80, 50.72, 56.20, 56.23, 60.97, 104.33, 105.56,
115.46, 115.68, 127.94, 128.02, 128.91, 129.51, 140.12,
140.86, 144.31, 149.89, 153.52, 161.52, 166.14, 169.49;
DART-MS: 443.20 [Mþ1]; Anal. calcd. for
C22H23FN4O5
(442.17): C, 59.72; H, 5.24; N, 12.66. Found: C, 59.80; H,
5.14; N, 12.34.
[3-(4-Methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-
yl]acetic acid (4-methoxybenzylidene)hydrazide 6
White powder in 69% yield; mp 157–158°C; 1H NMR (400 MHz,
[3-(4-Methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-
yl]acetic acid (3,4,5-trimethoxybenzylidene)hydrazide 10
White powder in 62% yield; mp 212–213°C; 1H NMR (400 MHz,
–
–
CDCl ) d (ppm): 2.61 (t, 2H, J ¼ 7.60 Hz, CH –C N), 2.93 (t, 2H,
3
2
–
–
J ¼ 7.60 Hz, CH –C O), 3.74 (s, 3H, OCH ), 3.76 (s, 3H, OCH ),
2
3
3
–
–
5.00 (s, 2H, CH2–N), 6.74–6.94 (m, 4H, Ar–H), 7.46 (d, 2H,
J ¼ 8.00 Hz, Ar–H), 7.55 (d, 1H, J ¼ 8.00 Hz, Ar–H), 7.61 (d, 2H,
J ¼ 8.70 Hz, Ar–H), 9.66 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d
(ppm): 23.04, 26.94, 50.74, 55.37, 113.87, 113.96, 114.00,
114.17, 126.36, 127.57, 127.77, 128.12, 128.76, 129.39, 144.36,
150.66, 160.90, 161.23, 166.41, 169.79; DART-MS: 395.20
[Mþ1]. Anal. calcd. for C21H22N4O4 (394.16): C, 63.95; H, 5.62;
N, 14.20. Found: C, 63.86; H, 5.99; N, 14.22.
CDCl ) d (ppm): 2.61 (t, 2H, J ¼ 8.10 Hz, CH –C N), 2.94 (t, 2H,
3
2
–
–
J ¼ 8.10 Hz, CH –C O), 3.75 (s, 3H, OCH ), 3.78 (s, 6H, 2 OCH ),
2
3
3
3.81 (s, 3H, OCH3), 5.03 (s, 2H, CH2–N), 6.80 (s, 2H, Ar–H),
6.82–6.89 (m, 2H, Ar–H), 7.58–7.62 (m, 3H, Ar–H), 9.62 (s, 1H,
NH); 13C NMR (100 MHz, CDCl3) d (ppm): 23.05, 26.94, 50.70,
55.38, 56.18, 56.25, 60.99, 104.22, 104.80, 106.69, 113.89,
114.00, 127.76, 128.01, 128.98, 139.95, 144.19, 148.95,
150.87, 153.37, 160.97, 166.35, 169.63; DART-MS: 455.20
[Mþ1] and 477.20 [MþNa]. Anal. calcd. for C23H26N4O6
(454.19): C, 60.78; H, 5.77; N, 12.33. Found: C, 60.55; H, 5.75;
N, 12.05.
[3-(4-Fluorophenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-
acetic acid (2,3-dimethoxybenzylidene)hydrazide 7
White powder in 70% yield; mp 128–129°C; 1H NMR (400 MHz,
–
CDCl ) d (ppm): 2.63 (t, 2H, J ¼ 8.20Hz, CH –C N), 2.95 (t, 2H,
[3-(4-Fluorophenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-
acetic acid [1-(3,4,5-trimethoxyphenyl)ethylidene]-
hydrazide 11
–
3
2
–
–
J ¼ 8.20Hz, CH –C O), 3.74(s, 3H,OCH ),3.82(s, 3H, OCH ),5.01
2
3
3
(s, 2H, CH–N), 6.85 (d, 1H, J ¼ 8.00Hz, Ar–H), 6.93–7.07 (m, 3H,
Ar–H), 7.38 (d, 1H, J ¼ 8.00 Hz, Ar–H), 7.62–7.66 (m, 2H, Ar–H),
White powder in 66% yield; mp 208–209°C; 1H NMR (400 MHz,
CDCl3) d (ppm): 2.20 (s, 3H, CH3), 2.72 (t, 2H, J ¼ 8.00 Hz,
8.05 (s, 1H, CH N), 9.61 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d
–
–
–
–
–
(ppm): 23.13, 26.77, 50.78, 55.82, 61.71, 113.78, 115.37, 115.59,
117.74, 124.29, 127.41, 127.94, 131.78, 140.38, 148.54, 149.78,
152.81, 162.43, 164.91, 166.28, 169.64; DART-MS: 413.20 [Mþ1]
and 435.20 [MþNa]. Anal. calcd. for C21H21FN4O4 (412.15): C,
61.16; H, 5.13; N, 13.59. Found: C, 60.98; H, 5.52; N, 13.23.
CH –C N), 3.03 (t, 2H, J ¼ 8.00 Hz, CH –C O), 3.90 (s, 3H,
–
2
2
OCH3), 3.92 (s, 6H, 2 OCH3), 5.13 (s, 2H, CH2–N), 6.80 (s, 2H,
Ar–H), 7.11 (t, 2H, J ¼ 2.80 Hz, Ar–H), 7.72–7.77 (m, 2H, Ar–H),
9.35 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d (ppm): 12.98,
23.16, 26.81, 50.93, 56.26, 60.98, 103.63, 115.47, 115.68,
127.93, 128.01, 131.73, 131.76, 133.35, 139.55, 147.94,
149.74, 153.11, 162.45, 166.25, 169.86; DART-MS: 457.20
[Mþ1] and 479.20 [MþNa]. Anal. calcd. for C23H25FN4O5
(456.18): C, 60.52; H, 5.52; N, 12.27. Found: C, 60.13; H, 5.60;
N, 12.14.
[3-(4-Methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazine-1-
yl]acetic acid (2,3-dimethoxybenzylidene)hydrazide 8
White powder in 64% yield; mp 217–218°C; 1H NMR (400 MHz,
–
CDCl ) d (ppm): 2.62 (t, 2H, J ¼ 8.10 Hz, CH –C N), 2.95 (t, 2H,
–
3
2
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H. M. Abdel-Rahman et al.
Archiv der Pharmazie
[3-(4-Methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-
yl]acetic acid [1-(3,4,5-trimethoxyphenyl)ethylidene]-
hydrazide 12
range of 10 concentrations with at least two replicates per
concentration. Dose–response curves were analyzed using
PrismTM 4 software (GraphPad) to calculate IC50 values using a
four parameter logistic equation.
White powder in 60% yield; mp 175–176°C; 1H NMR (400 MHz,
CDCl3) d (ppm): 2.12 (s, 3H, CH3), 2.61 (t, 2H, J ¼ 8.00 Hz,
–
–
–
Molecular modeling studies
CH –C N), 2.95 (t, 2H, J ¼ 8.00 Hz, CH –C O), 3.76 (s, 3H,
–
2
2
OCH3), 3.81 (s, 3H, OCH3), 3.83 (s, 6H, 2 OCH3), 5.03 (s, 2H,
CH2–N), 6.82 (d, 2H, J ¼ 8.88 Hz, Ar–H), 6.89 (s, 2H, Ar–H), 7.61
(d, 2H, J ¼ 8.88 Hz, Ar–H), 9.03 (s, 1H, NH). 13C NMR (100 MHz,
CDCl3) d (ppm): 12.88, 23.08, 26.93, 50.90, 55.38, 56.27, 56.31,
60.98, 103.27, 103.61, 105.78, 113.87, 127.53, 127.78, 128.11,
133.34, 139.51, 147.63, 150.73, 153.11, 160.93, 166.41, 169.79;
DART-MS: 469.20 [Mþ1] and 491.20 [MþNa]. Anal. calcd. for
All the molecular modeling studies were carried out on Dell
precision T3600 workstation with Intel Xeon1 CPU-1650.0 @
3.20 GHz and Windows 7 operating system using molecular
operating environment (MOE 2014, Chemical Computing
Group, Canada) [20] as the computational software.
The X-ray crystallographic structure of PDE4B complexed
with 5-carbamoyl-2-phenylpyrimidine derivative (PDB ID:
3WD9) [21] was obtained from the Protein Data Bank. The
enzyme was prepared for docking studies where (i) ligand
molecule was removed from the enzyme active site; (ii)
hydrogen atoms were added to the structure with their
standard geometry; (iii) MOE Alpha Site Finder was used for
the active sites search in the enzyme structure and dummy
atoms were created from the obtained a-spheres; (iv) docking
was performed using the Alpha Triangle placement. The
London dG scoring function estimates the free energy of
binding of the ligand from a given pose. The output database
dock file was created with different poses for each ligand and
arranged according to the final score function (S); and (v) the
obtained model was then used in predicting the ligand–
enzyme interactions at the active site.
C
24H28N4O6 (468.20): C, 61.53; H, 6.02; N, 11.96. Found: C,
61.22; H, 6.32; N, 11.80.
[3-(4-Fluorophenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-
acetic acid [1-(5-fluoro-2-hydroxyphenyl)ethylidene]-
hydrazide 13
Off-white powder in 90% yield; mp 171–172°C; 1H NMR
(400 MHz, CDCl3) d (ppm): 2.15 (s, 3H, CH3), 2.64 (t, 2H,
–
–
J ¼ 8.00 Hz, CH –C N), 2.96 (t, 2H, J ¼ 8.00 Hz, CH –C O), 4.62
–
–
2
2
(s, 1H, CH), 4.92 (s, 1H, CH), 6.73–6.82 (m, 1H, Ar–H), 6.83–6.86
(m, 1H, Ar–H), 6.87–7.01 (m, 3H, Ar–H), 7.62–7.68 (m, 2H,
Ar–H), 9.70 (s, 1H, NH), 10.08 (s, 1H, OH); 13C NMR (100 MHz,
CDCl3) d (ppm): 12.87, 22.85, 26.68, 50.71, 115.54, 115.70,
115.76, 115.92, 118.88, 118.95, 127.95, 128.03, 128.18, 128.27,
153.29, 155.29, 164.06, 166.29, 167.11, 168.80; DART-MS:
401.10 [Mþ1] and 423.10 [MþNa]. Anal. calcd. for
The authors have declared no conflicts of interest.
C
20H18F2N4O3 (400.13): C, 60.00; H, 4.53; N, 13.99. Found: C,
59.79; H, 4.82; N, 13.74.
References
[3-(4-Methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-
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hydrazide 14
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