New racemic annulated pyrazolo[1,2-b]phthalazines as tacrine-like AChE inhibitors with potential use in Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.07.072

A novel series of tacrine-like compounds 7a-u possessing a fused pyrazolo[1,2-b]phthalazine structure were designed and synthesized as potent and selective inhibitors of AChE. The in-vitro biological assessments demonstrated that several compounds had hig

Full text of DOI:10.1016/j.ejmech.2017.07.072

Accepted Manuscript
New racemic annulated pyrazolo[1,2-b]phthalazines as tacrine-like AChE inhibitors
with potential use in Alzheimer's disease
Leili Jalili-Baleh, Hamid Nadri, Alireza Moradi, Syed Nasir Abbas Burkhart, Mojtaba
Shakibaie, Mandana Jafari, Mostafa Golshani, Farshad Homayouni Moghadam,
Loghman Firoozpour, Ali Asadipour, Saeed Emami, Mehdi Khoobi, Alireza Foroumadi
PII:
S0223-5234(17)30594-9
10.1016/j.ejmech.2017.07.072
EJMECH 9635
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 December 2016
Revised Date: 23 April 2017
Accepted Date: 29 July 2017
Please cite this article as: L. Jalili-Baleh, H. Nadri, A. Moradi, S.N. Abbas Burkhart, M. Shakibaie,
M. Jafari, M. Golshani, F.H. Moghadam, L. Firoozpour, A. Asadipour, S. Emami, M. Khoobi, A.
Foroumadi, New racemic annulated pyrazolo[1,2-b]phthalazines as tacrine-like AChE inhibitors with
potential use in Alzheimer's disease, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.07.072.
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ACCEPTED MANUSCRIPT
Graphical abstract
New racemic annulated pyrazolo[1,2-b]phthalazines as tacrine-like AChE inhibitors
with potential use in Alzheimer’s disease
Leili Jalili-Baleh, Hamid Nadri, Alireza Moradi, Syed Nasir Abbas Burkhart, Mojtaba
Shakibaie, Mandana Jafari, Mostafa Golshani, Farshad Homayouni Moghadam,
Loghman Firoozpour, Ali Asadipour, Saeed Emami, Mehdi Khoobi, Alireza Foroumadi
NH2
O
O
NH2
N
N
N
N
O
Tacrine
Compound 7l
AChE IC50= 49 nM
High Selectivity
Low Toxicity on HepG2
Neuroprotective
AChE IC50= 365 nM
Low Selectivity
High Toxicity on HepG2
β-Amyloid Aggregation Inhibitor
A series of tacrine-like compounds 7a-u were designed and synthesized as potent AChE
inhibitors with high selectivity, low toxicity on hepatocytes, neuroprotective and and
amyloid aggregation inhibitory activity.
β-

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New racemic annulated pyrazolo[1,2-b]phthalazines as tacrine-like AChE
inhibitors with potential use in Alzheimer’s disease
a
b
b
c
Leili Jalili-Baleh , Hamid Nadri , Alireza Moradi , Syed Nasir Abbas Burkhart ,
d
d
a
Mojtaba Shakibaie , Mandana Jafari , Mostafa Golshani , Farshad Homayouni
Moghadam , Loghman Firoozpour , Ali Asadipour , Saeed Emami , Mehdi Khoobi
a,i*, Alireza Foroumadi a,g**
e
f
g
h
aDepartment of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran, Iran
bFaculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
cDepartment of Pharmaceutical Chemistry, College of Pharmacy, Aljouf University, Aljouf, Sakaka 2014, Saudi
Arabia
dPharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences,
Kerman, Iran
eDepartment of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology,
ACECR, Isfahan, Iran
fDrug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
gDepartment of Medicinal Chemistry, Faculty of Pharmacy and Neuroscience Research Center, Institute of
Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
hDepartment of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
iDepartment of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of
Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Abstract: A novel series of tacrine-like compounds 7a-u possessing a fused pyrazolo[1,2-b]phthalazine
structure were designed and synthesized as potent and selective inhibitors of AChE. The in-vitro biological
assessments demonstrated that several compounds had high anti-AChE activity at nano-molar level. The
more promising compound 7l with IC50 of 49 nM was 7-fold more potent than tacrine and unlike tacrine, it was
highly selective against AChE over BuChE. The cell-based assays against hepatocytes (HepG2) and neuronal
cell line (PC12) revealed that 7l had significantly lower hepatotoxicity compared to tacrine, with additional
neuroprotective activity against H2O2-induced damage in PC12 cells. This compound could also inhibit AChE-
induced and self-induced Aβ peptide aggregation. The advantages including synthetic accessibility, high
potency and selectivity, low toxicity, adjunctive neuroprotective and Aβ aggregation inhibitory activity, make
this compound as a new multifunctional lead for Alzheimer’s disease drug discovery.
Keywords: Alzheimer’s disease; Acetylcholinesterase; Neuroprotective activity; Tacrine; Phthalazine; β
-
amyloid aggregation
1

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* Corresponding author. Tel/Fax: +98-21-64121113. E-mail address: Mehdi.khoobi@gmail.com (M. Khoobi)
** Corresponding authors: Tel: +98-21-66954708; Fax: +98 21 66461178. E-mail addresses:
aforoumadi@yahoo.com (A. Foroumadi)
1. Introduction
Alzheimer’s disease (AD) as a most common cause of dementia is a neurodegenerative
disorder with complex pathological mechanisms. World health organization (WHO) predicted
that over the next century AD will be more prevalent than AIDS, cancer and cardiovascular
diseases [1]. The most important factors involved in AD are acetylcholine (ACh) decline,
accumulation of insoluble forms of amyloid-
followed by inflammatory actions ].
β (Aβ) and the hyperphosphorylated tau protein
[2
Because of multi-factorial nature of AD, and despite extensive research for understanding the
mechanism of the disease, development of new therapeutics is still challenging [3]. In
particular, design of multi-target directed ligands which act on multiple pathophysiological
pathways of AD has gained special attention [4, 5]. The main approved AD therapy has been
focused on increasing cholinergic transmission by inhibition of acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE) enzymes [6, 7]. By inhibiting cholinesterases, the amount
of ACh is enhanced in the neuronal synaptic cleft. Besides of cholinergic hypothesis, it was
approved that the peripheral anionic site (PAS) of AChE contributes to aggregation of
amyloid fibrils [8]. Also, recent evidences demonstrated that oxidative damage is an early
occurrence in initial progress of the neurodegenerative process in AD pathology [9, 10].
Therefore, protection against oxidative stress by neuroprotective agents will be a suitable
additional therapy for AD [11].
Tacrine, an aminoacridine derivative (Fig. 1,
A), was the first AChE inhibitor approved for
treatment of AD [12, 13]. This compound was withdrawn from the market due to its
hepatotoxicity [14]. In spite of tacrine’s side effects, it is still an attractive lead compound for
medicinal chemists due to its synthetic accessibility, low molecular weight and relatively easy
modification [15]. To develop new less toxic tacrine derivatives with high anticholinesterase
and selective peripheral binding potency, the replacement of benzene ring in the tacrine
structure with different heterocyclic systems has been considered more extensively [16, 17].
Previously, we described a number of pyrazolo[4′,3′:5,6]pyrano[2,3-b]quinolines as novel
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tacrine-derived AChE inhibitors [18, 19]. Furthermore, Barreiro et al. have reported a series of
tacrine-like compounds containing pyrazolo-quinoline scaffold (Fig.1). The rat brain
cholinesterases inhibition assay revealed that compound
B (Fig.1) had the most potent AChE
inhibitory activity [20]. On the other hand, some phthalazinone derivatives were also
synthesized and evaluated for their AChE and BuChE inhibitory activities [21-24].
In the light of above-mentioned reports, we were encouraged to design and synthesize a novel
series of tacrine-based compounds through replacement of the benzene ring in tacrine with
pyrazolo[1,2-b]phthalazine scaffold (Fig. 1). Thus we describe here the synthesis, in vitro and
in silico studies of compounds 7a-u as multifunctional agents for AD therapy.
2. Chemistry
The straightforward synthetic route to target compounds 7a-u was illustrated in Scheme 1.
The key intermediates pyrazolo[1,2-
b
]phthalazines 5a-u was synthesized via multi-
), hydrazine hydrate ( ), malononitrile ( ) and
in the presence of NiCl2 .6H2O as catalyst in
]phthalazines underwent AlCl3 catalyzed
) to obtain final products 7a-u [26].
component reaction of phthalimide (
appropriate benzaldehyde derivative
1
2
3
4
refluxing ethanol [25]. The pyrazolo[1,2-
b
5
-
Friedländer cyclization with cyclohexanone (
6
3. Results and discussion
3.1. Anti-cholinesterase activity and SAR discussion
The synthesized tacrine-like compounds 7a-u were evaluated in vitro for their anti-
cholinesterase activity against AChE and BuChE in comparison to tacrine as reference
The obtained IC50 values
drug.
presented in Table 1
(IC50 values 3.37 µM). Notably, compounds 7b-d
of compounds 7a-u against AChE and BuChE were
. In general, all compounds exhibited
potent activity against AChE
≤
,
7l and 7o possessing IC50 values in the
range of 23-80 nM were several times more potent than tacrine. The 3-fluorophenyl derivative
7o was the most potent compound against AChE being 15-fold more potent than tacrine.
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The IC50 values of 4-substituted phenyl derivatives 7b-f were less than that of unsubstituted
analog 7a. Thus, introduction of substituents such as halogen, Me and NO2 at the para-position
improves the anti-AChE activity. However, halogen group at the para-position of phenyl ring
is more favorable than other substituents. Although the substitution of halo group at ortho
-
position of phenyl ring could not improve inhibitory activity, but insertion of 2-Me, 2-NO2 and
2-MeO had positive effect on anti-AChE potency. In particular, 2-methoxyphenyl analog 7l
showed high potency with IC50 of 49 nM. Also, the meta-substituent on the phenyl ring could
positively modulate the inhibitory potential against AChE. However, introduction of
additional methoxy group at meta (3,5-dimethoxy) led to decrease of the activity against
AChE. The effect of 3-F substituent was more significant as observed with compound 7o as
the most potent derivative in the series.
Based on the results gathered in the Table 1, the anti-AChE potency was significantly
affected by the substitution on the C-14 of the pentacyclic framework. Replacing the 14-aryl
moiety with aliphatic groups such as methyl and propyl decreased the activity. However, the
pentyl derivative 7u was as potent as unsubstituted-phenyl analog 7a
.
As observed in Table 1, most of compounds were inactive against BuChE enzyme (IC50s
>100 µM). However, the most potent compound against AChE (7o), showed mild activity
against BuChE (IC50= 37.2 µ M). Unlike tacrine, all compounds exhibited high selectivity for
AChE over BuChE.
3.2. Kinetic study of AChE inhibition
The kinetics of AChE inhibition was studied for the active compound 7l with IC50 value of 49
nM. For this purpose, the rate of AChE inhibition by different concentrations (0, 25, 50 and
100 nM) of the potent inhibitor 7l was measured in the presence of substrate (ATCh). The
cholinesterase inhibition was determined as described in the experimental section. For each
inhibitor concentration, the initial velocity was measured at different substrate concentrations
(S) and the reciprocal of the initial velocity (1/v) was plotted respect to the reciprocal of
substrate concentration (1/[S]). Accordingly, the Lineweavere-Burk plot for the inhibition of
AChE and the secondary plot for calculation of steady-state inhibition constant (Ki) for
compound 7l were illustrated in Fig. 2. The Lineweavere-Burk plot showed a mixed-type
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inhibition pattern for compound 7l against AChE. The Ki value was 12.64 nM as calculated
from the secondary plot in Fig. 2.
3.3. Inhibition of AChE-induced and self-induced Aβ aggregation
Compound 7l as a potent AChE inhibitor was selected to assess its ability to inhibit Aβ1–42
peptide self-aggregation employing the thioflavin T (ThT) fluorescence method and
compared with donepezil as reference compound. The obtained inhibition for compound 7l
at 10
further explore the dual action of this compound, the capacity to inhibit the AChE-induced
β1-40 peptide aggregation was examined employing the same ThT-based fluorometric assay.
As seen in Table 2, compound 7l exhibited a significant AChE-induced A aggregation
µM concentration was 25.5% being higher than that of donepezil (15.4%, Table 2). To
A
β
inhibitory effect with percentage of inhibition 32% more effective than tacrine and donepezil,
indicating that compound 7l binds to PAS of AChE.
3.4. Cytotoxicity on HepG2 cells
As described in the Introduction section, the well-known drug tacrine suffers from
hepatotoxicity [14]. Thus, the tacrine-derived compounds may be associated with the
cytotoxicity on hepatocytes [27, 28]. In order to verify the hepatotoxicity of the selected
compounds (7b-d, 7l and 7o), an in vitro cytotoxicity assay was conducted on HepG2 cell
line. The effect of selected compounds on the viability of HepG2 cells at different
concentrations was outlined in Table 3. The obtained results showed that the cytotoxicity of
all tested compounds was significantly lower than that of tacrine. Particularly, 4-
bromophenyl derivative 7c showed no significant cytotoxicity even at the concentration of
50 µM. However, tacrine decreased the cell viability to 66.4% at this concentration. The 4-
fluoro- and 3-fluoro-phenyl analogs (7d and 7o, respectively) were somehow more toxic
than 4-chloro-, 4-bromo- and 2-methoxy-phenyl derivatives (7b, 7c and 7l).
3.5. Neuroprotective activity of compound 7l against H2O2-induced cell death in
PC12 cells
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Besides cholinesterases inhibition, other strategies such as protection against oxidative stress
may be useful in the management of AD [29]. Thus, the capability of the representative
compound 7l in the protection of neuronal cell line PC12 against H2O2-induced damage
was investigated in vitro. The differentiated PC12 cells were treated with compound 7l at
the concentrations of 1, 10 and 100 µM, prior to treatment with H2O2 (375 µM). The cell
viability was measured in comparison to quercetin by using MTT assay and the obtained
results were depicted in Fig. 3. As seen in Fig. 3, compound 7l (100 µM) significantly increased
the viability of PC12 cells under the ROS-mediated apoptosis condition. The neuroprotective
activity of compound 7l at 100 µM was comparable to quercetin (10 µM). It should be noted
that compound 7l at the 1 and 10 µM concentrations was neither neurotoxic nor
neuroprotective.
3.6. ADMET prediction
The ADMET (absorption, distribution, metabolism, excretion and toxicity) properties of the
target compounds 7a-u were predicted using admetSAR web-based application [30]. Based
on the predicted values (Table 4), all compounds are able to penetrate brain with a probability
of more than 90%, therefore they are considered as CNS-active compounds. Moreover, all
compounds were predicted to have no carcinogenic effect. Finally all compounds were
examined for oral activity using HIA and Caco-2 method. It was revealed that all compounds
may show desired oral absorption.
3.7. Ligand-protein docking study
The most promising compound 7l was selected for docking study with acetylcholinesterase
(1EVE) by using the AutoDock software. Since compound 7l have a chiral center at the 14-
position of pyrazoline ring, they are racemic mixture. Thus, both (
were studied in docking simulations. The binding energy of ( )-enantiomer was low and the
interactions were unfavorable but the ( )-enantiomer was fitted well in the active site. As
R)- and (S)-enantiomers
S
R
depicted in Figs 4 and 5, compound 7l is positioned in the enzyme active site in such a way to
interact with the amino acids at the rim of the cavity, and to act as a barrier preventing the
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entry of the substrates into the active site. The fused-rings part of the compound, which has an
approximately planar structure, makes
of the active site (PAS) including tyrosine 120 and tryptophan 278. Moreover, the
cyclohexene ring binds to tryptophan 278 by a -alkyl interaction. In this situation, one
π-π stacking with the aromatic amino acids at the rim
π
carbonyl group along with nitrogen of the pyridine ring bind to the hydroxyl group of tyrosine
69 by forming a network of hydrogen bonds. The pendent aryl group connected to 14-position
of the fused rings is positioned almost perpendicularly to the plane of the fused rings and
forms an intramolecular bond with the amine group.
4. Conclusion
A multi-component reaction followed by Friedländer reaction were applied to prepare a novel
tacrine analogs 7a-u bearing pyrazolo[1,2-b]phthalazines as selective anti-acetycholinesterase
agents. The anti-AChE activity of the designed compounds was optimized through altering
the pendent group on C-14 position of the pentacyclic skeleton. Most of compounds showed
selective anti-AChE activity at sub-micromolar levels. The best result was obtained by 3-
fluorophenyl in compound 7o with IC50 of 23 nM against AChE. This compound was 15 times
more potent than tacrine against AChE. Besides compound 7o, the 2-methoxyphenyl
derivative 7l with IC50 of 49 nM showed high potency against AChE. The promising
compounds 7o and 7l were highly selective for AChE over BuChE, and significantly less
toxic than reference drug tacrine. Since compound 7l showed less cytotoxic effect on HepG2
cells, thus this compound was selected for further studies. The biological results indicated
that compound 7l had neuroprotective activity against H2O2-induced damage in PC12 cells at
higher concentrations. Moreover, compound 7l was found to be an inhibitor of AChE-
induced A
study of compound 7l with AChE enzyme revealed that the fused rings component of the
molecule made stacking with the aromatic amino acids at the peripheral anionic site of
the enzyme (PAS). The straightforward synthesis, high potency and selectivity against AChE,
inhibition of AChE-induced A aggregation as well as A self-aggregation, relatively low
toxicity, along with significant neuroprotective effects of newly designed tacrine-like
β aggregation, which can also interfere with Aβ self-aggregation. The docking
π-π
β
β
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compounds 7a-u introduce them as attractive lead compounds towards discovery of effective
disease-modifying drugs for AD therapy.
5. Experimental
5.1. General chemistry
All commercially available reagents and starting materials were used without further
purification. TLC was conducted on silica gel 250 micron, F254 plates (Merck). Melting
points were measured on a Kofler hot stage apparatus and are uncorrected. The IR
1
spectra were taken using Nicolet FT-IR Magna 550 spectrograph (KBr disks). H NMR
spectra were recorded on Brucker 500 MHz NMR instrument. The chemical shifts (
coupling constants ( ) are presented in parts per million (ppm) and Hertz (Hz), respectively.
Scheme 1 shows the atoms numbering of the final compounds 7a-u useful in the H NMR
δ) and
J
1
interpretation. Elemental analyses were carried out by a CHN-Rapid Heraeus Elemental
Analyzer. The results of elemental analyses (C, H, N) were within ±0.4% of the
calculated values.
5.2. General procedure for the preparation of pyrazolo[1,2-b]phthalazine derivatives 5a-u
This procedure was carried out according to the reported method [25]. A mixture of
phthalimide (
ethanol (3 mL) was refluxed for 2 h. Afterward, malononitrile (
aldehyde (1 mmol) were added and the mixture was refluxed for 2 h. The precipitated solid
1
, 1 mmol), hydrazine hydrate (
2
, 1 mmol) and NiCl2 .6H2O (0.1 mmol) in
3
, 1 mmol) and approperiate
was separated by filtration and recrystallized from ethanol to obtain pure product 5a-u
.
5.3. General procedure for the preparation of compounds 7a-u
This procedure was carried out according to our previously reported method [26]. Briefly,
aluminium chloride (1.5 equiv.) was suspended in dry 1,2-dichloroethane (10 mL) at
room temperature under argon atmosphere. After stirring of the suspension for a few
minutes, the corresponding pyrazolo[1,2-
b]phthalazine (5, 1 equiv.) and cyclohexanone
(
6
, 1.5 equiv.) were added to the mixture and the reaction mixture was heated under
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reflux for 24 h. Progress of the reaction was monitored by TLC. After completion of the
reaction, an aqueous saturated solution of ammonium chloride was added to the mixture
until the aqueous solution became basic. After stirring for 30 min, the precipitate was
filtered, washed with water and recrystallized with acetonitrile.
5.3.1.
15-Amino-14-phenyl-2,3,4,14-tetrahydro-1H-quinolino[2',3':3,4]pyrazolo[1,2-
b]phthalazine-7,12-dione (7a). Yield 90%; off white solid; mp >250 ºC; IR (KBr, cm-1)
1
1642 (C=O), 1581 (C=N). H NMR (DMSO-d6, 500 MHz)
δ
:
νmax: 3421 and 3373 (NH2),
8.28 (d, 1H, = 7.5 Hz, H8
(m, 2H, H9,10
J
1H, J = 7.5 Hz, H11
), 7.91
phthalazine
), 8 . 0 1 ( d ,
phthalazine
), 7.44 (d, 2H,
phthalazine
J
= 7.5 Hz, H2,6
), 7.28 (m, 3H, H
phenyl
3,4,5
), 6.73 (s, 1H, H ), 5.63 (S, 2H, NH2), 2.73 (bs, 2H,
), 2.39-2.26 (m, 2H,
phenyl
cyclohexyl
14
13
), 1.75 (bs, 4H,
). C NMR (DMSO-d6, 100 MHz)
δ: 155.35, 153.5,
cyclohexyl
cyclohexyl
152.7, 148.3, 147.7, 137.5, 133.46, 133.34, 130.2, 128.19, 127.92, 127.36, 127.28,
126.44, 113.8, 100.2, 95.32, 61.08, 32.6, 22.3, 21.86. Anal. Calcd for C24H20N4O2: C, 72.1;
H, 5.09; N, 14.13. Found: C, 72.53; H, 6.93; N, 15.01.
5.3.2.
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7b
solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3421 and 3373 (NH2),
15-Amino-14-(4-chlorophenyl)-2,3,4,14-tetrahydro-1H
). Yield 85%; off white
1629 (C=O), 1581 (C=N).
1H
δ: 8.45 (d, 1H,
J
= 7.3 Hz, H
J
NMR (DMSO-d6, 500 MHz)
), 8.24 (d, 1H,
8 phthalazine
= 7.4 Hz,
H
), 7.82 (m, 2H, H
= 7.95 Hz, H3,4,5
), 7.46 (d, 2H, J = 8.05 Hz, H2,6
phthalazine
11 phthalazine
9,10
), 7.35 (d, 2H,
J
), 6.6 (s, 1H, H ), 4.3 (S, 2H, NH2), 3.0
14
phenyl
phenyl
(bs, 2H,
), 2.23 (bs, 2H,
), 1.85 (bs, 4H,
). 13C NMR
cyclohexyl
cyclohexyl
cyclohexyl
(DMSO-d6, 100 MHz)
δ: 155.6, 153.5, 152.6, 148.4, 147.8, 147.71, 136.9, 133.6,
133.5, 131.2, 130.2, 129.9, 128.2, 127.4, 126.5, 121.3, 113.8, 101.5, 60.6, 45.1, 32.7, 22.8,
22.3, 21.9. Anal. Calcd for C24H19ClN4O2: C, 66.9; H, 4.44; N, 13. Found: C, 66.02; H, 3.9;
N, 14.01.
5.3.3.
15-Amino-14-(4-bromophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7c
mp >250 ºC; IR (KBr, cm-1) νmax: 3461 and 3394 (NH2),
)
. Yield 90%; off white solid;
1
1626 (C=O), 1500 (C=N). H
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NMR (DMSO-d6, 500 MHz)
6.8 Hz,
δ
: 8.27 (d, 1H,
J
= 7.4 Hz, H8
J =
), 8.08 (d, 1H,
phthalazine
H11
), 7.93 (m, 2H, H
), 7.49 (d, 2H,
phthalazine
J
= 7.6 Hz, H2,6
phthalazine
9,10
), 7.42 (d, (d, 2H,
J
= 7.7 Hz, H
3,5 phenyl
), 6.7 (s, 1H, H ), 5.7 (S, 2H, NH2), 2.73
phenyl
14
(bs, 2H, cyclohexyl), 2.40-2.26 (m, 2H, cyclohexyl), 1.75 (bs, 4H,
). 13C NMR
cyclohexyl
δ:155.51, 153.47, 152.55, 148.34, 147.76, 136.73, 133.46,
(DMSO-d6, 100 MHz)
133.38, 131.08, 130.14, 129.77, 128.15, 127.28, 126.43, 121.18, 113.83, 101.35, 60.48,
44.88, 32.59, 22.74, 22.27, 21.85. Anal. Calcd for C24H19BrN4O2: C, 66.64; H, 4.03; N,
11.79. Found: C, 66.12; H, 3.95; N, 11.11.
5.3.4.
15-Amino-14-(4-fluorophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7d). Yield 92%; off white
solid; mp >300 ºC; IR (KBr, cm-1) νmax: 3454 and 3421 (NH2),
1633 (C=O), 1507 (C=N).
1H NMR (DMSO-d6, 500 MHz)
δ: 8.27 (d, 1H,
J
= 8 Hz, H
J
= 7
), 8.1 (d, 1H,
8 phthalazine
Hz,
H11
), 7.9 (m, 2H, H
phthalazine
), 7.48 (m, 2H, H
9,10 phthalazine 2,6 phenyl
), 7.1 (t, 2H,
J
= 9 Hz, H3,4,5
), 6.75 (s, 1H, H ), 5.65 (S, 2H, NH2), 2.73 (bs, 2H,
),
cyclohexyl
phenyl
14
13
2.4-2.27 (m, 2H,
), 1.76 (bs, 4H,
). C NMR (DMSO-d6, 100 MHz)
cyclohexyl
cyclohexyl
δ: 155.47, 153.46, 152.58, 148.4, 147.76, 147.2, 133.48, 133.37, 130.13, 129.77,
129.71, 128.22, 127.28, 126.44, 115.02, 114.85, 113.81, 101.56, 60.37, 32.60, 22.75, 22.28,
21.86. Anal. Calcd for C24H19FN4O2: C, 69.55; H, 4.62; N, 13.52. Found: C, 69.41; H, 4.43;
N, 13.51.
5.3.5.
15-Amino-14-(p-tolyl)-2,3,4,14-tetrahydro-1H-quinolino[2',3':3,4]pyrazolo[1,2-
b]phthalazine-7,12-dione (7e). Yield 95%; off white solid; mp >250 ºC; IR (KBr, cm-1)
1
1632 (C=O), 1490 (C=N). H NMR (DMSO-d6, 500 MHz)
δ
:
νmax: 3446 and 3360 (NH2),
8.28 (d, 1H, = 7.5 Hz, H8
J
J
= 7 Hz,
H11
), 8.08 (d, 1H,
phthalazine
), 7.9 (m,
phthalazine
J
= 7.5 Hz,
H
2H, H
), 7.3 (d, 2H,
9,10 phthalazine
), 7.08 (d, 2H,
J
= 7.5 Hz, H3,5
2,6 phenyl
), 6.69 (s, 1H, H ), 5.58 (S, 2H, NH2), 2.73 (bs, 2H,
), 2.42-2.29 (bs, 2H,
phenyl
cyclohexyl
). 13C NMR (DMSO-d6, 100
cyclohexyl
14
), 2.23 (bs, 3H, Me), 1.75 (bs, 4H,
cyclohexyl
MHz)
δ: 155.30, 153.35, 152.54, 148.29, 147.67, 137.29, 134.46, 133.46, 133.31,
130.07, 128.75, 128.29, 127.35, 127.26, 126.42, 113.77, 101.94, 60.91, 32.58, 22.74, 22.30,
21.88, 20.55. Anal. Calcd for C25H22N4O2: C, 73.15; H, 5.40; N, 13.65. Found: C, 72.95; H,
4.80; N, 13.33.
10

ACCEPTED MANUSCRIPT
5.3.6.
15-Amino-14-(4-nitrophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7f). Yield 80%; off white solid;
1
mp >250 ºC; IR (KBr, cm-1) νmax: 3435 and 3358 (NH2),
1633 (C=O), 1530 (C=N). H
NMR (DMSO-d6, 500 MHz)
δ
: 8.28-7.9 (m, 6H, H8,
), 7.7 (t,
9,10,11 phthalazine, H2,6 phenyl
2H,
J
= 8.5 Hz, H
), 6.8 (s, 1H, H ), 5.7 (s, 2H, NH2), 2.73 (bs, 2H, cyclohexyl),
3,5 phenyl
14
13
2.40-2.23 (m, 2H,
), 1.75 (bs, 4H,
). C NMR (DMSO-d6, 100 MHz)
cyclohexyl
cyclohexyl
δ: 156.97, 155.76, 150.85, 148.42, 147.90, 147.12, 134.57, 133.73, 133.53, 130.25,
128.88, 127.99, 127.35, 126.62, 123.67, 123.40, 113.97, 101.03, 60.40, 32.61, 22.77, 22.25,
21.83. Anal. Calcd for C24H19N5O4: C, 65.30; H, 4.34; N, 15.86. Found: C, 66.08; H, 4.35;
N, 14.91.
5.3.7.
15-Amino-14-(2-chlorophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7g). Yield 80%; off white solid;
1
mp >250 ºC; IR (KBr, cm-1) νmax: 3472 and 3410 (NH2),
1661 (C=O), 1556 (C=N). H
NMR (DMSO-d6, 500 MHz)
δ
: 8.34 (d, 1H, H
,
), 8.15 (d, 1H H
),
),
8 phthalazine 11 phthalazine
8.04-7.96 (m, 2H, 2H9,10
), 7.88 (1H, H3 phenyl), 7.43 (m, 3H, H
4,5,6
phthalazine
phenyl
7.03 (s, 1H, H14), 6.9 (S, 2H, NH2), 2.92 (bs, 2H,
), 2.47-2.30 (m, 2H,
cyclohexyl
). 13C NMR (DMSO-d6, 100 MHz)
cyclohexyl
), 1.75 (bs, 4H,
δ: 154.35,
cyclohexyl
135.79, 134.64, 134.01, 132.55, 131.34, 130.53, 130.6, 130.42, 127.51, 127.44, 126.89,
114.97, 100.83, 61.48, 22.59, 22.50, 21.19, 21.00. Anal. Calcd for C24H19ClN4O2: C, 66.90;
H, 4.44; N, 13. Found: C, 65.93; H, 4.63; N, 13.81.
5.3.8.
15-Amino-14-(2-bromophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7h). Yield 85%; off white
solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3424 and 3360 (NH2),
1638 (C=O), 1496 (C=N).
1H NMR (DMSO-d6, 500 MHz)
= 67.5 Hz, H ), 7.95 (m, 1H, H
δ
: 8.31 (d, 1H,
, H
= 7.5 Hz
,
), 8.07 (d, 1H J
8 phthalazine
J
), 7.88 (m, 2H, H
), 7.60
9,10 phthalazine
11 phthalazine
3 phenyl
1H,
,
7.24
1H,
,
(d, 1H, H5
), 7.37 (t,
J
= 7 Hz H6
),
(td,
cyclohexyl
). 13C NMR (DMSO-d6, 100 MHz)
cyclohexyl
J
= 8 Hz H ),
phenyl
6.85 (s, 1H, H14), 5.2 (S, 2H, NH2), 2.73 (bs, 2H,
), 1.75 (bs, 4H,
phenyl
4 phenyl
), 2.39-2.27 (m, 2H,
: 155.73,
153.74, 152.77, 148.57, 135.53, 133.55, 133.20, 132.49, 130.26, 130.18, 128.36, 172.93, 127,
δ
cyclohexyl
11

ACCEPTED MANUSCRIPT
35, 126.44, 113.98, 62.25, 32.57, 22.7, 22.24, 21.8. Anal. Calcd for C24H19BrN4O2: C,
60.64; H, 4.03; N, 11.79. Found: C, 61.38; H, 4.79; N, 10.11.
5.3.9.
15-Amino-14-(2-fluorophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7i). Yield 80%; off white solid;
1
mp >250 ºC; IR (KBr, cm-1) νmax: 3478 and 3356 (NH2),
1658 (C=O), 1530 (C=N). H
NMR (DMSO-d6, 500 MHz)
δ: 8.31 (d, 1H,
, H
= 7.5 Hz
,
), 8.08 (d, 1H J
phthalazine
J
=
8
7.5 Hz, H ), 7.94 (m, 1H, H
), 7.88 (m, 2H, H
), 7.33 (t,
9,10 phthalazine
11 phthalazine
3 phenyl
1H,
,
7.19
1H,
,
1H,
,
J
= 7 Hz H5
),
(t,
J
= 8 Hz H6
), 7.07 (t,
J
= 8 Hz H4
phenyl
phenyl
), 6.8 (s, 1H, H ), 5.53 (s, 2H, NH2), 2.72 (bs, 2H,
), 2.4-2.26 (m, 2H,
phenyl
cyclohexyl
14
), 1.75 (bs, 4H,
). 13C NMR (DMSO-d6, 100 MHz)
δ: 155.51,
cyclohexyl
cyclohexyl
152.63, 152.62, 148.59, 148.58, 133.68, 132.57, 131.04, 130.22, 130.21, 128.02, 127.42,
126.56, 124.34, 116.05, 115.89, 100.79, 95.06, 58.09, 32.65, 22.85, 22.34, 21,92. Anal.
Calcd for C H FN O2: C, 69.55; H, 4.62; N, 13.52. Found: C, 69.35; H, 4.33; N, 13.47.
4
24 19
5.3.10.
15-Amino-14-(o-tolyl)-2,3,4,14-tetrahydro-1H-quinolino[2',3':3,4]pyrazolo[1,2-
b]phthalazine-7,12-dione (7j). Yield 90%; off white solid; mp >250 ºC; IR (KBr, cm-1)
1
1657 (C=O), 1600 (C=N). H NMR (DMSO-d6, 500 MHz)
δ:
νmax: 3481 and 3365 (NH2),
8.28 (dd, 1H,
(m, 2H, H9,10
, H
= 6.5 Hz
,
), 8.07 (dd, 1H J
phthalazine
J
= 7 Hz, H11
), 7.92
phthalazine
8
1H,
,
7.17
3H,
H
), 7.23 (d,
phthalazine
J
= 7 Hz H3
),
(m,
),
4,5,6 phenyl
phenyl
6.74 (s, 1H, H14), 5.05 (S, 2H, NH2), 2.74 (bs, 2H, H2
), 2.43 (S, 3H, Me), 2.35-
cyclohexyl
). 13C NMR (DMSO-d6, 100 MHz)
δ:
2.28 (m, 2H,
), 1.76 (bs, 4H,
cyclohexyl
cyclohexyl
155.57, 153.60, 152.66, 148.52, 147.51, 136.29, 134.76, 133.56, 133.43, 130.76, 130.03,
128.24, 128.12, 127.29, 126.46, 114.11, 101.73, 59.94, 32.58, 22.72, 22.26, 21.83, 18.67.
Anal. Calcd for C25H22N4O2: C, 73.15; H, 5.40; N, 13.65. Found: C, 72.97; H, 5.32; N,
13.55.
5.3.11.
15-Amino-14-(2-nitrophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7k). Yield 85%; off white
solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3483 and 3419 (NH2),
1632 (C=O), 1522 (C=N).
1H NMR (DMSO-d6, 500 MHz)
δ: 8.49 (m, 1H, H8
,
), 8.18 (m, 1H H
phthalazine
11
), 7.78 (m, 3H, H9,10
phthalazine
and H
), 7.42 (m, 3H, H
),
phenyl
phthalazine
phenyl
3
4,5,6
12

ACCEPTED MANUSCRIPT
7.13 (s, 1H, H ), 4.77 (S, 2H, NH2), 2.98 (bs, 2H,
), 2.4 (bs, 2H,
),
cyclohexyl
cyclohexyl
14
1.86 (bs, 4H,
). 13C NMR (DMSO-d6, 100 MHz)
δ: 156.03, 153.57, 152.62,
cyclohexzyl
149.90, 148.86, 134.07, 133.56, 133.51, 131.53, 130.31, 129.61, 128.75, 127.35, 126.36,
123.67, 116.05, 115.89, 100.79, 95.06, 56.31, 32.60, 22.74, 22.23, 21,78. Anal. Calcd for
C24H19N5O2: C, 69.30; H, 4.34; N, 15.86. Found: C, 70.01; H, 4.83; N, 15.71.
5.3.12.
15-Amino-14-(2-methoxyphenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7l). Yield 75%; off white solid;
1
mp >250 ºC; IR (KBr, cm-1) νmax: 3442 and 3350 (NH2),
1668 (C=O), 1595 (C=N). H
NMR (DMSO-d6, 500 MHz)
δ: 8.32 (d, 1H,
, H
= 7.5 Hz
,
), 8.08 (d, 1H J
phthalazine
J
=
8
7.5 Hz, H11 ), 7.96 (m, 2H, H
), 7.47 (d, 1H, H
), 7.27 (td,
phthalazine
9,10 phthalazin
3 phenyl
1H,
1H,
,
7.04
1H,
,
,
J
= 7 Hz H5
),
(d,
J
= 8.2 Hz H6
), 6.91 (t,
phenyl
J
= 8 Hz H4
),
cyclohexyl
phenyl
), 6.82 (s, 1H, H ), 5.97 (S, 2H, NH2), 3.72 (S, 3H, OMe), 2.76 (bs, 2H,
phenyl
14
13
2.4-2.25 (m, 2H,
), 1.73 (bs, 4H,
). C NMR (DMSO-d6, 100 MHz)
cyclohexyl
cyclohexyl
δ: 155. 97, 153.37, 134.12, 133.75, 131.44, 129.67, 128.62, 128.39, 127.46, 126.74, 124.55,
121.29, 114.13, 112.03, 101.87, 58.12, 56.07, 26.20, 22.70, 21.91, 21.60. Anal. Calcd for
C25H22N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C, 69.97; H, 4.62; N, 12.84.
5.3.13.
15-Amino-14-(3,5-dimethoxyphenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7m). Yield 85%; off white
solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3440 and 3420 (NH2),
1633 (C=O), 1462 (C=N).
1H NMR (DMSO-d6, 500 MHz)
7 Hz, H ), 7 . 9 4 (m, 2H, H
δ
: 8.29 (d, 1H,
, H
= 7 Hz
,
), 8.11 (d, 1H J
8 phthalazine
J
=
), 6.63 (m, 3H, H
), 6.41 (s,
11 phthalazine
9,10 phthalazin
2,4,6 phenyl
1H, H ), 5.71 (s, 2H, NH2), 3.68 (s, 6H, 2OMe), 2.71 (bs, 2H,
), 2.42-2.27 (m,
cyclohexyl
). 13C NMR (DMSO-d6, 100 MHz)
δ: 160.34,
14
2H,
), 1.75 (bs, 4H,
cyclohexyl
cyclohexyl
155. 42, 152.73, 147.72, 140.34, 133.46, 133.36, 130.10, 127.30, 126.47, 113.94, 105.64,
99.02, 95.36, 61.05, 55.07, 32.57, 22.73, 22.28, 21.87. Anal. Calcd for C26H24N4O4: C,
68.41; H, 5.30; N, 12.27. Found: C, 67.93; H, 5.21; N, 13.1.
5.3.14.
15-Amino-14-(3-bromophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7n). Yield 90%; off white
solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3421 and 3354 (NH2),
1637 (C=O), 1600 (C=N).
13

ACCEPTED MANUSCRIPT
1H NMR (DMSO-d6, 500 MHz)
δ: 8.28 (d, 1H,
, H
= 7.5 Hz
,
), 8.09 (d, 1H J
8 phthalazine
J
= 7.5 Hz, H11 ), 7.93 (m, 2H, H
), 7.82 (s, 1H, H
phthalazine
), 7.46
phenyl
phthalazine
9,10
2
1H,
,
7.33
1H,
,
7.24
1H, ,
J = 8 Hz H5
(d,
J
= 7 Hz H
),
(d,
J
= 8 Hz H
),
cyclohexyl
). 13C NMR (DMSO-d6, 100 MHz)
(d,
), 2.43-2.26 (m, 2H,
: 155.63, 153,
4 phenyl
6 phenyl
), 6.7 (S, 1H, H ), 5.78 (S, 2H, NH2), 2.73 (bs, 2H,
phenyl
14
), 1.75 (bs, 4H,
δ
cyclohexyl
cyclohexyl
60, 148.39, 140.18, 133. 62, 130.97, 130.88, 130.69, 130.32, 128.21, 127.42, 126.57, 126.06,
121.36, 117.36, 113.91, 101.38, 95.50, 60.53, 32.68, 22.87, 22.35, 21.93. Anal. Calcd for
C24H19BrN4O2: C, 60.64; H, 4.03; N, 11.79. Found: C, 61.38; H, 4.79; N, 10.11.
5.5.15
15-Amino-14-(3-fluorophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7o). Yield 80%; off white
solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3485 and 3375 (NH2),
1659 (C=O), 1587 (C=N).
1H NMR (DMSO-d6, 500 MHz)
δ: 8.28 (d, 1H,
, H
= 7.5 Hz
,
), 8.09 (d, 1H J
8 phthalazine
J
= 7.5 Hz, H11 ), 7.9 (m, 2H, H
), 7.42 (d, 1H,
phthalazine
J
= 9.5 Hz, H4
7.09 1H,
phthalazine
1H,
9,10
,
7.19
1H,
,
), 7.32 (q,
J
= 7 Hz H
),
(d,
J
= 7 Hz H
),
(t,
J
phenyl
2 phenyl
6 phenyl
,
= 8 Hz H
), 6.73 (s, 1H, H ), 5.73 (s, 2H, NH2), 2.73 (bs, 2H, cyclohexyl), 2.4-2.29
5 phenyl
14
(m, 2H,
), 1.75 (bs, 4H,
). 13C NMR (DMSO-d6, 100 MHz)
δ:
cyclohexyl
cyclohexyl
155.62, 152.91, 152.60, 148.09, 147.75, 145.54, 140.31, 133.51, 130.25, 130.26, 127.34,
126.50, 122.99, 114.98, 114.89, 102.9, 60.77, 32.61, 22.77, 22.29, 21.87. Anal. Calcd for
C24H19FN4O2: C, 69.55; H, 4.62; N, 13.52. Found: C, 68.89; H, 4.68; N, 13.2.
5.5.16
15-Amino-14-(3-nitrophenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7p). Yield 80%; off white
solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3435 and 3358 (NH ),
1633 (C=O), 1530 (C=N).
2
1H NMR (DMSO-d6, 500 MHz)
δ
: 8.54 (s, 1H, H phenyl), 8.28 (d, 1H,
, H
= 7.5 Hz
8
J
2
1H,
,
,
J
), 8.13 (d,
phthalazine
J
= 8 Hz H
), 8.08 (d, 1H
= 7.5 Hz, H11
),
phthalazine
4 phenyl
1H,
,
7.92 (m, 2H, H
), 7.78 (d, 1H,
9,10 phthalazine
J
= 7.6 Hz, H
), 7.57 (t,
J
= 8 Hz
6 phenyl
H5
), 6.86 (S, 1H, H ), 5.86 (S, 2H, NH2), 2.73 (bs, 2H,
cyclohexyl
14
), 2.4-2.27 (m,
phenyl
2H,
), 1.75 (bs, 4H,
). 13C NMR (DMSO-d6, 100 MHz)
δ: 155.86,
cyclohexyl
cyclohexyl
153.77, 152.78, 148.57, 148.04, 147.44, 139.58, 133.86, 133.67, 130.42, 130.17, 128.19,
14

ACCEPTED MANUSCRIPT
127.47, 126.60, 123.42, 123.23, 114.01, 101.06, 60.49, 32.74, 22.93, 22.39, 21.96. Anal.
Calcd for C24H19N5O4: C, 65.30; H, 4.34; N, 15.86. Found: C, 64.56; H, 4.21; N, 15.08.
5.5.17.
15-Amino-14-(m-tolyl)-2,3,4,14-tetrahydro-1H-quinolino[2',3':3,4]pyrazolo[1,2-
b]phthalazine-7,12-dione (7q). Yield 95%; off white solid; mp >250 ºC; IR (KBr, cm-1)
1
1678 (C=O), 1600 (C=N). H NMR (DMSO-d6, 500 MHz)
δ
:
νmax: 3482 and 3350 (NH2),
8.28 (d, 1H, , H
,
J
= 7.5 Hz
), 8.08 (d, 1H
phthalazine
J
= 7.5 Hz, H11
), 7.93
phthalazine
8
1H,
,
(m, 2H, H
), 7.25 (m, 2H, H
9,10 phthalazine
), 7.18 (t,
J
= 7.5 Hz H5
),
phenyl
phenyl
2,4
,
J = 7.5 Hz H6
phenyl
7.06 (d, 1H,
), 6.67 (S, 1H, H ), 5.66 (s, 2H, NH2), 2.72 (bs, 2H,
14
), 2.4-2.27 (m, 2H,
), 2.23 (s, 3H, 3-Me), 1.75 (bs, 4H, ).
cyclohexyl
cyclohexyl
cyclohexyl
13C NMR (DMSO-d6, 100 MHz)
δ: 155.35, 152.75, 147.77, 137.48, 133.63, 133.49, 130.21,
128.77, 128.36, 128.24, 127.80, 127.40, 126.56, 124.56, 113.86, 102.16, 61.20, 32.66, 22.87,
22.38, 21.95, 20.99. Anal. Calcd for C25H22N4O2: C, 73.15; H, 5.40; N, 13.65. Found: C,
73.22; H, 5.03; N, 12.99.
5.5.18.
15-Amino-14-(3-hydroxyphenyl)-2,3,4,14-tetrahydro-1H-
quinolino[2',3':3,4]pyrazolo[1,2-b]phthalazine-7,12-dione (7r). Yield 75%; off white solid;
1
mp >250 ºC; IR (KBr, cm-1) νmax: 3481 and 3449 (NH2),
1641 (C=O), 1577 (C=N). H
NMR (DMSO-d6, 500 MHz)
δ
: 9.38 (s, 1H, OH), 8.28 (d, 1H,
, H
= 7.5 Hz
J
),
=
8 phthalazine
,
8.09 (d, 1H
J
= 7.5 Hz, H11
), 7.93 (m, 2H, H
), 7.08 (t, 1H,
J
phthalazine
9,10 phthalazine
1H,
,
7.9 Hz, H5
), 6.92 (d,
J
= 7.7 Hz H
), 6.76 (bs, H
), 6.66 (S, 1H,
phenyl
6 phenyl
2 phenyl
,
H14), 6.64 (d, 1H,
J
= 8 Hz H
), 5.72 (S, 2H, NH2), 2.72 (bs, 2H,
), 2.42-
4 phenyl
cyclohezyl
). 13C NMR (DMSO-d6, 100 MHz)
δ:
2.26 (m, 2H, cyclohexyl), 1.75 (bs, 4H,
cyclohexyl
157.33, 155.60, 153.55, 148.28, 139.05, 133.66, 133.49, 129.24, 128.35, 127.37, 126.57,
118.28, 117.07, 115.16, 113.87, 102.07, 61.04, 32.63, 22.81, 22.34, 21.93. Anal. Calcd for
C24H20N4O3: C, 69.89; H, 4.89; N, 13.58. Found: C, 70.11; H, 5.06; N, 13.83.
5.3.19.
15-Amino-14-methyl-2,3,4,14-tetrahydro-1H-quinolino[2',3':3,4]pyrazolo[1,2-
b]phthalazine-7,12-dione (7s). Yield 70%; off white solid; mp >250 ºC; IR (KBr, cm-1)
1
1641 (C=O), 1588 (C=N). H NMR (DMSO-d6, 500 MHz)
δ
:
νmax: 3480 and 3349 (NH2),
8.23 (d, 2H, , H
J
= 2 Hz
), 7.94 (bs, 2H, H
8,11 phthalazine
), 6.14 (s, 2H, NH2),
9,10 phthalazine
5.79 (S, 1H, H14), 2.67 (bs, 2H,
), 2.4-2.1 (m, 2H, cyclohexyl), 1.75 (bs, 4H,
cyclohexyl
15

ACCEPTED MANUSCRIPT
), 1.55 (d, 3H, ,
J
= 6Hz, CH3). 13C NMR (DMSO-d6, 100 MHz)
δ: 154.95,
cyclohexyl
153.75, 152.77, 149.48, 133.63, 133.40, 130.21, 128.77, 127.32, 126.53, 113.86, 100.16,
55.23, 32.68, 23.01, 22.49, 22.12, 16.54. Anal. Calcd for C19H18N4O2: C, 68.25; H, 5.43; N,
16.76. Found: C, 68.71; H, 5.10; N, 16.62.
5.3.20.
15-Amino-14-propyl-2,3,4,14-tetrahydro-1H-quinolino[2',3':3,4]pyrazolo[1,2-
b]phthalazine-7,12-dione (7t). Yield 70%; off white solid; mp >250 ºC; IR (KBr, cm-1)
1
2980 (CH aliphatic), 1661 (C=O), 1567 (C=N). H NMR
νmax: 3478 and 3415 (NH2),
(DMSO-d6, 500 MHz) : 8.24 (d, 2H,
), 6.09 (s, 2H, NH2), 5.93 (s, 1H, H ), 2.69 (bs, 2H, cyclohexyl ), 2.43-2.31 (m,
δ
, H
= 2 Hz
J
), 7.95 (bs, 2H, H
phthalazine
8,11
9,10
phthalazine
2H,
14
), 1.99 (t, 2H, CH2), 1.76 (bs, 4H, cyclohexyl), 0.94-0.85 (m, 2H, CH2),
cyclohexyl
0.71 (t, 3H, ,
13
J
= 6.9Hz, CH3). C NMR (DMSO-d6, 100 MHz)
δ
: 154.94, 153.85, 152.64,
149.02, 147.99, 133.69, 133.46, 130.09, 128.31, 127.34, 126.63, 113.52, 99.85, 58.95, 32.68,
30.23, 22.98, 22.48, 22.11, 15.71, 13.80. Anal. Calcd for C21H22N4O2: C, 69.59; H, 6.12; N,
15.46. Found: C, 68.03; H, 5.50; N, 16.58.
5.3.21.
15-Amino-14-pentyl-2,3,4,14-tetrahydro-1H-quinolino[2',3':3,4]pyrazolo[1,2-
b]phthalazine-7,12-dione (7u). Yield 75%; off white solid; mp >250 ºC; IR (KBr, cm-1)
1
2980 (CH aliphatic), 1656 (C=O), 1563 (C=N). H NMR
νmax: 3478 and 3374 (NH2),
(DMSO-d6, 500 MHz) : 8.24 (m, 2H, H
δ
), 7.95 (m, 2H, H
8,11 phthalazine 9,10 phthalazine
),
6.08 (s, 2H, NH2), 5.93 (s, 1H, H14), 2.69 (bs, 2H, 2H9), 2.43-2.31 (m, 4H, cyclohexyl),
2.02 (m, 2H, CH2), 1.77 (bs, 4H, cyclohexyl), 1.08 (m, 2H, CH2), 0.94-0.8 (m, 2H, CH2),
13
0.68 (t, 3H,
J
= 6.6 Hz, CH3). C NMR (DMSO-d6, 100 MHz)
δ: 154.87, 153.81, 152.58,
148.97, 147.94, 133.63, 133.39, 130.02, 128.25, 127.29, 126.57, 113.45, 99.75, 58.93, 32.62,
30.99, 27.72, 22.91, 22.42, 22.05, 21.74, 13.71. Anal. Calcd for C23H26N4O2: C, 70.75; H,
6.71; N, 14.35. Found: C, 70.74; H, 6.51; N, 14.12.
5.4. In vitro AChE/BuChE inhibition assay
The anti-cholinesterase activity of target compounds 7a-u was assessed in vitro against
AChE from Electrophorus electricus
(eelAChE) and horse serum butyrylcholinesterase
(eqBuChE) by using the spectrophotometric method of Ellman [32]. In order to obtain a
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range of 20-80% enzyme inhibition, five different concentrations of each compound were
tested. A mixture of phosphate buffer (0.1 mol/L-1, pH 8.0, 3 mL),
nitrobenzoic acid) , acetylcholinesterase
or butyrylcholinesterase
5,5
′
-dithio-bis(2-
(100 L,
and compound solution (100 µL) was pre-incubated for 10 min. Then, the
µ
(DTNB, 100
µ
L)
2.5 IU/mL)
substrate (acetylthiocholine iodide or butyrylthiocholine iodide) was added. In parallel, a
blank containing all components without enzyme was used in order to account the non-
enzymatic reaction. Changes in absorbance were measured at 412 nm for 6 min at 25 °C by
using an UV Unico Double Beam spectrophotometer. The IC50 values were determined
graphically from log concentration vs. % of inhibition curves. All experiments were
performed in triplicate.
5.5. Determination of the inhibitory potency on Aβ1-42 self-aggregation
In order to investigate the Aβ1-42 self-aggregation, a ThT-based fluorometric assay was
performed [33, 34]. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) pre-treated A
(Bachem company, Switzerland) was diluted in assay buffer to have a stock solution ([A
42]= 50 mM). Experiments were performed by incubating the peptide in phosphate buffer
(pH 7.4), at 37 °C for 48 h (final A concentration = 25 mM, 10 mL) with and without
β
sample
β
1-
β
inhibitor. The inhibitor was dissolved in DMSO and diluted in the assay buffer at a final
concentration of 10 µM. Blanks containing inhibitor and ThT were also prepared and
evaluated to account for quenching and fluorescence properties. After incubation, samples
were diluted to a final volume of 200 mL with 180 mL ThT (5 mM in 50 mM glycine-NaOH
buffer, pH 8.5). The fluorescence intensity was carried out with multi-mode plate reader
(EnSpire, PerkinElmer Waltham, Massachusetts, United States) at (λex = 446 nm; λem = 490
nm), each assay was run in triplicate and each reaction was repeated at least three
independent times, values at plateau were averaged after subtracting the background
fluorescence of 5 mM ThT solution. The fluorescence intensities were compared and the %
inhibition was calculated by the following equation: 100 – [(IFi - IFb)/(IFo - IFb) × 100] where
IFi, IFo and IFb are the fluorescence intensities obtained for A
β aggregation in the presence of
inhibitors, in the absence of inhibitors and the blanks, respectively.
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5.6. Determination of the inhibitory potency on Aβ1-40 aggregation induced by AchE
For co-incubation experiments [35, 36] of Aβ (1-40) trifluoroacetate salt (Bachem company,
Switzerland) and AChE from (Sigma, Electrophorus electricus), the mixtures of Aβ1-40
peptide and AChE in presence or absence of the test inhibitor were incubated for 24 h at
room temperature. The final concentrations of Aβ (dissolved in DMSO and diluted 0.215 M
sodium phosphate buffer, pH 8), AChE (dissolved in 0.215 M sodium phosphate buffer, pH
8.0) and the tested compound are 200 µM, 2 µM and 100 µM respectively. To analyze co-
aggregation inhibition, the ThT fluorescence method was used and the fluorescence was
measured with multi-mode plate reader (EnSpire, PerkinElmer Waltham, Massachusetts,
United States) at (λex = 446 nm; λem = 490 nm). After co-incubation, 20 µL of the mixture
solutions was diluted to a final volume of 2 mL with ThT (1.5 µM in 50 mM glycine-NaOH
buffer, pH 8.5). Blanks containing Aβ, AChE, Aβ plus the tested compound, and AChE plus
the tested compound in 0.215 M sodium phosphate buffer (pH 8.0) were prepared. The
percent inhibition of the AChE-induced aggregation due to the presence of inhibitor was
calculated by the following expression: 100 – [(IFi - IFb)/(IFo - IFb) × 100] where IFi, IFo and
IFb are the fluorescence intensities obtained for Aβ aggregation in the presence of inhibitors,
in the absence of inhibitors and the blanks, respectively. Each assay was run in triplicate and
each reaction was repeated at least three independent times.
5.7. Neuroprotection assay against H2O2-induced cell death in PC12 cells
The neuroprotection assay was conducted by using rat differentiated PC12 cells [37]. The
differentiated PC12 cells were treated with different concentrations (1, 10, 50 and 100 µM)
of the compound and incubated for 3 h before treatment with H2O2 (350 µM). The DAPI
staining was used for checking the occurrence of apoptosis. The cell viability was measured
after 24 h by using the MTT assay. Briefly, MTT solution (10 µ L, 5 mg/mL, Sigma) was
added to the cultured PC12 cells (150
µ L) and incubated in a CO2 incubator for 3.5 h.
Thereafter, medium was removed and DMSO (150 µ l) was added into the each well for
dissolving the precipitated formazan. The dye dissolution was facilitated by shaking the plate
for 10 min at speed of 120 rpm. Finally, optical density (OD) was determined at 560 nm on
the microplate reader (BioTek synergy HT). Results were adjusted considering OD measured
in the blank [38].
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5.8. In vitro cytotoxicity assay against HepG2 cells
In order to investigate the effect of selected compounds 7b-d and 7o on cell viability, the
MTT assay was performed using HepG2 cell line. The cell line was provided from Iranian
Biological Resource Center (IBRC, Tehran, Iran) and subcultured in DMEM medium
containing FBS (10%) at 37 °C under 5% CO2 in a humidified chamber. The cell was
harvested in exponential phase of growth and seeded into 96-well tissue culture microplate
(1.5×104 cells/well) and incubated at 37 °C for 24 h to adhere. The medium was then removed
and cells were exposed (for further 24 h) to desired concentrations (1–300 µM) of each
compound which previously dissolved in FBS-free DMEM medium. Thereafter, the medium
of each well was replaced by MTT solution (5 mg/mL) and incubated for 4 h to form
formazan crystal which was then dissolved by addition of DMSO (100 µL). The related
absorbance (540 nm) was consequently recorded using a Synergy 2 multi-mode plate reader
apparatus (Biotek, Winooski, VT, USA) and applied to determine the viability percent
regarding to that of the control. Three independent replicates of each experiment were
performed and mean of the obtained results was reported.
5.9. Docking simulations
The program Autodock Vina was used for docking simulations [39]. The crystal structure of
Torpedo californica acetylcholinesterase (1EVE) was retrieved from Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do). To create receptor and ligand structures for
docking, the following procedure was conducted. First, the co-crystallized ligand and water
molecules were removed from the protein. After that the atomic coordinates of the ligands
was prepared using Marvinsketch, 2012, ChemAxon (http://www.chemaxon.com), the 3D
structures were constructed using Openable [40]. Then, the receptor and optimized structure
of the ligands were converted to required pdbqt format using Autodock Tools [41]. The
Autodock Vina parameters were set as follow; box size: 15×15×15 Å, the center of box:
x = 2.023, y = 63.295, z = 67.062 (geometrical center of co-crystallized ligand), the
exhaustiveness: 100, and the remaining parameters were left unchanged. The calculated
geometries were ranked in terms of free energy of binding and the best poses were selected
19

ACCEPTED MANUSCRIPT
for further analysis. All molecular visualizations were carried out in DS Viewer Pro
(Accelrys, Inc., San Diego, CA).
Acknowledgments
This study was funded and supported by Tehran University of Medical Sciences (TUMS);
and Iran National Science Foundation (INSF).
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Captions:
Figure 1. The structures of tacrine, previously reported pyrazolo-quinoline analog of tacrine
and newly designed tacrine-like compounds 7a-u bearing an annulated pyrazolo[1,2-
b
]phthalazine nucleus.
Figure 2. Left: Lineweavere-Burk plot for the inhibition of AChE by compound 7l at
different concentrations of substrate (ATCh), Right: Secondary plot for calculation of steady-
state inhibition constant (Ki) of compound 7l
.
Figure 3. Neuroprotective effect of compound 7l against H2O2-induced cell death in
PC12 cells. Data are expressed as Mean ± SD (
P<0.01 all vs. control group.
n = 8), ****: P<0.0001, ***: P<0.001, *:
Figure 4. The proposed orientation of compound 7l in the AChE active site.
Figure 5. The binding mode of compound 7l in the active site of AChE.
Scheme 1. Synthesis of compounds 7a-u
.
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Table
1
Inhibitory activity of the target compounds 7a-u against AChE and BuChE.
O
R
NH2
N
N
N
O
IC50 (µM)a IC50 (µM)
Compound
R
AChE
0.614
0.060
0.080
0.069
0.100
0.310
0.592
0.599
0.618
0.193
0.280
0.049
3.37
BuChE
>100
96.2
Phenyl
7a
7b
7c
4- Chlorophenyl
4- Bromophenyl
4- Fluorophenyl
>100
>100
>100
>100
>100
>100
>100
>100
>100
93.28
>100
>100
37.2
7d
7e
p
-Tolyl
4- Nitrophenyl
2- Chlorophenyl
2-Bromophenyl
2- Fluorophenyl
7f
7g
7h
7i
o
-Tolyl
7j
2-Nitrophenyl
7k
7l
2-Methoxyphenyl
3,5-Dimethoxyphenyl
3-Bromophenyl
3-Fluorophenyl
3-Nitrophenyl
7m
7n
7o
7p
7q
7r
0.271
0.023
0.140
0.467
0.160
2.15
>100
>100
>100
>100
>100
>100
0.013
m
-Tolyl
3-Hydroxophenyl
Methyl
Propyl
Pentyl
-
7s
1.63
7t
0.612
0.365
7u
Tacrine
aData are expressed as mean ± S.E. of at least three different experiments.
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Table 2
Inhibition of AChE-induced and self-induced A
with tacrine and donepezil.
β aggregation by compound 7l in comparison
Inhibition of Aβ aggregation (%)
Compound
7l
Self-induceda
AChE-inducedb
25.5 ± 2.9
NTc
32.0 ± 4.0
7.0 ± 0.5
Tacrine
15.4 ± 4.2
25.0 ± 2.0
Donepezil
a
Inhibition of self-induced Aβ(1-42) aggregation (25 mM) produced by the tested compound at 10 µM
concentration. Values are expressed as means ± SEM of three experiments.
b
Co-aggregation inhibition of Aβ(1-40) and AChE (2 µM, ratio 100:1) by the tested compound at 100 µM
concentration was detected by ThT assay. Values are expressed as means ± SEM of three experiments.
c Not tested.
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Table 3
The effect of selected compounds 7b-d
different concentrations.
Compound
, 7l and 7o on the viability of HepG2 cell line at
Viability (%) of HepG2 cells
1 µM
5 µM
10 µM
25 µM
50 µM
100 µM
300 µM
98.6±1.3 ns 96.2±0.9 ns 95.1±2.2 ns 94.4±1.1 *
92.4±1.1*
95.7±2.2ns
91.3±1.2*
92.8±1.5*
89.3±0.4*
90.0±1.3*
7b
99.8±1.2ns
91.2±1.6 *
93.5±1.7*
96.3±0.8ns
94.2±1.3*
98.3±0.5ns
97.6±1.2ns
96.4±1.4ns
7c
87.5±1.3 ** 83.3±1.3 ** 80.0±0.7 ** 75.9±2.2 ** 71.4±1.4 ** 66.4±2.1 **
90.9±1.2 * 86.1±2.1 ** 82.7±1.6 ** 78.8±1.4 ** 74.8±2.1 ** 68.2±1.4 **
95.0±1.6 ns 95.2±0.5 ns 90.2±1.1 * 87.3±0.2 * 86.4±1.4 ** 83.0±0.7 **
90.3±0.9** 77.3±1.1** 73.3±0.7** 66.4±1.2** 58.3±2.2** 43.2±0.9**
7d
7o
7l
Tacrine
Cell viability was determined using MTT assay protocol. Data are expressed as the mean ± SEM of three
independent replicates. The significant (** 0.01, * 0.05) and not significant (ns) values with respect to
p
≤
p ≤
control group were obtained after one-way ANOVA analysis followed by Newman-Keuls post hoc test.
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