Design and synthesis of novel anti-Alzheimer's agents: Acridine-chromenone and quinoline-chromenone hybrids

Article abstract of DOI:10.1016/j.bioorg.2016.06.001

A novel series of acridine-chromenone and quinoline-chromenone hybrids were designed, synthesized, and evaluated as anti-Alzheimer's agents. All synthesized compounds were evaluated as cholinesterases (ChEs) inhibitors and among them, 7-(4-(6-chloro-2,3-d

Full text of DOI:10.1016/j.bioorg.2016.06.001

Accepted Manuscript
Design and synthesis of novel anti-Alzheimer’s agents: acridine-chromenone
and quinoline-chromenone hybrids
Zahra Najafi, Mina Saeedi, Mohammad Mahdavi, Reyhaneh Sabourian,
Mahnaz Khanavi, Maliheh Barazandeh Tehrani, Farshad Homayouni
Moghadam, Najmeh Edraki, Elahe Karimpor-Razkenari, Mohammad
Sharifzadeh, Alireza Foroumadi, Abbas Shafiee, Tahmineh Akbarzadeh
PII:
S0045-2068(16)30055-4
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.06.001
YBIOO 1915
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 February 2016
31 May 2016
1 June 2016
Please cite this article as: Z. Najafi, M. Saeedi, M. Mahdavi, R. Sabourian, M. Khanavi, M.B. Tehrani, F.H.
Moghadam, N. Edraki, E. Karimpor-Razkenari, M. Sharifzadeh, A. Foroumadi, A. Shafiee, T. Akbarzadeh, Design
and synthesis of novel anti-Alzheimer’s agents: acridine-chromenone and quinoline-chromenone hybrids,
Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.06.001
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Design and synthesis of novel anti-Alzheimer’s agents: acridine-chromenone and quinoline-
chromenone hybrids
Zahra Najafi a, Mina Saeedi b,c, Mohammad Mahdavi d, Reyhaneh Sabourian c, Mahnaz Khanavi
a
f
g
e, Maliheh Barazandeh Tehrani , Farshad Homayouni Moghadam , Najmeh Edraki , Elahe
c
h
i
i
Karimpor-Razkenari , Mohammad Sharifzadeh , Alireza Foroumadi , Abbas Shafiee ,
Tahmineh Akbarzadeh a,c
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
bMedicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
cPersian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
dDrug Design and Development Research Center, Tehran University of Medical Sciences,
Tehran, Iran
eDepartment of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran
fDepartment of Cellular Biotechnology at Cell Science Research Center, Royan Institute for
Biotechnology, ACECR, Isfahan, Iran
 Corresponding author. Tel.: +98-21-66419413; Fax: +98-21-66461178.
E-mail address: akbarzad@tums.ac.ir (T. Akbarzadeh).
1

gMedicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
hDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
iDepartment of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran, Iran
Key words: Acridine-Chromenone, Alzheimer’s disease, Anti-Cholinesterase, Docking study,
Neuroprotective activity, Quinoline-Chromenone, β-Secretase inhibitor.
Abstract: A novel series of acridine-chromenone and quinoline-chromenone hybrids were
designed, synthesized, and evaluated as anti-Alzheimer’s agents. All synthesized compounds
were evaluated as cholinesterases (ChEs) inhibitors and among them, 7-(4-(6-chloro-2,3-
dihydro-1H-cyclopenta[b]quinolin-9-ylamino)phenoxy)-4-methyl-2H-chromen-2-one
(8e)
exhibited the most potent anti-acetylcholinesterase (AChE) inhibitory activity (IC50 = 16.17
µM) comparing with rivastigmine (IC50 = 11.07 µM) as the reference drug. Also, compound 8e
was assessed for its β-secretase (BACE1) inhibitory and neuroprotective activities which
demonstrated satisfactory results. It should be noted that both kinetic study on the inhibition of
AChE and molecular modeling revealed that compound 8e interacted simultaneously with both
the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE.
1. Introduction
2

Alzheimer’s disease (AD) is the most common cause of dementia which is characterized by the
progressive cognitive impairment in the elderly people. The classic characteristics found in the
brains of AD patients confirmed that the following factors are involved in the disease: i) loss of
cholinergic neurons in areas of the brain associated with memory and cognition, ii)
accumulation of the β-amyloid peptide (βA), and iii) formation of neurofibrillary tangles
(NFTs) of hyperphosphorylated tau protein [1-2]. Moreover, other factors such as
mitochondrial dysfunction, hormone imbalance, inflammation, mitotic dysfunction, calcium
mishandling, and genetic components play important roles in the development of disease [3].
Among factors involved in AD, the cholinergic hypothesis is related to the low concentration
of acetylcholine (ACh) in hippocampus and cortex which are associated with learning and
memory functions [4]. ACh is rapidly hydrolyzed by acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) enzymes into choline and acetic acid leading to the termination
of neurotransmission signal. AChE has shown a very high catalytic efficiency for ACh
hydrolysis and it is widely found in cholinergic synapses. However, BChE has exhibited low
activity in the hydrolysis reaction and it is mainly distributed in plasma and tissues playing an
supplementary role in synaptic transmission [5]. It should be considered that in patients with
AD, the activity of AChE decreases or does not change but the activity of BChE gradually
increases leading to imbalance between AChE and BChE [6]. It sees that both AChE and
BChE are probably to be involved in the regulation of the ACh level [7] and ChE inhibitors
increase ACh levels in cholinergic neurons which can be considered as an efficient strategy for
the treatment of AD symptoms [8-9].
Deposition of βA (amyloid plaques) in the brain has emerged as one of the most important
origin of AD. Consequently, development of compounds possessing both AChE and βA
3

aggregation inhibitory activities, provides additional advantages in AD therapeutic approach
[10]. In this respect, one of the versatile routes for the inhibition of βA aggregation is
prevention the hydrolysis of amyloid precursor protein (APP) via inhibition of β-secretase
(BACE1) [11]. Also, it has been reported that development of amyloid plaques and
neurofibrillary (NFT) tangles in the brain may be accelerated by oxidative damages [12].
Accordingly, neuroprotective agents can be helpful in the treatment of AD by inhibiting the
formation of free radicals or protecting against oxidative stress [13].
AChE possesses two binding sites including catalytic site (CS which involves catalytic anionic
site (CAS) and catalytic triad) and the peripheral anionic site (PAS) mainly characterized by
two tryptophan residues, Trp84 at the active site and Trp279 at the mouth of the gorge [14]. In
this respect, several dual-binding inhibitors have been synthesized and evaluated for anti-
AChE activity [15-18]. Tacrine, donepezil, rivastigmine, and galantamine are well-known
AChE inhibitors. Tacrine is the first cholinesterase inhibitor which was approved by the FDA
in 1993. However, it soon showed hepatotoxicity and was subsequently abandoned [19, 20]. In
this respect, lots of efforts were made to design and synthesize tacrine-based agents with low
side effects and better pharmacokinetics properties. Also, tacrine hybrids such as tacrine-4-
aminoquinoline, tacrine-huperzine A, tacrine-ferulic acid, tacrine-phenylphenanthridinium,
tacrine-indole were efficiently developed [21-25]. To establish further tacrine-based inhibitors,
we also focused on the anti-ChE activity of chromenones (A-D) (Fig. 1) [26-30], and in
continuation of our research program on the synthesis of anti-ChE agents [31], novel series of
acridine-chromenone and quinoline-chromenone hybrids 8 (Fig. 2) were designed, synthesized,
and evaluated for their anti-Alzheimer’s activities.
2. Results and Discussion
4

2.1. Chemistry
Synthetic route for the preparation of acridine-chromenone and quinoline-chromenone hybrids
8a–q is depicted in Scheme 1. Initially, two required starting materials, compounds 4 and 7
were synthesized. For this purpose, compounds 3 were prepared through the reaction of 7-
hydroxycoumarins (1) and 1-fluoro-4-nitrobenzene (2) in the presence of anhydrous K2CO3 in
◦
DMF at 80 C overnight. Then, the latter compound 3 tolerated reduction reaction using
Zn/NH4Cl in H2O/EtOH at room temperature for 3 h to obtain the corresponding amine 4.
Compounds 7 including 9-chloro-1H,2H,3H-cyclopenta[b]quinolines 7a-c, 9-chloro-1,2,3,4-
tetrahydroacridines 7d-f, 11-chloro-6H,7H,8H,9H,10H-cyclohepta[b]quinolines 7g-i were
prepared by condensation of commercially available anthranilic acids 5a-c and appropriate
cycloketones 6a-c in the presence of POCl3 under reflux [32]. Finally, compounds 7a-i reacted
with amines 4a-b in the presence of KI in refluxing n-propanol for 12–24 h affording target
compounds 8a-q in good yields (60–80%).
2.2. Biological activity
2.2.1. The anti-AChE activity of compounds 8a-q
In vitro anti-AChE and anti-BChE activities of the synthesized compounds 8a-q were
evaluated according to the modified Ellman’s method [31, 33] comparing with rivastigmine as
the reference drug. All results are presented in Table 1. Based on the IC50 values, compounds
8a, 8c-g, 8j-l, and 8o-q showed anti-AChE activity in the range of micromolar concentrations
(IC50 = 16.17-83.10 µM) in comparison to rivastigmine (IC50 = 11.07 µM). As can be seen in
Table 1, compounds 8a-q can be divided into three categories, 8a-f, 8g-l, 8m-q (Fig. 2)
according to the cycloalkyl fused to quinolone moiety. Among the synthesized compounds,
5

compound 8e possessing methyl and chlorine groups at 4-position of chromenone and 6-
position of cyclopenta[b]quinoline moieties, showed the most potent inhibitory activity with
IC50 = 16.17 µM. Replacing five-membered ring (in compound 8e) by six-membered ring
leading to the formation of tetrahydroacridine scaffold (compound 8k), reduced inhibitory
activity (IC50 = 18.03 µM). Further replacement by seven-membered ring (compound 8p) led
to decrease of inhibitory activity (IC50 = 31.81 µM).
Compound 8g with tetrahydroacridine moiety and no other substituents showed IC50 = 19.07
µM. Introduction of methyl group into the 4-position of chromenone moiety in compound 8j
afforded lower anti-AChE activity (IC50 = 22.10 µM) in comparison to its counterpart 8g. Also,
our results revealed that introduction of chlorine into 7-position of tetrahydroacridine core
(compound 8l) reduced activity (IC50 = 30.80 µM) comparing with 8j and 8k. It seems that
chlorine and its position on the tetrahydroacridine moiety plays important role since the
following order was observed in the inhibitory activity of compounds 8k>8j>8l. These results
are in good agreement with those obtained for compounds 8e (IC50 = 16.17 µM) and 8f (IC50 =
36.65 µM) as well as literature describing that 6-chlorotacrine has shown more potent activity
comparing with tacrine [34, 35]. Moreover, it was perceived that in the case of similar
substituents on the chromenone and quinoline moieties, fused five-membered ring induced
more activity comparing with six-membered ring since 8e was more potent than 8k.
Another instructive point is related to compounds 8p (IC50 = 31.81 µM) having seven-
membered ring fused to quinoline moiety. It showed lower activity comparing with its
counterpart, 8e and 8k (IC50 = 16.17, 18.03 µM, respectively).
6

Compounds 8f and 8c both having five-membered ring showed IC50s = 36.65 and 38.50 µM,
respectively. It is clear that the introduction of methyl to 4-position of coumarin moiety did not
significantly affect inhibitory activity. Compound 8d showed the inhibitory activity with IC50 =
45.27 µM. Deletion of chlorine in this compound led to the reduction of activity and it was less
active than its compartments 8e and 8f.
Compound 8q having chlorine at 2-position of cyclohepta[b]quinoline (IC50 = 54.12 µM) was
less active than 8p having chlorine at 3-position of cyclohepta[b]quinoline (IC50 = 31.81 µM),
the same results were observed for 8e/8f and 8k/8l pairs. Also, it was found that the presence
of seven-membered in compound 8q induced less activity in comparison to its compartments
8f and 8l as obtained for 8e>8k>8p. The result obtained for compound 8o (IC50 = 64.42 µM)
confirmed that deletion of chlorine reduced activity comparing with 8p and 8q.
Most of synthesized compounds exhibited no activity against BChE and compounds 8b, 8g, 8j,
and 8k showed IC50 = 11.65-38.63 µM. Among them, 8b lacking methyl group at 4-position of
chromenone and having chlorine at 6-position of cyclopenta[b]quinolone moiety was the most
potent compound (IC50 = 11.65) comparing with rivastigmine as the reference drug (IC50 =
7.72 µM). The remained three active compounds were belonged to the tetrahydroacridine
group. Compound 8g showed IC50 = 28.67 µM and insertion of methyl group into 4-position of
chromenone moiety led to reduction of inhibitory activity in compounds 8j and 8k.
2.2.2. Kinetic studies
In order to obtain insight into the inhibition mode of AChE by the most potent compound 8e, a
kinetics study was carried out using the modified Ellman’s method [31, 33]. As can be seen in
Fig. 3, graphical analysis of the Lineweaver-Burk reciprocal plots confirmed a mixed type of
7

inhibition suggesting that compound 8e can bind to the both free enzyme and enzyme-substrate
complex. It seems that compound 8e interacts with the both peripheral anionic site (PAS) and
the catalytic anionic site (CAS) of enzyme. The inhibition constant (Ki = 34 µM) was
calculated using secondary replots of the slope versus concentrations of compound 8e.
2.2.3. BACE1 enzyme inhibitory activity of compound 8e
BACE-1 inhibitory activity of the most potent anti-AChE compound 8e was evaluated via a
fluorescence resonance energy transfer (FRET) based BACE-1 kit including BACE-1 enzyme
and specific APP based peptide substrate (Rh-EVNLDAEFK-quencher). Experiments were
repeated for three times and compared with the reference compound OM99-2. Results are
shown in Table 2 indicating that compound 8e possesses good inhibitory activity with IC50 =
7.99 µM in comparison to OM99-2 (IC50 = 0.014 µM). It is worth mentioning that non-peptide
structure of compound 8e is desired and acts as dual inhibitor of AChE and BACE1.
2.2.4. Neuroprotective effect of compound 8e against H2O2-induced cell death in PC12
neurons
The PC12 cell line is a useful model for studying neuronal differentiation [36]. Consequently,
differentiated PC12 cells and H2O2 were selected as in vitro model and oxidative agent,
respectively [37]. Neuroprotective activity of the most potent compound 8e against AChE was
assessed by subjecting PC12 cells to H2O2-induced damage. As depicted in Fig. 4, the percent
of cell viability was calculated in comparison to control group. Compound 8e demonstrated
remarkable neuroprotective activity at 50 and 100 µM (cell viability = 80.03 and 80.07 %,
respectively and P <0.001 vs. H2O2 treatment alone). However, compound 8e showed
8

moderate neuroprotective activity at 10 µM (cell viability = 66.15 % and P <0.05 vs. H2O2
treatment alone).
2.2.3. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
Compounds 8b, 8e, 8i, 8l, 8n and 8q were evaluated for their 1,1-diphenyl-2-picrylhydrazyl
radical scavenging activity (DPPH) according to the method described in the literature [38].
The test compounds were evaluated at 33, 167, 350, 700 µg/mL. All results are shown in Table
3. The inhibition percentage values were compared to butylated hydroxyanisole (BHA) as a
standard antioxidant. All the tested compounds showed no remarkable antioxidant activities.
2.2.4. Docking studies
To understand a possible binding mode of the most active compound 8e, a molecular modeling
study was performed with Torpedo Californica AChE (PDB code: 2CMF) using the docking
program, AutoDock 4.2 package with Discovery Studio 4.0 Client. Docking result showed that
100 GA runs for compound 8e were categorized in 8 clusters and the mean binding energy
scored by Autodock ranged from -11.82 to -14.58 (in kcal/mol). The lowest energy
conformation of the highest populated cluster was selected for analysis (first cluster).
Docking results indicated that the most active compound 8e is located in the binding site of
AChE as the same as bis-(5)-tacrine. Also, it was clear that compound 8e bound to both the
CAS and the PAS. It has occupied the mid-gorge site between these two active sites of enzyme
(Fig. 5 and Fig. 6). As it is shown in Fig. 6, there are two π-π interactions between quinoline
moiety and Trp84 and Phe330 residues (in the CAS). Another one is hydrophobic interaction
of chlorine at 6-position of compound 8e with Ile439, Met436, Tyr442 and Trp432 residues (in
the CAS). The phenoxy group has also occupied the mid-gorge making π-anion interaction
9

with Asp72 and π-π interaction with Phe330. Moreover, the 4-methylchromenone moiety
established π-π and hydrophobic interaction with Trp279 (in the PAS).
2.2.5. Virtual calculation of pharmacokinetic properties
To qualify compound 8e as drug candidate, some pharmacokinetics aspects, physicochemical
and topological properties such as octanol-water partition coefficient (Clog P), number of H-
bond donors (HBD), number of H-bond acceptors (HBA), rotatable bonds count (RBC), and
topological polar surface area (tPSA) were calculated (Table 4). According to the Lipinski's
rule-of-five including MW≤ 500, HBD ≤ 5, HBA ≤ 10, and Clog P ≤ 5 [39, 40] and as the
compounds had rotatable bonds ≤ 10 and polar surface area ≤ 140 Å2, it seems that these
compounds are relatively orally active drugs in humans. The obtained results revealed that
compound 8e had 1 H-bond donor and 4 H-bond acceptors and it possessed tPSA 60.45 Å2 and
3 RBC. Compound 8e had molecular weight 468.9.93 Da with Clog P value of 8.23.
3. Conclusion
In summary, new acridine-chromenone and quinoline-chromenone hybrids were designed,
synthesized and evaluated as novel anti-alzheimer’s agents. Most of these compounds showed
moderate potency for inhibition of AChE and compound 8e indicated the most anti-AChE
activity (IC50 = 16.17 µM). This compound contained a 4-methylchromenone moiety as a
peripheral site interacting unit and quinoline moiety as a catalytic site binding unit. The kinetic
studies and molecular modeling showed that compound 8e had enough length to bind to both
the CAS and the PAS of AChE as a dual binding inhibitor. The quinoline moiety of compound
8e showed parallel π–π stacking with Trp84 at the gorge’s bottom in the CAS, while the 4-
10

methylchromenone moiety of compound 8e depicted parallel π–π stacking with Trp279 at the
gorge’s mouth in the PAS. Also, compound 8e was evaluated for its neuroprotective and
BACE1 inhibitory activities. The neuroprotective effect was moderate in comparison with
quercetin. It should be noted that compound 8e showed good BACE1 inhibitory activity (IC50
= 7.99 µM) which can be considered as a non-peptide agent. Our study presented advantageous
results for AD drug discovery focusing on the novel acridine-chromenone, quinoline-
chromenone hybrids and dual AChE/BACE1 inhibitors.
4. Methods and Materials
4.1. General chemistry
1
Melting points were measured on a Kofler hot stage apparatus and are uncorrected. H and 13C
NMR spectra as well as 2D HSQC experiment were recorded on a Bruker FT-500, using TMS
as an internal standard. IR spectra were obtained on a Nicolet Magna FTIR 550
spectrophotometer (KBr disks). Elemental analysis was performed on an Elementar
Analysensystem GmbH VarioEL CHNS mode. All chemical were obtained from Merck and
Aldrich. 4-Methyl-7-hydroxycoumarin was prepared by Pechmann reaction via the
condensation of resorcinol and ethylacetoacetate in the presence of concentrated sulfuric acid
[41].
4.2. General procedure for the synthesis of compounds 3
K2CO3 (6 mmol) was added to a mixture of coumarin derivative 1 (10 mmol) and 1-fluoro-4-
nitrobenzene (10 mmol) in DMF and heated at 80 °C overnight. After completion of reaction,
the reaction mixture was cooled down to room temperature, poured into ice water; the
precipitates were filtered off and washed with water to afford products 3 as cream solids.
11

4.3. General procedure for the synthesis of compounds 4
Zinc powder (10 mmol) was gradually added to a suspension of compound 3 (5 mmol) and
NH4Cl (10 mmol) in H2O/EtOH (20 mL, 1:1). Then, the mixture was stirred at room
temperature for 3 h. Upon completion of the reaction (checked by TLC), the mixture was
diluted with H2O, extracted with ethyl acetate, and dried over anhydrous Na2SO4. The solvent
was evaporated under vacuum to give pure compounds 4 as off-cream solids.
4.4. General procedure for the synthesis of compounds 7
POCl3 (25 mL) was added to a mixture of anthranilic acid derivative 5 (23 mmol) and
cycloketones derivative 6 (25 mmol) in an ice bath. The mixture was heated at reflux for 3 h.
Then, it was cooled to room temperature and concentrated to give slurry. The residue was
diluted with ethyl acetate, neutralized with aqueous K2CO3, the organic layer was dried over
anhydrous Na2SO4 and concentrated under vacuum affording a pale brown solid which was
purified by flash chromatography on silica gel using petroleum ether/ ethyl acetate (9:1) to give
7 in 80-90 % yield.
4.5. General procedure for the synthesis of compounds 8
KI (50 mg) was added to a mixture of compound 7 (1.1 mmol) and compound 4 (1 mmol) in n-
propanol. The mixture was heated at 100 ◦C for 12-24 h. Next, it was cooled room temperature;
the mixture was dissolved in 10% NaOH, and extracted with ethyl acetate. The organic layer
was dried over anhydrous Na2SO4 and the solvent was removed under vacuum. Finally, the
residue was purified by column chromatography on silica gel using petroleum ether/ethyl
12

acetate (1:9) to give 8 in 60-85 % yield. The structure of all compounds 8 was elucidated using
1H and 13C NMR as well as elemental analysis. Also, 2D HSQC experiment was performed for
compound 8i to describe chemical shift correlation between directly-bonded 1H and 13C (Table
5).
4.5.1. 7-(4-((2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)phenoxy)-2H-chromen-2-one
(8a). Yellow solid; yield: 68%, mp 212-214 °C. IR (KBr): 3391, 3065, 2953, 2922, 2840,
1
1726, 1616 cm-1. H NMR (500 MHz, CDCl3): 8.02 (d, J = 8.0 Hz, 2H, H5', H8'), 7.67 (d, J =
9.5 Hz, 1H, H4), 7.61 (t, J = 8.0 Hz, 1H, H6'), 7.44-7.42 (m, 2H, H5, H7'), 7.04 (d, J = 8.7 Hz,
2H, OC6H4), 6.99 (d, J = 8.7 Hz, 2H, OC6H4), 6.94 (dd, J = 8.5, 2.1 Hz, 1H, H6), 6.83 (s, 1H,
H8), 6.30 (d, J = 9.5 Hz, 1H, H3), 3.15 (t, J = 7.0 Hz, 2H, CH2), 2.57 (t, J = 7.0 Hz, 2H, CH2),
2.12 (quin, J = 7.0 Hz, 2H, CH2). 13C NMR (125 MHz, DMSO-d6): 168.2 (C3'a), 161.1 (C=O),
159.7 (C7), 154.9 (C8a), 150.5 (C4'a), 148.3 (C-O), 143.9 (C4), 141.5 (C9'), 140.0 (C-NH), 129.8
(C6'), 128.2 (C5'), 128.1 (C5), 126.6 (C7'), 124.1 (C8'), 122.7 (C8'a), 121.4 (C9'a), 120.5 (2C,
OC6H4), 120.4 (2C, OC6H4), 113.7 (C4a), 113.3 (C6), 113.6 (C3), 103.8 (C8), 34.2 (C3'), 30.4
(C1'), 22.9 (C2'). Anal. Calcd for C27H20N2O3: C, 77.13; H, 4.79; N, 6.66. Found: C, 77.31; H,
4.96; N, 6.82.
4.5.2.
7-(4-((6-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)phenoxy)-2H-
chromen-2-one (8b). Cream solid; yield: 75%, mp 217-219 °C. IR (KBr): 3343, 3071, 2944,
1
2855, 1719, 1615, 1561 cm-1. H NMR (500 MHz, CDCl3): 7.99 (d, J = 2.0 Hz, 1H, H5'), 7.88
(d, J = 8.9 Hz, 1H, H8'), 7.67 (d, J = 9.4 Hz, 1H, H4), 7.44 (d, J = 8.5 Hz, 1H, H5), 7.32 (dd, J =
8.9, 2.0 Hz, 1H, H7'), 7.03 (d, J = 8.8 Hz, 2H, OC6H4), 6.96-6.93 (m, 3H, OC6H4, H6), 6.79 (d,
J = 2.3 Hz, 1H, H8), 6.29 (d, J = 9.4 Hz, 1H, H3), 3.12 (t, J = 7.5 Hz, 2H, CH2), 2.63 (t, J = 7.5
13

13
Hz, 2H, CH2), 2.14 (quin, J = 7.5 Hz, 2H, CH2). C NMR (125 MHz, CDCl3): 169.9 (C3'a),
161.6 (C=O), 160.8 (C7), 155.5 (C8a), 149.9 (C4'a), 148.7 (C-O), 143.2 (C4), 141.9 (C9'), 139.3
(C-NH), 134.6 (C6'), 129.0 (C5), 127.3 (C5'), 125.5 (C8'), 123.4 (C7'), 122.5 (C8'a), 121.4 (2C,
OC6H4), 121.2 (2C, OC6H4), 119.7 (C9'a), 114.2 (C6), 114.0 (C3), 113.8 (C4a), 104.5 (C8), 34.8
(C3'), 30.4 (C1'), 23.2 (C2'). Anal. Calcd for C27H19ClN2O3: C, 71.29; H, 4.21; N, 6.16. Found:
C, 77.45; H, 4.40; N, 6.32.
4.5.3.
7-(4-((7-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)phenoxy)-2H-
chromen-2-one (8c). Cream solid; yield: 79%, mp 234-236 °C. IR (KBr): 3342, 2941, 2901,
2852, 1720, 1619, 1503 cm-1. 1H NMR (500 MHz, CDCl3): 7.96 (d, J = 8. 9, 1H, H5'), 7.95 (s,
1H, H8' ), 7.67 (d, J = 9.0 Hz, 1H, H4), 7.39 (dd, J = 8.7, 2.0 Hz, 1H, H6'), 7.43 (d, J = 8.5, 1H,
H5), 7.04 (d, J = 8.6 Hz, 2H, OC6H4), 6.95 (d, J = 8.6 Hz, 2H, OC6H4), 6.93 (d, J = 8.5, 1H,
H6), 6.82 (d, J = 2.2, 1H, H8), 6.67 (bs, 1H, NH), 6.30 (d, J = 9.0 Hz, 1H, H3), 3.14 (t, J = 7.5
Hz, 2H, CH2), 2.63 (t, J = 7.5 Hz, 2H, CH2), 2.14 (quin, J = 7.5 Hz, 2H, CH2). 13C NMR (125
MHz, CDCl3): 169.1 (C3'a), 161.6 (C=O), 160.8 (C7), 155.5 (C8a), 149.9 (C4'a), 146.4 (C-O),
143.1 (C4), 140.9 (C9'), 139.2 (C-NH), 130.7 (C7'), 130.0 (C6'), 129.5 (C5'), 129.0 (C5), 123.3
(C8'), 122.2 (C8'a), 121.2 (2C, OC6H4), 121.0 (2C, OC6H4), 119.1 (C9'a), 114.2 (C6), 114.0 (C3),
113.8 (C4a), 104.5 (C8), 34.8 (C3'), 30.5 (C1'), 23.3 (C2'). Anal. Calcd for C27H19ClN2O3: C,
71.29; H, 4.21; N, 6.16. Found: C, 71.11; H, 4.38; N, 6.29.
4.5.4.
7-(4-((2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)phenoxy)-4-methyl-2H-
chromen-2-one (8d). Off-green solid; yield: 60%, mp 161-163 °C. IR (KBr): 3360, 3037, 2969,
1
2923, 1723, 1621, 1498 cm-1. H NMR (500 MHz, DMSO-d6): 8.74 (s, 1H, NH), 8.18 (d, J =
8.0 Hz, 1H, H5'), 7.85 (d, J = 8.0 Hz, 1H, H8'), 7.74 (d, J = 9.0 Hz, 1H, H5), 7.61 (t, J = 8.0 Hz,
1H, H H6'), 7.44 (t, J = 8.0 Hz, 1H, H7'), 7.06 (d, J = 8.9 Hz, 2H, OC6H4), 6.95-6.94 (m, 3H, H6,
14

OC6H4), 6.84 (J = 2.0 Hz, 1H, H8), 6.24 (s, 1H, H3), 2.98 (t, J = 7.2 Hz, 2H, CH2), 2.54 (t, J =
13
7.2 Hz, 2H, CH2), 2.40 (s, 3H, CH3), 2.02 (quin, J = 7.2 Hz, 2H, CH2). C NMR (125 MHz,
DMSO-d6): 168.4 (C3'a), 161.1 (C=O), 159.8 (C7), 154.4 (C8a), 153.1 (C4'a), 148.3 (C-O), 148.2
(C4), 141.5 (C9'), 140.0 (C-NH), 128.4 (C6'), 128.3 (C5'), 127.0 (C5), 124.2 (C7'), 122.9 (C8'),
121.4 (C8'a), 121.3 (C9'a), 120.7 (2C, OC6H4), 120.5 (2C, OC6H4), 114.6 (C4a), 113.3 (C6),
112.0 (C3), 103.9 (C8), 34.3 (C3'), 30.7 (C1'), 23.1(C2'), 18.1 (Me). Anal. Calcd for C28H22N2O3:
C, 77.40; H, 5.10; N, 6.45. Found: C, 77.65; H, 5.21; N, 6.59.
4.5.5 7-(4-((6-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)phenoxy)-4-methyl-
2H-chromen-2-one (8e). Cream solid; yield: 75%, mp 217-219 °C. IR (KBr): 3355, 2961,
1
1737, 1610, 1581, 1539 cm-1. H NMR (500 MHz, DMSO-d6): 8.85 (s, 1H, NH), 8.19 (d, J =
9.0 Hz, 1H, H8'), 7.85 (d, J = 2.0 Hz, 1H, H5'), 7.75 (d, J = 8.5 Hz, 1H, H5), 7.46 (dd, J = 9.0,
2.0 Hz, 1H, H7'), 7.08 (d, J = 8.8 Hz, 2H, OC6H4), 6.97-6.94 (m,3H, H6, OC6H4), 6.85 (d, J =
2.5 Hz, 1H, H8), 6.25 (s, 1H, H3), 2.97 (t, J = 7.5 Hz, 2H, CH2), 2.50 (t, J = 7.5 Hz, 2H, CH2),
13
2.40 (s, 3H, CH3), 2.01 (quin, J = 7.5 Hz, 2H, CH2). C NMR (125 MHz, DMSO-d6): 170.2
(C3'a), 161.0 (C=O), 159.8 (C7), 154.4 (C8a), 153.1 (C4'a), 149.1 (C-O), 148.6 (C4), 141.6 (C9'),
139.6 (C-NH), 132.8 (C6'), 127.5 (C5'), 127.0 (C5), 124.9 (C8'), 124.4 (C7'), 121.4 (C8'a), 120.9
(2C, OC6H4), 120.6 (2C, OC6H4), 120.0 (C9'a), 114.6 (C4a), 113.4 (C6), 112.0 (C3), 104.0 (C8),
34.4 (C3'), 30.7 (C1'), 23.0 (C2'), 18.1 (Me). Anal. Calcd for C28H21ClN2O3: 77.72; H, 4.51; N,
5.97. Found: C, 77.85; H, 4.35; N, 6.12.
4.5.6. 7-(4-((7-Chloro-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)amino)phenoxy)-4-methyl-
2H-chromen-2-one (8f). Cream solid; yield: 74%, mp 133-135 °C. IR (KBr): 3351, 2953,
2911, 1718, 1610, 1563, 1508 cm-1. 1H NMR (500 MHz, DMSO-d6): 8.91 (s, 1H, NH), 8.27 (d,
J = 2.2 Hz, 1H, H8'), 7.86 (d, J = 8.9 Hz, 1H, H5'), 7.77 (d, J = 8.8 Hz, 1H, H5), 7.63 (dd, J =
15

8.9, 2.2 Hz, 1H, H6'), 7.10 (d, J = 8.8 Hz, 2H, OC6H4), 6.96 (m, 3H, H6, OC6H4), 6.87 (d, J =
2.4 Hz, 1H, H8), 6.28 (s, 1H, H3), 2.97 (t, J = 7.5 Hz, 2H, CH2), 2.50 (t, J = 7.5 Hz, 2H, CH2),
13
2.36 (s, 3H, CH3), 2.03 (quin, J = 7.5 Hz, 2H, CH2). C NMR (125 MHz, DMSO-d6): 169.4
(C3'a), 161.1 (C=O), 159.8 (C7), 154.4 (C8a), 153.2 (C4'a), 148.6 (C-O), 146.9 (C4), 140.7 (C9'),
139.5 (C-NH), 130.5 (C7'), 128.7 (C6'), 128.6 (C5), 127.1 (C5'), 122.3 (C8'), 121.9 (C8'a), 121.8
(C9'a), 120.9 (2C, OC6H4), 120.7 (2C, OC6H4), 114.6 (C4a), 113.4 (C6), 112.0 (C3), 104.0 (C8),
34.4 (C3'), 30.9 (C1'), 23.1 (C2'), 18.1 (Me). Anal. Calcd for C28H21ClN2O3: C, 72.72; H, 4.51;
N, 5.97. Found: C, 72.88; H, 4.70; N, 6.18.
4.5.7 7-(4-((1,2,3,4-Tetrahydroacridin-9-yl)amino)phenoxy)-2H-chromen-2-one (8g). Off-
cream solid; yield: 71%, mp 130-132 °C. IR (KBr): 3251, 3080, 2931, 2864, 1716, 1615 cm-1.
1H NMR (500 MHz, CDCl3): 8.02 (d, J = 8.2 Hz, 1H, H8'), 7.81 (d, J = 8.2 Hz, 1H, H5'), 7.64
(d, J = 9.0 Hz, 1H, H4), 7.60 (t, J = 8.2 Hz, 1H, H7'), 7.39 (d, J = 8.5 Hz, 1H, H5), 7.36 (t, J =
8.2, 1H, H7'), 6.93 (d, J = 8.7 Hz, 2H, OC6H4), 6.90 (d, J = 8.5 Hz, 1H, H6), 6.77 (d, J = 2.3 Hz,
1H, H8), 6.75 (d, J = 8.7 Hz, 2H, OC6H4), 6.26 (d, J = 9.0 Hz, 1H, H3), 3.16 (t, J = 6.0 Hz, 2H,
CH2), 2.63 (t, J = 6.0 Hz, 2H, CH2), 1.97 (quin, J = 6.0 Hz, 2H, CH2), 1.87 (quin, J = 6.0 Hz,
13
2H, CH2). C NMR (125 MHz, CDCl3): 162.1 (C4'a), 160.9 (C=O), 159.6 (C7), 155.5 (C8a),
148.4 (C10'a), 146.6 (C-O), 143.7 (C4), 143.2 (C9'), 141.8 (C-NH), 129.0 (C6'), 128.1 (C5), 125.2
(C5'), 123.16 (C7'), 123.10 (C8'), 122.7 (C8'a), 122.5 (C9 ), 121.5 (2C, OC6H4), 118.1 (2C,
OC6H4), 114.1 (C6), 113.7 (C3), 113.6 (C4a), 104.2 (C8), 33.5 (C4'), 25.3 (C1'), 22.6 (C2'), 22.6
(C3'). Anal. Calcd for C28H22N2O3: C, 77.40; H, 5.10; N, 6.45. Found: C, 77.29; H, 5.26; N,
6.60.
4.5.8. 7-(4-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)phenoxy)-2H-chromen-2-one (8h
Off-orange solid; yield: 85%, mp 134-136 °C. IR (KBr): 3524, 3245, 3085, 2933, 2864, 1718,
16

1613, 1558 cm-1. 1H NMR (500 MHz, CDCl3): 8.04 (s, 1H, H5' ), 7.74 (d, J = 9.0 Hz, 1H, H8'),
7.66 (d, J = 9.5 Hz, 1H, H4), 7.42 (d, J = 8.5 Hz, 1H, H5), 7.30 (d, J = 9.0 Hz, 1H, H7' ), 6.96
(d, J = 8.8 Hz, 2H, OC6H4), 6.93 (dd, , J = 8.5, 2.3 Hz, 1H, H6), 6.78 (m, 3H, H8, OC6H4), 6.29
(d, J = 9.5 Hz, 1H, H3), 3.12 (t, J = 6.0 Hz, 2H, CH2), 2.63 (t, J = 6.0 Hz, 2H, CH2), 1.97 (quin,
13
J = 6.0 Hz, 2H, CH2), 1.91 (quin, J = 6.0 Hz, 2H, CH2) . C NMR (125 MHz, CDCl3): 161.9
(C4'a), 160.9 (C=O), 160.8 (C7), 155.5 (C8a), 148.7 (C10'a), 147.3 (C-O), 143.7 (C9'), 143.2 (C4),
141.5 (C-NH), 134.7 (C6'), 128.9 (C5'), 127.3 (C5), 125.9 (C8'), 124.7 (C7'), 123.0 (C8 ), 121.6
(2C, OC6H4), 120.9 (C9'a), 118.3 (2C, OC6H4), 114.1 (C6), 113.8 (C3), 113.6 (C4a), 104.5 (C8),
33.7 (C4'), 25.2 (C1'), 22.5 (C2' , C3'). Anal. Calcd for C28H21ClN2O3: C, 71.72; H, 4.51; N, 5.97.
Found: C, 71.51; H, 4.60; N, 6.05.
4.5.9. 7-(4-((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)phenoxy)-2H-chromen-2-one (8i).
Cream solid; yield: 65%, mp 228-230 °C. IR (KBr): 3357, 3047, 2925, 2858, 1718, 1611, 1554
cm-1. 1H NMR (500 MHz, CDCl3): 7.98 (d, J = 9.0 Hz, 1H, H5'), 7.76 (d, J = 2.0 Hz, 1H, H8'),
7.65 (d, J = 9.5 Hz, 1H, H4), 7.51 ( dd, J= 9.0, 2.0 Hz, 1H, H6'), 7.40 (d, J = 8.5 Hz, 1H, H5),
6.97 (d, J = 8.8 Hz, 2H, OC6H4), 6.90 (dd, J = 8.5, 2.3 Hz, 1H, H6), 6.80 (d, J = 2.3 Hz, 1H,
H8), 6.78 (d, J = 8.8 Hz, 1H, OC6H4), 6.28 (d, J = 9.5 Hz, 1H, H3), 3.15 (t, J = 6.2 Hz, 2H,
CH2), 2.68 (t, J = 6.2 Hz, 2H, CH2), 1.93 (quin, J = 6.2 Hz, 2H, CH2), 1.86 (quin, J = 6.2 Hz,
13
2H, CH2). C NMR (125 MHz, CDCl3): 161.9 (C3'a), 161.0 (C=O), 159.7 (C7), 155.5 (C8a),
149.1 (C4'a), 144.2 (C-O), 143.8 (C9'), 143.3 (C4), 141.0 (C-NH), 131.0 (C7'), 130.1 (C6'), 129.0
(C5', C5), 123.4 (C8'a), 123.4 (C9'a), 123.1 (C8'), 121.7 (2C, OC6H4), 118.9 (2C, OC6H4), 114.1
(C6), 113.9 (C3), 113.7 (C4a), 104.4 (C8), 32.8 (C4'), 25.5 (C1'), 22.3 (C2', C3'). Anal. Calcd for
C28H21ClN2O3: C, 71.72; H, 4.51; N, 5.97. Found: C, 71.53; H, 4.46; N, 6.08.
17

4.5.10.
4-Methyl-7-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)phenoxy)-2H-chromen-2-one
(8j). Cream solid; yield: 77%, mp 202-204 °C. IR (KBr): 3343, 3054, 2934, 2859, 1692, 1602,
1
1558 cm-1. H NMR (500 MHz, DMSO-d6): 8.29 (s, 1H, NH), 7.91 (d, J = 8.0 Hz, 1H, H8'),
7.86 (d, J = 8.0 Hz, 1H, H5'), 7.72 (d, J = 8.8 Hz, 1H, H5), 7.60 (t, J = 8.0 Hz, 1H, H H6') 7.41 (t,
J = 8.0 Hz, 1H, H7'), 6.97 (d, J = 8.9 Hz, 2H, OC6H4), 6.91 (dd, J = 8.8, 2.5 Hz, 1H, H6), 6.79
(d, J = 2.5 Hz, 1H, H8), 6.68 (d, J = 8.9 Hz, 2H, OC6H4), 6.67 (d, J = 1.2 Hz, 1H, H3), 3.02 (t, J
= 6.3 Hz, 2H, CH2), 2.69 (t, J = 6.3 Hz, 2H, CH2), 2.38 (d, J = 1.2 Hz, 3H, CH3), 1.87 (quin, J
= 6.3 Hz, 2H, CH2), 1.76 (quin, J = 6.3 Hz, 2H, CH2). 13C NMR (125 MHz, DMSO-d6): 161.4
(C4'a), 159.8 (C=O), 159.5 (C7), 154.3 (C8a), 153.1 (C10'a), 146.9 (C-O), 146.6 (C9'), 143.0 (C4),
142.6 (C-NH), 128.4 (C6'), 128.3 (C5'), 126.9 (C5), 124.6 (C7'), 124.06 (C8'a), 123.4 (C8'), 123.2
(C9'a), 121.1 (2C, OC6H4), 116.5 (2C, OC6H4), 114.3(C4a), 113.1 (C6), 111.8 (C3), 103.6 (C8),
33.4 (C4'), 25.3 (C1'), 22.3 (C2'), 22.1 (C3'), 18.0 (Me). Anal. Calcd for C29H24N2O3: C, 77.66;
H, 5.39; N, 6.25. Found: C, 77.78; H, 5.21; N, 6.42.
4.5.11. 7-(4-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)phenoxy)-4-methyl-2H-chromen-
2-one (8k). Off-cream solid; yield: 68%, mp 193-195 °C. IR (KBr): 3367, 2933, 1727, 1610,
1
1556 cm-1. H NMR (500 MHz, DMSO-d6): 8.43 (s, 1H, NH), 7.93 (d, J = 9.0 Hz, 1H, H8'),
7.89 (s, 1H, H5'), 7.72 (d, J = 8.8 Hz, 1H, H5), 7.44 (d, J = 9.0 Hz, 1H, H7'), 6.99 (d, J = 8.5 Hz,
2H, OC6H4), 6.91 (dd, J = 8.8, 2.2 Hz, 1H, H6), 6.80 (d, J = 2.2 Hz, 1H, H8), 6.73 (d, J = 8.5
Hz, 2H, OC6H4), 6.25 (s, 1H, H3), 3.02 (t, J = 6.0 Hz, 2H, CH2), 2.66 (t, J = 6.0 Hz, 2H, CH2),
13
2.39 (s, 3H, CH3), 1.87 (quin, J = 6.0 Hz, 2H, CH2), 1.76 (quin, J = 6.0 Hz, 2H, CH2). C
NMR (125 MHz, DMSO-d6): 161.3 (C4'a), 161.1 (C=O), 159.8 (C7), 154.4 (C8a), 153.1 (C10'a),
147.3 (C-O), 147.1 (C9'), 143.5 (C4), 142.2 (C-NH), 133.0 (C6'), 127.2 (C5'), 126.9 (C5), 125.7
(C8'), 125.1 (C7'), 124.08 (C8'a), 121.6 (C9'a), 121.2 (2C, OC6H4), 117.1 (2C, OC6H4), 114.4
18

(C4a), 113.2 (C6), 111.9 (C3), 103.6 (C8), 33.4 (C4'), 25.4 (C1'), 22.2 (C2'), 22.0 (C3'), 18.1. Anal.
Calcd for C29H23ClN2O3: C, 72.12; H, 4.80; N, 5.80. Found: C, 71.91; H, 4.93; N, 5.61.
4.5.12. 7-(4-((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)phenoxy)-4-methyl-2H-chromen-
2-one (8l). Orange solid; yield: 85%, mp 258-260 °C. IR (KBr): 3355, 2934, 2857, 1713, 1607,
1555, 1493cm-1. 1H NMR (500 MHz, DMSO-d6): 8.44 (s, 1H, NH), 7.97 (s, 1H, H8'), 7.86 (d, J
= 8.9 Hz, 1H, H5'), 7.73 (d, J = 8.8 Hz, 1H, H5), 7.48 (d, J = 8.9 Hz, 1H, H6'), 7.01 (d, J = 8.7
Hz, 2H, OC6H4), 6.93 (dd, J = 8.8, 2.3 Hz, 1H, H6), 6.82 (d, J = 2.3 Hz, 1H, H8), 6.72 (d, J =
8.7 Hz, 2H, OC6H4), 6.26 (s, 1H, H3), 3.02 (t, J = 6.0 Hz, 2H, CH2), 2.96 (t, J = 6.0 Hz, 2H,
CH2), 2.63 (s, 3H, CH3), 1.88 (quin, J = 6.0 Hz, 2H, CH2), 1.76 (quin, J = 6.0 Hz, 2H, CH2) .
13C NMR (125 MHz, DMSO-d6): 161.4 (C4'a), 160.3 (C=O), 159.8 (C7), 154.4 (C8a), 153.2
(C10'a), 147.1 (C-O), 145.4 (C9'), 142.5 (C4), 142.0 (C-NH), 130.6 (C7'), 129.2 (C6'), 128.9 (C5'),
127.0 (C5), 124.6 (C8'a), 123.8 (C9'a), 122.2 (C8'), 121.2 (2C, OC6H4), 117.2 (2C, OC6H4), 114.4
(C4a), 113.2 (C6), 111.9 (C3), 103.7 (C8), 33.3 (C4'), 25.6 (C1'), 22.3 (C2'), 22.0 (C3'), 18.1 (Me).
Anal. Calcd for C29H23ClN2O3: C, 72.12; H, 4.80; N, 5.80. Found: C, 72.30; H, 4.64; N, 6.10.
4.5.13.
7-(4-((7,8,9,10-Tetrahydro-6H-cyclohepta[b]quinolin-11-yl)amino)phenoxy)-2H-
chromen-2-one (8m). Off-cream solid; yield: 77%, mp 204-206 °C. IR (KBr): 3264, 2965,
2912, 2864, 1743, 1617, 1559 cm-1. 1H NMR (500 MHz, DMSO-d6): 8.29 (s, 1H, NH), 8.00 (d,
J = 9.5 Hz, 1H, H4), 7.92 (d, J = 8.1 Hz, 1H, H1'), 7.89 (d, J = 8.1 Hz, 1H, H4'), 7.65 (d, J = 8.6
Hz, 1H, H5) 7.60 (t, J = 8.1 Hz, 1H, H3'), 7.45 (t, J = 8.1 Hz, 1H, H2'), 6.96 (d, J = 8.8 Hz, 2H,
OC6H4), 6.89 (dd, J = 8.6, 2.3 Hz, 1H, H6), 6.77 (d, J = 2.3 Hz, 1H, H8), 6.66 (d, J = 8.8 Hz,
2H, OC6H4), 6.32 (d, J = 9.5 Hz, 1H, H3), 3.17 (bs, 2H, CH2), 2.89 (bs, 2H, CH2), 1.80 (bs, 2H,
CH2), 1.71 (bs, 2H, CH2), 1.57 (bs, 2H, CH2). 13C NMR (125 MHz, CHCl3): 166.0 (C5'a), 162.3
(C=O), 160.8 (C7), 155.6 (C8a), 147.6 (C4'a), 147.3 (C-O), 143.5 (C11'), 143.1 (C4), 141.4 (C-
19

NH), 131.4 (C4'), 129.2 (C3'), 128.9 (C5), 128.7 (C2'), 125.9 (C1'), 124.5 (C1'a), 122.9 (C10'a),
121.7 (2C, OC6H4), 116.3 (2C, OC6H4), 114.0 (C6), 113.7 (C3), 113.5 (C4a), 104.2 (C8), 40.4
(C6'), 31.9 (C10'), 28.1 (C7'), 28.0 (C9'), 26.9 (C8'). Anal. Calcd for C29H24N2O3: C, 77.66; H,
5.39; N, 6.25. Found: C, 77.51; H, 5.74; N, 6.02.
4.5.14. 7-(4-((3-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-yl)amino)phenoxy)-
2H-chromen-2-one (8n). Cream solid; yield: 73%, mp 183-185 °C. IR (KBr): 3227, 2917,
1
2849, 1742, 1616, 1557cm-1. H NMR (500 MHz, DMSO-d6): 8.37 (s, 1H, NH), 8.00 (d, J =
9.5 Hz, 1H, H4), 7.93 (d, J = 9.0 Hz, 1H, H1'), 7.93 (s, 1H, H4'), 7.66 (d, J = 8.6 Hz, 1H, H5),
7.48 (d, J = 9.0 Hz, 1H, H2'), 6.96 (d, J = 8.5 Hz, 2H, OC6H4), 6.89 (d, J = 8.6 Hz, 1H, H6),
6.77 (s, 1H, H8), 6.66 (d, J = 8.5 Hz, 2H, OC6H4), 6.32 (d, J = 9.5 Hz, 1H, H3), 3.16 (bs, 2H,
13
CH2), 2.88 (bs, 2H, CH2), 1.80 (bs, 2H, CH2), 1.70 (bs, 2H, CH2), 1.56 (bs, 2H, CH2). C
NMR (125 MHz, CDCl3): 167.1 (C5'a), 161.5 (C=O), 159.9 (C7), 154.9 (C8a), 147.1 (C4'a),
146.3 (C-O), 144.0 (C11'), 143.8 (C4), 142.2 (C-NH), 132.9 (C3'), 131.3 (C4'), 129.8 (C5), 127.1
(C1'), 125.8 (C1'a),, 125.7 (C2'), 122.8 (C10'a), 121.3 (2C, OC6H4), 115.6 (2C, OC6H4), 113.5
(C4a), 113.3 (C6), 113.2 (C3), 103.4 (C8), 39.8 (C6'), 31.2 (C10'), 27.5 (C7'), 27.4 (C9'), 26.4 (C8').
Anal. Calcd for C29H23ClN2O3: C, 71.12; H, 4.80; N, 5.80. Found: C, 71.35; H, 4.69; N, 5.62.
4.5.15. 4-Methyl-7-(4-((7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-yl)amino)phenoxy)-
2H-chromen-2-one (8o). Cream solid; yield: 69%, mp 226-228 °C. IR (KBr): 3335, 2925,
2848, 1721, 1608, 1561, 1498 cm-1. 1H NMR (500 MHz, DMSO-d6): 8.29 (s, 1H, NH), 7.92 (d,
J = 8.1 Hz, 1H, H1'), 7.89 (d, J = 8.1 Hz, 1H, H4'), 7.71 (d, J = 8.8 Hz, 1H, H5), 7.62 (t, J = 8.1
Hz, 1H, H2') 7.45 (t, J = 8.1 Hz, 1H, H3'), 6.94 (d, J = 8.7 Hz, 2H, OC6H4), 6.91 (dd, J = 8.8,
2.3 Hz, 1H, H6), 6.76 (d, J = 2.3 Hz, 1H, H8), 6.24 (d, J = 8.7 Hz, 2H, OC6H4), 6.24 (s, 1H,
H3), 3.17 (bs, 2H, CH2), 2.89 (bs, 2H, CH2), 2.37 (s, 3H, CH3), 1.81 (bs, 2H, CH2), 1.72 (bs,
20

2H, CH2), 1.57 (bs, 2H, CH2). 13C NMR (125 MHz, CDCl3): 166.0 (C5'a), 162.1 (C=O), 160.9
(C7), 155.0 (C8a), 152.2 (C4'a), 147.7 (C-O), 147.3 (C11'), 143.1 (C4), 141.6 (C-NH), 131.4 (C3'),
129.2 (C4'), 128.6 (C5), 125.8 (C2'), 125.6 (C1'), 124.5 (C1'a), 123.0 (C10'a), 121.7 (2C, OC6H4),
116.3 (2C, OC6H4), 114.6 (C4a), 113.7 (C6), 112.5 (C3), 104.2 (C8), 40.4 (C6'), 31.9 (C10'),
28.1(C7'), 28.0 (C9'), 26.9 (C8'), 18.4 (Me). Anal. Calcd for C30H26N2O3: C, 77.90; H, 5.67; N,
6.06. Found: C, 77.78; H, 5.89; N, 5.89.
4.5.16. 7-(4-((3-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-yl)amino)phenoxy)-
4-methyl-2H-chromen-2-one (8p). Cream solid; yield: 64%, mp 198-200 °C. IR (KBr): 3257,
1
3053, 2918, 1736, 1619, 1559, 1499 cm-1. H NMR (500 MHz, DMSO-d6): 8.40 (s, 1H, NH),
7.94 (d, J = 9.1 Hz, 1H, H1'), 7.93 (s, 1H, H4'), 7.73 (d, J = 8.7 Hz, 1H, H5), 7.50 (d, J = 9.1
Hz, 1H, H2'), 6.96 (d, J = 8.6 Hz, 2H, OC6H4), 7.92 (d, J = 8.7 Hz, 1H, H6), 6.78 (s, 1H, H8),
6.67 (d, J = 8.6 Hz, 2H, OC6H4), 6.25 (s, 1H, H3), 3.18 (bs, 2H, CH2), 2.89 (bs, 2H, CH2), 2.39
(s, 2H, CH3), 1.82 (bs, 2H, CH2), 1.72 (bs, 2H, CH2), 1.57 (bs, 2H, CH2). 13C NMR (125 MHz,
CHCl3): 167.3 (C5'a), 162.0 (C=O), 160.9 (C7), 155.0 (C8a), 152.2 (C4'a), 148.0 (C-O), 147.7
(C11'), 143.2 (C4), 141.6 (C-NH), 134.5 (C3'), 131.5 (C4'), 128.2 (C5), 126.6 (C2'), 125.7 (C1'),
124.7 (C1'a), 124.7 (C10'a), 121.7 (2C, OC6H4), 116.5 (2C, OC6H4), 114.6 (C4a), 113.7 (C6),
112.5 (C3), 104.2 (C8), 40.3 (C6'), 31.8 (C10'), 27.9 (C7'), 27.8 (C9'), 26.8 (C8'), 18.7 (Me). Anal.
Calcd for C30H25ClN2O3: C, 72.50; H, 5.07; N, 5.64. Found: C, 72.73; H, 4.85; N, 5.49.
4.5.17. 7-(4-(2-Chloro-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-11-ylamino)phenoxy)-4-
methyl-2H-chromen-2-one (8q). Cream solid; yield: 71%, mp 262-264 °C. IR (KBr): 3338,
1
3062, 2916, 2854, 1721, 1608, 1558, 1501 cm-1. H NMR (500 MHz, DMSO-d6): 8.35 (s, 1H,
NH), 7.95 (s, 1H, H1'), 7.90 (d, J = 8.9 Hz, 1H, H4'), 7.72 (d, J = 8.8 Hz, 1H, H5), 7.48 (d, J =
8.9 Hz, 1H, H3'), 6.91 (d, J = 8.5 Hz, 2H, OC6H4), 6.90 (dd, J = 8.8, 2.2 Hz, 1H, H6), 6.78 (d, J
21

= 2.2 Hz, 1H, H8), 6.68 (d, J = 8.5 Hz, 2H, OC6H4), 6.24 (s, 1H, H3), 3.16 (bs, 2H, CH2), 2.86
(bs, 2H, CH2), 2.38 (bs, 3H, CH3), 1.80 (bs, 2H, CH2), 1.71 (bs, 2H, CH2), 1.71 (bs, 2H, CH2).
13C NMR (125 MHz, DMSO-d6): 166.2 (C5'a), 161.5 (C=O), 159.8 (C7), 154.4 (C8a), 153.1
(C4'a), 146.5 (C-O), 145.2 (C11'), 143.7 (C4), 141.4 (C-NH), 131.9 (C2'), 130.8 (C4'), 129.9 (C3'),
128.9 (C5), 126.9 (C1'), 125.0 (C1'a), 122.4 (C10'a), 121.3 (2C, OC6H4), 115.8 (2C, OC6H4),
114.3 (C4a), 113.1 (C6), 111.9 (C3), 103.5 (C8), 39.8 (C6'), 31.3 (C10'), 27.7 (C7'), 27.5 (C9'), 26.5
(C8'), 18.1 (Me). Anal. Calcd for C30H25ClN2O3: C, 72.50; H, 5.07; N, 5.64. Found: C, 72.78;
H, 4.88; N, 5.52.
4.6. AChE and BChE inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder, from electric eel,
1000 unit), butylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum), acetylthiocholine
iodide (ATCI), and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-
Aldrich. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium
hydroxide, and sodium hydrogen carbonate were purchased from Fluka. In vitro AChEI
activity was performed based on the modified Ellman’s method [31, 33] as previously reported
by our group using a 96-well plate reader (BioTek ELx808). For this purpose, compounds 8
were dissolve in a mixture of DMSO (5 mL) and methanol (5 mL) and diluted in 0.1 M
KH2PO4/K2HPO4 buffer (pH 8.0). Each well contained 50 µL potassium phosphate buffer
(KH2PO4/ K2HPO4, 0.1 M, pH 8), 25µL prepared sample as described above, 25µL enzyme
with final concentration of 0.22 U/mL in buffer. They were preincubated for 15 min at rt, and
then 125 µL DTNB (3 mM in buffer) was added. Characterization of the hydrolysis of ATCI
catalyzed by AChE was performed spectrometrically at 405 nm followed by the addition of
substrate (ATCI 3 mM in water). The change in absorbance was measured at 405 nm after 15
22

min. The IC50 values were determined graphically from inhibition curves (log inhibitor
concentration vs. percent of inhibition). A control experiment was performed under the same
conditions without inhibitor and the blank contained buffer, water, DTNB, and substrate. The
described method was also used for BChE inhibition assay. For all compounds, four different
concentrations were tested for each compound in triplicate to obtain the range of 20%-80%
inhibition for AChE.
4.7. Kinetic studies of AChE inhibition
Estimates of the inhibition model and inhibition constant Ki, reciprocal plots of 1/V versus
1/[S] were obtained using different concentrations of the substrate (0.33–5.33 mM)
acetylthiocholine. For this purpose, experiments were performed similar to enzyme inhibition
assay [31-33]. The rate of enzymatic reaction was obtained with different concentrations of
inhibitor (5, 10, and 15 µM) and in the absence of inhibitor. For each experiment, reaction was
started by adding substrate (acetylthiocholine) and progress curves were recorded at 412 nm
over 2 min. Next, double reciprocal plots (1/v vs. 1/[s]) were made using the slopes of progress
curves to obtain the type of inhibition. Slopes of these reciprocal plots were then plotted
against the concentration of compound 8e in a weighted analysis, and Ki was determined as the
intercept on the negative x-axis. All rate measurements were performed in triplicate and data
analysis was performed with Microsoft Excel 2003.
4.8. BACE1 enzymatic assay
A FRET-based BACE1 enzyme assay kit was purchased from Invitrogen (former Pan Vera
Corporation, Madison, WI) and the assay was carried out according to the manufacturers’
instructions (Invitrogen. http://tools.invitrogen.com/content/sfs/manuals/L0724.pdf). Stock
23

solution of compound 8e was prepared in DMSO. It was further diluted in assay buffer to
prepare a 3X concentration of the test compounds. Ten μL of BACE1 substrate (3X
concentration) was added to 10 μL of 3X concentration of the test compound in separate wells
of a black 96-well microplate and gently mixed. Ten μL of 3X BACE1 enzyme was added to
each well to start the reaction. Plates were incubated for 90 minutes at room temperature in the
dark. Then, ten μL stop buffer (sodium acetate) was added to each well to stop the reaction.
Finally, fluorescence measurements were performed with a multimode microplate reader
(BMG Labtech) at excitation and emission wavelength of 545 nm and 585 nm, respectively.
Experiments were repeated three to five times and mean percent of enzyme inhibitory activities
at 10 and 50 µM concentrations of the test compounds were calculated.
4.8. Neuroprotection assay
Rat differentiated PC12 cells were provided as described in the literature [42]. Differentiated
PC12 cells were incubated with different concentrations of the compound 8e for 3 h before
treatment with H2O2 (300 mM). Cell death by apoptosis was established after staining with
DAPI, and cell viability was measured after 24 h by using the MTT assay. Briefly, 10 mL of
MTT solution (5 mg/mL, Sigma) was added to the cell culture media (150 mL) and incubated
in a CO2 incubator for 3.5 h. Then, medium was removed and DMSO (150 mL) was added into
the each well and the formazan precipitates were dissolved 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.
4.9. DPPH radical scavenging activity (DPPH)
24

Antioxidant activity of compounds 8b, 8e, 8i, 8l, 8n, and 8q were determined using DPPH
(1,1-diphenyl-2-picrylhydrazyl) [38]. Several concentrations of the above mentioned
compounds (33, 167, 350, 700 µg/mL) in DMSO were prepared. The compound solution (0.5
mL) was added to the methanolic DPPH solution (1.0 mL, 0.1 mM), and the mixture was kept
in the dark for 30 min. Then, the absorbance at 517 nm was measured by an UV⁄visible
spectrophotometer. The percent scavenging activity was calculated: (%) = 100× [(Abscontrol
–
Abssample) ⁄ Abscontrol] since Abscontrol = AbsDPPH + Abssolvent.
4.10. Molecular docking study
The crystallographic structure of tacrine was retrieved from RCSB protein data bank (PDB
code: 2CMF). Molecular docking studies were carried out with AUTODOCK 4.2 software
(http://autodock.scripps.edu/). Additionally, the program Cygwin (version 1.7.
http://www.cygwin.com) was used to properly run the Autogrid and Autodock applications. To
prepare AChE template, after eliminating the original inhibitors and water molecules, all polar
hydrogens were added and Kollman charges were assigned using Autodock Tools (ADT,
version 1.5.4). The structure of 8e was built and converted to 3D using MarvineSketch 5.10.4,
2012, ChemAxon (http://www.chemaxon.com). To prepare the compound 8e for docking
purpose, all hydrogens were added and Gasteiger charges were assigned. The AutoDockTools
(ADT) was used to merge nonpolar hydrogens and define which bonds should be rotatable.
The scoring grid box for Autodock was approximately centered between the CAS and PAS (x-
center: 3.381, y-center: 65.434, z-center: 63.34). The grid size was set to 60 × 60 × 60 points
with a spacing value of 0.375 Å. The prepared compound was docked to the AChE template
using a Lamarckian genetic algorithm of an initial population of 150 randomly placed
individuals, the maximum number of 2.5 × 106 energy evaluations, the maximum number of
25

27,000 generations and the number of 100 GA runs. A cluster analysis was performed on the
docking results using a root mean square (RMS) tolerance of 2.0 and the lowest energy
conformation of the highest populated cluster was selected for analysis. Graphic visualizations
were done by Discovery Studio 4.0 client software.
4.11. Computational methods
The log P values, tPSA, HBD, and HBA were calculated by the means of MarvineSketch 5.8.3.
RBC was calculated using Autodock Tools (ver.1.5.4).
4.12. Statistical analysis
Results for neuroprotective effects are expressed as the means ± SD value. The significant
differences among various groups were compared by one-way analysis of variance (ANOVA)
followed by Tukey’s test using Graphpad prism software version 5. P<0.05 was considered to
be statistically significant.
Acknowledgment
This work was supported by Research Council of Tehran University of Medical Sciences.
Conflict of interest
The authors have declared no conflict of interest.
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