New tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: Efficient synthesis, biological assessment and docking simulation study

Article abstract of DOI:10.1016/j.ejmech.2014.10.049

A new series of tacrine-based acetylcholinesterase (AChE) inhibitors 7a-l were designed by replacing the benzene ring of tacrine with aryl-dihydropyrano[2,3-c]pyrazole. The poly-functionalized hybrid molecules 7a-l were efficiently synthesized through mul

Full text of DOI:10.1016/j.ejmech.2014.10.049

Accepted Manuscript
New tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: Efficient synthesis,
biological assessment and docking simulation study
Mehdi Khoobi, Farzaneh Ghanoni, Hamid Nadri, Alireza Moradi, Morteza Pirali
Hamedani, Farshad Homayouni Moghadam, Saeed Emami, Mohsen Vosooghi, Reza
Zadmard, Alireza Foroumadi, Abbas Shafiee
PII:
S0223-5234(14)00977-5
10.1016/j.ejmech.2014.10.049
EJMECH 7457
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 July 2014
Revised Date: 15 October 2014
Accepted Date: 16 October 2014
Please cite this article as: M. Khoobi, F. Ghanoni, H. Nadri, A. Moradi, M.P. Hamedani, F.H. Moghadam,
S. Emami, M. Vosooghi, R. Zadmard, A. Foroumadi, A. Shafiee, New tetracyclic tacrine analogs
containing pyrano[2,3-c]pyrazole: Efficient synthesis, biological assessment and docking simulation
study, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.10.049.
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ACCEPTED MANUSCRIPT
Graphical abstract
New tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: efficient synthesis, biological assessment
and docking simulation study
Mehdi Khoobi, Farzaneh Ghanoni, Hamid Nadri, Alireza Moradi, Morteza Pirali Hamedani, Farshad Homayouni
Moghadam, Saeed Emami, Mohsen Vosooghi, Reza Zadmard, Alireza Foroumadi, Abbas Shafiee*
OMe
MeO
NH2
NH2
N
HN
N
N
O
Tacrine
7h
A new series of tacrine-based compounds 7a-l were designed and synthesized as AChE inhibitors. The most potent
compound 7h bearing a 3,4-dimethoxyphenyl group was more active than reference drug tacrine.

ACCEPTED MANUSCRIPT
New tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: efficient
synthesis, biological assessment and docking simulation study
Mehdi Khoobia, Farzaneh Ghanonib, Hamid Nadric, Alireza Moradi c, Morteza Pirali Hamedania,
Farshad Homayouni Moghadamd, Saeed Emamie, Mohsen Vosooghia, Reza Zadmardb, Alireza
Foroumadia, Abbas Shafieea,*
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b
Chemistry and Chemical Engineering Research Center of Iran (CCERCI), P. O. BOX 14335-
186, Tehran, Iran
c Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
d Neurobiomedical Research Center, School of Medicine, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
e Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Abstract: A new series of tacrine-based acetylcholinesterase (AChE) inhibitors 7a-l were designed by replacing the
benzene ring of tacrine with aryl-dihydropyrano[2,3-c]pyrazole. The poly-functionalized hybrid molecules 7a-l were
efficiently synthesized through multi-component reaction and subsequent Friedländer reaction between the obtained
pyrano[2,3-c]pyrazoles and cyclohexanone. Most of target compounds showed potent and selective anti-AChE
activity at sub-micromolar range. The most potent compound 7h bearing a 3,4-dimethoxyphenyl group was more
active than reference drug tacrine. The representative compound 7h could significantly protect neurons against
oxidative stress as potent as quercetin at low concentrations. The docking study of compound 7h with AChE
enzyme revealed that the (R)-enantiomer binds preferably to CAS while the (S)-enantiomer prone to be a PAS
binder.
Keywords: Alzheimer’s disease; Acetylcholinesterase; Docking study; Neuroprotective activity;
Tacrine
* Corresponding author. Tel: +98 21 66406757; Fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
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1. Introduction
Alzheimer's disease (AD) is a complex and multifactorial age-related neurodegenerative disorder
characterized by progressive loss of memory, attention, language skills, along with depression
[1]. Several factors including decline of acetylcholine (ACh), β-amyloid (Aβ) deposits, τ-protein
(τ) aggregation, oxidative stress, and imbalance of bio-metals have been proposed to explain the
mechanism of AD pathogenesis [2].
The previous studies have demonstrated that the inhibition of AChE which resulted in rising the
ACh level in brain, is a desirable approach in the treatment of AD [3]. Also, it was proposed that
the peripheral anionic site (PAS) of AChE involves in the aggregation of amyloid fibrils [4].
Consequently, the finding of AChE inhibitors which simultaneously bind to both catalytic and
peripheral binding sites has become a very active area for design and development of anti-
Alzheimer's disease drugs [5]. On the other hand, oxidative damages may accelerate the
appearance of amyloid plaques and neurofibrillary (NFT) tangles in the brain [6]. Therefore,
preventing the formation of the free radicals or protecting against oxidative stress by
neuroprotective agents may be a suitable approach for AD therapy [7].
Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a AChE inhibitor that proved to have a beneficial
effect in patients with mild-to-moderate dementia of AD [8]. Unfortunately, the administration of
tacrine for the treatment of AD associated with high incidence of hepatotoxicity [9].
Modifications on the tacrine structure have been mainly focused on the replacement of the
benzene ring in tacrine by a heterocyclic ring, and also tacrine-based molecular hybridizations.
Thomae et al. reported different analogs where the benzene ring in tacrine was replaced by
thiophene, thiazole, 4-azaisoindole, or selenophene heterocycles [10-13]. Futhermore, Tzeng and
co-workers synthesized a few substituted benzofuro[2,3-b]quinoline derivatives as tetracyclic
analogs of tacrine [14].
In the search for finding multi-targeted AChE inhibitors, Marco-Contelles et al. have designed a
series of 1,8-naphthyridine derivatives related to tacrine. [15]. Tacrine analog A (Fig. 1), the best
AChE inhibitor of this series, exhibited neuroprotective activity against oxidative stress induced
by two toxic insults of the mitochondrial chain. The same group also reported tacrine analogs,
such as pyrazolo[3,4-b]quinoline and benzo[b]pyrazolo[4,3-g][1,8]naphthyridine derivatives, as
potent and selective AChE inhibitors. Particularly, compound B was able to protect SH-SY5Y
cells against two toxic molecules (rotenone/oligomicin-A) [16].
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Recently, a series of benzochromene-based tacrines namely 7-aryl-9,10,11,12-tetrahydro-7H-
benzo[7,8]chromeno[2,3-b]quinolin-8-amines were synthesized and evaluated as potent and
selective inhibitors of AChE [17,18]. Among them, tacrine-like compound C had an excellent
antioxidant profile and significant neuroprotective effects in cortical neurons against
mitochondrial chain blocker-induced cell death.
As described above, AD is a complex disease which could be better managed by agents acting
upon more than one target. Considering this strategy and in continue of our previous works on
anti-AChE agents [19], we designed a novel series of tacrine-based compounds consisting
pyrano[2,3-c]pyrazole substructure. Since the pyrazole and 4-substituted pyran were found in the
reported compounds B and C, they were incorporated in the tacrine scaffold to achieve new
entity 2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amines (7a-l). Thus we
described here, synthesis, in vitro anti-cholinesterase and neuroprotective evaluation and in silico
study of tacrine-based compounds 7a-l containing pyrano[2,3-c]pyrazole system.
2. Chemistry
As illustrated in Scheme 1, target compounds 7 were easily prepared in two steps. Initially,
preparation of pyrano[2,3-c]pyrazoles 5 [20] via one-pot four-component reaction of
ethylacetoacetate (1), hydrazine hydrate (2), malononitrile (3) and 4-methylbenzaldehyde (4a)
was studied under several conditions. Several catalysts [(S)-proline, piperidine, pyridine, NaOH,
K2CO3] and various environmentally benign solvents (water, EtOH and polyethylene glycol)
were investigated to find the best condition. After optimization, the best results in terms of yield
were achieved by using of (S)-proline in water-ethanol medium (1:1) under reflux condition
(isolated yield was 81%). Ultrasonic is an alternative mode of energy to accelerate the reaction
time and even improve the yield of the reaction. Therefore, the model reaction was screened
under ultrasonic irradiation to find the best reaction condition. The results of this comparative
experiment showed that application of ultrasonic irradiation cause to shorten the reaction time up
to 20 min and improve the yield of the reaction up to 92% at room temperature. In the next step,
AlCl3 catalyzed Friedländer reaction between the obtained pyrano[2,3-c]pyrazoles 5 and
cyclohexanone (6) resulted in the formation of target products 7.
It should be noted that intermediates 5a-l and target compounds 7a-l were obtained as a mixture
of enantiomers, having no detectable optical activity.
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3. Results and discussion
3.1. Pharmacology
3.1.1. Anti-cholinesterase activity
The anti-cholinesterase activity of target compounds 7a-l was assessed in vitro against AChE
from Electrophorus electricus (eelAChE) and horse serum butyrylcholinesterase (eqBuChE) in
comparison to tacrine as reference drug. The results were presented in Table 1, as IC50 values in
µM. The obtained IC50 values revealed that all tested compounds with exception of 7f showed
significant inhibitory activity against AChE (IC50 values = 0.19-3.27 µM). The most active
compound was 3,4-dimethoxyphenyl derivative 7h with IC50 value of 0.19 µM. Its anti-AChE
activity was superior to that of reference drug tacrine. Furthermore, 4-methoxyphenyl analog 7e
and 1-naphthyl derivative 7k were practically as potent as tacrine against AChE.
The IC50 values of 4-substituted phenyl analogs 7a, 7c and 7e revealed that the methoxy group
on para-position of phenyl ring is more favorable than methyl or fluoro substituents. The
introduction of second methoxy group on 4-methoxyphenyl residue of compound 7e, resulted in
compound 7h with improved anti-AChE activity. However, the insertion of third methoxy group
on phenyl ring diminished the activity, as observed in compound 7i. Surprisingly, while the 2-
methoxyphenyl analog 7f was inactive against AChE, but 2,5-dimethoxyphenyl derivative 7g
had potent activity with IC50 value of 0.5 µM. The sub-micromolar activity of compound 7k
showed that the bicyclic 1-naphthyl group is truly tolerated on the pyran part of the molecule.
In terms of anti-BuChE activity, only compounds 7a-c, 7k and 7l showed inhibitory activity and
remaining compounds were inactive. 4-Tolyl derivative 7a with IC50 value of 4.5 µM was the
most potent compound against BuChE. The data obtained for inhibition of AChE/BuChE by
target compounds 7a-l demonstrated that most compounds had high selectivity for AChE over
BuChE. However, compound 7a with selectivity index of 6 had the lowest selectivity.
3.1.2. Kinetic analysis of AChE inhibition
The kinetic studies were performed using the most active compound 7h to understand the kinetic
of the inhibition on AChE. The inhibition kinetic of compound 7h using Lineweaver-Burk plot
was depicted in Fig. 2. As interpreted from the pattern of inhibition in Fig. 2, the mechanism of
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action on AChE for compound 7h is a mixed-type inhibition. The Ki value of compound 7h was
calculated (Ki= 0.49 µM) using Lineweaver-Burk secondary plot (Fig. 3).
3.1.3. Neuroprotection against H2O2-induced cell death in PC12 cells
The most potent compound against AChE (7h) was further subjected to in vitro neuroprotective
assay against H2O2-induced damage in PC12 cells. The data are summarized in Fig. 4 in which
the percent of cell viability was calculated in comparison with control group. The tested
concentrations were selected according to the non-toxic range (<15 µM) of the 7h against PC12
cells. Based on the results, compound 7h showed significant neuroprotective activity at 10 µM
(viability= 48.35%) that is comparable with quercetin at the same concentration (viability=
51.63%).
3.2. Ligand-protein docking simulation
The molecular docking study was performed to understand the binding interactions of designed
compounds at the molecular level. Since the final compounds have a chiral center at the 4-
position of pyran ring, they are racemic mixtures. Thus, both (R)- and (S)-enantiomers were
studied in docking simulations. The preliminary analysis of docking results by visual inspection
showed that the best poses of target compounds 7a-l were docked similarly. Therefore, the best
pose of compound (R,S)-7h as a promising compound was further analyzed in detail.
As shown in Fig. 5, the orientation of (R)- and (S)-enantiomers was different in the gorge of the
enzyme. The (R)-enantiomer was placed at the bottom of the gorge in the vicinity of catalytic
triad while the (S)-enantiomer occupied the bottle-neck of the gorge adjacent to peripheral
anionic site (PAS). Moreover, according to the interaction mode of the both (R)- and (S)-
enantiomers, it seems that aminopyridine moiety play an important role in ligand recognition via
π–π stacking with Tyr334. In the case of (R)-enantiomer (Fig. 6), after anchoring of the ligand
through aforementioned interaction, the dimethoxyphenyl moiety oriented toward the catalytic
anionic subsite (CAS). In this position, the dimethoxyphenyl moiety was well accommodated in
the CAS through a T-shape π–π stacking with Phe330 and hydrogen binding with His440. In
addition, the second methoxy was set alongside the oxyanion hole. In such orientation, the (S)-
enantiomer could not well accommodate due to steric clash with Trp84. Thereupon, it turned
reversely toward PAS to relieve this clash leading to formation of the following interactions: a T-
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shape π–π stacking of dimethoxyphenyl with Trp279 and a hydrogen bond between pyrazole ring
and Ser286 (Fig. 7). It could be concluded that the (R)-enantiomer binds preferably to CAS while
the (S)-enantiomer prone to be a PAS binder.
4. Conclusion
In the search for finding new potential anti-cholinesterase agents, we replaced the benzene ring
of tacrine by aryl-dihydropyrano[2,3-c]pyrazole. The poly-functionalized hybrid molecules 7a-l
were efficiently synthesized through multi-component reaction and subsequent Friedländer
reaction between the obtained pyrano[2,3-c]pyrazoles and cyclohexanone. Most of target
compounds showed potent and selective anti-AChE activity at sub-micromolar levels.
The structure-activity relationship study revealed that the potency profile against AChE/BuChE
could be modulated properly by altering the substitutions on the phenyl group attached to the
dihydropyran substructure. Compound 7h bearing a 3,4-dimethoxyphenyl group was the most
potent compound against AChE, being more active than reference drug tacrine. The in vitro
neuroprotective assay of compound 7h against H2O2-induced damage in PC12 cells
demonstrated that the tested compound could significantly protect neurons against oxidative
stress as potent as quercetin at low concentrations. The docking study of compound 7h with
AChE enzyme revealed that the (R)-enantiomer binds preferably to CAS while the (S)-
enantiomer prone to be a PAS binder. The high potency and selectivity of the representative
compound 7h, along with its significant neuroprotective effects against oxidative stress and
potential binding of its (S)-enantiomer to the PAS reserved it as a lead for AD therapy.
5. Experimental
5.1. General chemistry
All commercially available reagents were used without further purification. TLC was conducted
on silica gel 250 micron, F254 plates. Melting points were measured on a Kofler hot stage
apparatus and are uncorrected. The IR spectra were taken using Nicolet FT-IR Magna 550
1
spectrograph (KBr discs). H NMR spectra were recorded on Brucker 400 MHz NMR
instruments. The chemical shifts (δ) and coupling constants (J) are expressed in parts per million
1
and Hertz, respectively. The atoms numbering of the target compounds used for H NMR data
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are shown in Scheme 1. Mass spectra of the products were obtained with an HP (Agilent
technologies) 5937 Mass Selective Detector. 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 pyrano[2,3-c]pyrazole derivatives (5)
In a 100 mL round bottom flask, a mixture of hydrazine hydrate (12 mmol), ethyl acetoacetate
(10 mmol), malononitrile (10 mmol), (S)-proline (10% mol) and water (20 mL) were taken and
the mixture was irradiated in the ultrasonic bath for 2 min. Afterward, aromatic aldehyde (10
mmol) was added to the mixture and the mixture was irradiated for 10-30 min. Progress of the
reaction was monitored by TLC. After completion of the reaction, the precipitated solid was
filtered and further purified by recrystallization from ethanol.
5.3. General procedure for the preparation of compounds 7a-l
This procedure was carried out according to our previous reported method [21]. Briefly,
aluminium chloride (1.5 equiv.) was suspended in dry 1,2-dichloroethane (10 mL) at room
temperature under argon atmosphere. After stirring the suspension for a few minutes, the
corresponding pyrano[2,3-c]pyrazole (1 equiv.) and cyclohexanone (1.5 equiv.) were added to
the mixture and the reaction mixture was heated under reflux for 24 h. Progress of the reaction
was monitored by TLC. After completion of the reaction, an aqueous solution of sodium
hydroxide (10%) was added dropwise to the mixture until the aqueous solution became basic.
After stirring for 30 min, the precipitate was filtered and washed with water.
5.3.1.
3-Methyl-4-(p-tolyl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (7a). Yield 92%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3391 and 3191 (NH2),
1581 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 12.04 (bs, 1H, NH), 7.12 (d, 2H, J = 8.0 Hz, H2,6
phenyl), 7.03 (d, 2H, J = 8.0 Hz, H3,5 phenyl), 5.30 (bs, 2H, NH2), 5.12 (s, 1H, H4), 2.57 (bs, 2H,
2H9), 2.50 (s, 3H, CH3 phenyl), 2.34-2.20 (bs, 2H, 2H6), 1.96 (s, 3H, CH3 pyrazol), 1.66 (bs, 4H, 2H7
13
and 2H8). C NMR (DMSO-d6, 100 MHz) δ: 175.4, 156.1, 152.6, 152.4, 142.5, 135.6, 129.3,
127.6, 112.4, 100.2, 99.0, 34.3, 32.4, 25.3, 23.3, 22.8, 22.5, 10.2. Anal. Calcd for C21H22N4O: C,
72.81; H, 6.40; N, 16.17. Found: C, 72.53; H, 6.63; N, 15.08.
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5.3.2.
4-(2-Fluorophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7b). Yield 86%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3494 and
3407 (NH2), 1585 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.90 (bs, 1H, NH), 7.36 (t, 1H, J
= 7.2 Hz, Hphenyl), 7.21-7.20 (m 1H, Hphenyl), 7.11-7.04 (m, 2H, Hphenyl), 5.34 (s, 1H, H4), 5.20
(bs, 2H, NH2), 2.57 (bs, 2H, 2H9), 2.33-2.16 (m, 2H, 2H6), 1.95 (s, 3H, CH3 pyrazol), 1.69 (bs, 4H,
2H7 and 2H8). 13C NMR (DMSO-d6, 100 MHz) δ: 161.4, 158.9, 157.1, 156.1, 152.8, 152.3,
135.4, 131.3, 131.2, 130.5, 130.4, 129.0, 128.9, 124.9, 116.3, 112.2, 98.5, 97.7, 32.4, 29.7, 23.3,
22.7, 22.5, 9.9. Anal. Calcd for C20H19FN4O: C, 68.56; H, 5.47; N, 15.99. Found: C, 68.21; H,
5.18; N, 15.72.
5.3.3.
4-(4-Fluorophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7c). Yield 88%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3505 and
3385 (NH2), 1586 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.90 (bs, 1H, NH), 7.28-7.25 (m,
2H, H3,5 phenyl), 7.08-7.04 (m, 2H, H2,6 phenyl), 5.41 (bs, 2H, NH2), 5.23 (s, 1H, H4), 2.57 (bs, 2H,
2H9), 2.33-2.16 (m, 2H, 2H6), 1.99 (s, 3H, CH3 pyrazol), 1.70 (bs, 4H, 2H7 and 2H8). 13C NMR
(DMSO-d6, 100 MHz) δ: 162.2, 159.8, 156.7, 156.0, 152.7, 152.2, 141.7, 141.6, 135.3, 129.5,
115.6, 115.4, 112.5, 100.1, 98.8, 33.7, 32.3, 23.3, 22.7, 22.5, 10.2. Anal. Calcd for C20H19FN4O:
C, 68.56; H, 5.47; N, 15.99. Found: C, 68.79; H, 5.79; N, 16.22.
5.3.4.
3-Methyl-4-(3-nitrophenyl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7d). Yield 94%; yellow solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3411 and
3368 (NH2), 1583 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.9 (bs, 1H, NH), 8.24 (s, 1H, H2
phenyl), 8.00 (d, 1H, J = 8.0 Hz, H4 phenyl), 7.60-7.51 (m, 2H, H5,6 phenyl), 5.57 (bs, 2H, NH2), 5.45
(s, 1H, H4), 2.58 (bs, 2H, 2H9), 2.35-2.13 (m, 2H, 2H6), 2.00 (s, 3H, CH3 pyrazol), 1.69 (bs, 4H,
2H7 and 2H8). 13C NMR (DMSO-d6, 100 MHz) δ: 156.8, 156.0, 153.4, 152.6, 148.0, 147.8,
135.6, 134.4, 130.5, 122.1, 121.0, 112.6, 99.4, 98.1, 33.8, 32.4, 23.3, 22.7, 22.5, 10.2. Anal.
Calcd for C20H19N5O3: C, 63.65; H, 5.07; N, 18.56. Found: C, 63.34; H, 5.26; N, 18.21.
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5.3.5.
4-(4-Methoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7e). Yield 93%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3493 and
3403 (NH2), 1574 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.84 (bs, 1H, NH), 7.16 (d, 2H, J
= 8.4 Hz, H2,6 phenyl), 6.80 (d, 2H, J = 8.4 Hz, H3,5 phenyl), 5.29 (bs, 2H, NH2), 5.10 (s, 1H, H4),
3.39 (s, 3H, OCH3), 2.57 (bs, 2H, 2H9), 2.32-2.19 (m, 2H, 2H6), 1.98 (s, 3H, CH3 pyrazol), 1.70
13
(bs, 4H, 2H7 and 2H8). C NMR (DMSO-d6, 100 MHz) δ: 158.0, 156.8, 156.0, 152.6, 137.5,
135.2, 128.7, 114.2, 112.4, 100.5, 99.1, 55.4, 33.8, 32.4, 23.3, 22.8, 22.5, 10.2. Anal. Calcd for
C21H22N4O2: C, 69.59; H, 6.12; N, 15.46. Found: C, 69.21; H, 6.48; N, 15.12.
5.3.6.
4-(2-Methoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7f). Yield 81%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3431 and
3375 (NH2), 1582 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.95 (bs, 1H, NH), 7.03-6.83 (m,
4Hphenyl), 5.31 (s, 1H, H4), 5.16 (bs, 2H, NH2), 3.69 (s, 3H, OCH3), 2.49 (bs, 2H, 2H9), 2.24-2.19
(m, 2H, 2H6), 1.96 (s, 3H, CH3 pyrazol), 1.68 (bs, 4H, 2H7 and 2H8). 13C NMR (DMSO-d6, 100
MHz) δ: 157.3, 156.3, 152.4, 150.8, 136.8, 134.6, 126.4, 125.1, 122.3, 114.2, 112.6, 100.0, 99.6,
54.8, 33.5, 32.2, 23.1, 22.6, 22.3, 10.1. Anal. Calcd for C21H22N4O2: C, 69.59; H, 6.12; N, 15.46.
Found: 69.76; H, 6.32; N, 15.68.
5.3.7. 4-(2,5-Dimethoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7g). Yield 88%; yellow solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3448 and
3374 (NH2), 1586 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.86 (bs, 1H, NH), 6.97 (d, 1H, J
= 8.5 Hz, H3 phenyl), 6.75-6.73 (m, 1H, H4 phenyl), 6.35 (bs, 1H, H6 phenyl), 5.27 (s, 1H, H4), 5.22
(bs, 2H, NH2), 3.83 (s, 3H, OCH3), 3.56 (s, 3H, OCH3), 2.51-2.26 (m, 4H, 2H9 and 2H6), 1.89 (s,
3H, CH3 pyrazol), 1.69 (bs, 4H, 2H7 and 2H8). 13C NMR (DMSO-d6, 100 MHz) δ: 157.9, 156.0,
154.0, 152.6, 152.2, 149.8, 135.2, 134.1, 116.2, 112.6, 112.2, 11.8, 99.6, 98.9, 56.8, 55.6, 45.5,
32.3, 23.2, 22.7, 22.5, 9.8. Anal. Calcd for C22H24N4O3: C, 67.33; H, 6.16; N, 14.28. Found: C,
67.58; H, 6.43; N, 14.01.
9

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5.3.8.
4-(3,4-Dimethoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7h). Yield 90%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3481 and
3389 (NH2), 1584 (C=N). ). 1H NMR (DMSO-d6, 400 MHz) δ: 11.86 (bs, 1H, NH), 7.05 (d, 1H, J
= 2.0 Hz, H2 phenyl), 6.80 (d, 1H, J = 8.4 Hz, H5 phenyl), 6.59 (dd, 1H, J = 8.4 and J = 2.0 Hz, H6
phenyl), 5.30 (bs, 2H, NH2), 5.08 (s, 1H, H4), 3.68 (s, 3H, OCH3), 3.66 (s, 3H, OCH3), 2.57 (bs,
2H, 2H9), 2.32-2.17 (m, 2H, 2H6), 2.02 (s, 3H, CH3 pyrazol), 1.70 (bs, 4H, 2H7 and 2H8). 13C
NMR (DMSO-d6, 100 MHz) δ: 156.7, 156.0, 152.7, 152.5, 148.7, 147.5, 138.0, 135.3, 119.6,
112.5, 112.3, 112.1, 100.3, 98.9, 55.9, 55.8, 34.3, 32.4, 23.3, 22.8, 22.5, 10.3. Anal. Calcd for
C22H24N4O3: C, 67.33; H, 6.16; N, 14.28. Found: C, 67.55; H, 5.89; N, 14.54.
5.3.9. 3-Methyl-4-(3,4,5-trimethoxyphenyl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7i). Yield 95%; yellow solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3483 and
1
3383 (NH2), 1590 (C=N). H NMR (DMSO-d6, 400 MHz) δ: 11.90 (bs, 1H, NH), 6.57 (s, 2H,
H2,6 phenyl), 5.46 (s, 1H, H4), 5.12 (bs, 2H, NH2), 3.72 (s, 6H, 2×OCH3), 3.65 (s, 3H, OCH3), 2.50
13
(bs, 2H, 2H9), 2.09 (bs, 2H, 2H6), 2.05 (s, 3H, CH3 pyrazol), 1.71 (bs, 4H, 2H7 and 2H8). C
NMR (DMSO-d6, 100 MHz) δ: 156.2, 155.4, 152.7, 140.6, 135.8, 134.9, 111.9, 104.7, 99.5, 98.1,
59.9, 55.8, 34.5, 31.6, 22.8, 22.2, 22.0, 9.9. Anal. Calcd for C23H26N4O4: C, 65.39; H, 6.20; N,
13.26. Found: C, 65.67; H, 6.51; N, 13.01.
5.3.10.
4-(2,3-Dichlorophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7j). Yield 82%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3477 and
3343 (NH2), 1586 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.99 (bs, 1H, NH), 7.62 (d, J = 8.9
Hz, H4 phenyl), 7.49 (t, J = 8.9 Hz, H5 phenyl), 7.28 (d, J = 8.9 Hz, H6 phenyl), 5.56 (s, 1H, H4), 5.07
(bs, 2H, NH2), 2.56 (bs, 2H, 2H9), 2.36 (bs, 2H, 2H6), 1.96 (s, 3H, CH3 pyrazol), 1.77 (bs, 4H,
2H7 and 2H8). 13C NMR (DMSO-d6, 100 MHz) δ: 157.1, 156.2, 153.3, 152.4, 135.7, 132.4, 130.7,
130.1, 129.7, 129.5, 129.2, 112.8, 98.1, 32.4, 23.3, 22.8, 22.4, 10.3. Anal. Calcd for
C20H18Cl2N4O: C, 59.86; H, 4.52; N, 13.96. Found: C, 59.51; H, 4.26; N, 13.68.
5.3.11.
3-Methyl-4-(naphthalen-1-yl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7k). Yield 78%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3419 and
1
3321 (NH2), 1585 (C=N). H NMR (DMSO-d6, 400 MHz) δ: 11.92 (bs, 1H, NH), 7.97 (bs, 1H,
10

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Hnaphtyl), 7.84-7.75 (m, 3H, Hnaphtyl), 7.49-7.42 (m, 2H, Hnaphtyl), 7.21 (d, 1H, J = 8.4 Hz, Hnaphtyl),
5.38 (s, 1H, H4), 5.36 (bs, 2H, NH2), 2.59 (bs, 2H, 2H9), 2.31-2.12 (m, 2H, 2H6), 1.97 (s, 3H, CH3
13
pyrazol), 1.68 (bs, 4H, 2H7 and 2H8). C NMR (DMSO-d6, 100 MHz) δ: 156.8, 156.1, 152,9,
152.6, 142.6, 135.6, 133.1, 132.2, 128.8, 128.0, 127.9, 126.7, 126.3, 126.0, 125.7, 112.5, 99.8,
98.6, 34.9, 32.4, 23.3, 22.7, 22.5, 10.2. Anal. Calcd for C24H22N4O: C, 75.37; H, 5.80; N, 14.65.
Found: C, 75.09; H, 5.61; N, 14.89.
5.3.12.
3-Methyl-4-(thiophen-2-yl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (7l). Yield 77%; white solid; mp >250 ºC; IR (KBr, cm-1) νmax: 3472 and
3362 (NH2), 1591 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ: 11.95 (bs, 1H, NH), 7.22 (dd, 1H, J
= 5.2 and J = 1.2 Hz, H5 thiophen), 7.11(dd, 1H, J = 4.8 and J = 1.2 Hz, H3 thiophen), 6.87-6.85 (m,
1H, H3 thiophen), 5.61 (s, 1H, H4), 5.56 (bs, 2H, NH2), 2.56 (bs, 2H, 2H9), 2.35-2.21 (m, 2H, 2H6),
2.10 (s, 3H, CH3 pyrazol), 1.71 (bs, 4H, 2H7 and 2H8). 13C NMR (DMSO-d6, 100 MHz) δ: 155.5,
154.6, 154.0, 150.6, 149.1, 136.4, 126.1, 125.3, 112.8, 99.7, 98.8, 30.4, 29.5, 23.0, 22.3, 10.1.
Anal. Calcd for C18H18N4OS: C, 63.88; H, 5.36; N, 16.56. Found: C, 63.61; H, 5.12; N, 16.90.
5.4. In vitro inhibition studies on AChE and BuChE
The inhibitory activities of the target compounds against AChE and BuChE were determined
using the spectrophotometric method of Ellman [22]. Five different concentrations of each
compound were tested to obtain the range of 20% to 80% enzyme inhibition for AChE and
BuChE. The assay solution consisted of 3 ml phosphate buffer (0.1 mol/L-1, pH 8.0), 100 µl of
5,5′-dithio-bis(2-nitrobenzoic acid), 100 µl of 2.5 IU/ml of acetylcholinesterase (AChE, E.C.
3.1.1.7, Type V-S, lyophilized powder, from electric eel, 1000 unit) or butyrylcholinesterase
(BuChE, E.C. 3.1.1.8, from equine serum) and 100 µl of each test compounds. The mixture was
pre-incubated for 10 min followed by the addition of substrate (acetylthiocholine iodide or
butyrylthiocholine iodide). Assays were carried out with a blank containing all components
except enzyme 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.
11

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The IC50 values were determined graphically from log concentration vs. % of inhibition curves.
All experiments were performed in triplicate.
5.5. Neuroprotection assay against H2O2-induced cell death in PC12 cells
Rat differentiated PC12 cells were provided as previously described method [23]. Differentiated
PC12 cells were incubated with different concentrations (1, 5 and 10 µM) of the compound for 3
h before treatment with H2O2 (300 µM). The occurrence of apoptosis was established after
staining with DAPI, and cell viability was measured after 24 h by using the MTT assay. Briefly,
10 µl of MTT solution (5 mg/ml, Sigma) was added to the cell culture media (150 µl) and
incubated in a CO2 incubator for 3.5 h. Thereafter, medium was removed and DMSO (150 µl)
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 [24].
5.6. Docking simulations
The program Autodock Vina (AV) 1.1.1 [25] was used for docking simulations. For docking
purpose, 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 5.8.3, 2012, ChemAxon (http://www.chemaxon.com),
the 3D structures were constructed using Openbabel 2.3.1 [26]. Then, the receptor and optimized
structure of the ligands were converted to required pdbqt format using Autodock Tools 1.5.4
[27]. 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 for
further analysis. All molecular visualizations were carried out in DS Viewer Pro (Accelrys, Inc.,
San Diego, CA).
12

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Acknowledgments
This work was supported by a grant from the Research Council of Tehran University of Medical
Sciences.
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Captions:
Fig. 1. The structures of tacrine and some previously reported tacrine-based anti-AChE agents
(A, B and C). Compounds 7a-l were designed in this work, as new tetracyclic tacrine analogs
containing pyrano[2,3-c]pyrazole.
Fig. 2. Inhibition kinetics of compound 7h using Lineweaver-Burk plot with eelAChE.
Fig. 3. The Lineweaver-Burk secondary plot of compound 7h, slope vs. various concentrations
of compound 7h.
Fig. 4. Neuroprotective effect of compound 7h against H2O2-induced cell death in PC12 cells.
###
Data are expressed as mean ± SD (n = 8),
**P <0.05 vs. H2O2 treated only.
P <0.001 vs. control, *** P <0.001 vs. H2O2 and
Fig. 5. The proposed orientation of R (green) and S (magenta) enantiomers of 7h in the AChE
active site.
Fig. 6. Illustration of (R)-7h binding mode in the AChE active site. The hydrogen bond is
presented as red dashed line and rings having π–π stacking are filled with yellow color.
Fig. 7. Illustration of (S)-7h enantiomer binding mode in the AChE active site. The hydrogen
bond is presented as red dashed line and rings having π–π stacking are filled with green color.
Scheme 1. Synthesis of compounds 7a-l.
16

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Table 1
Inhibitory activity of the target compounds 7a-l against AChE and BuChE
Ar
NH2
HN
N
O
N
IC50 (µM)a
IC50 (µM)
BuChE
4.5 ± 0.2
33.0 ± 2.1
25.0 ± 1.8
>100
Compound
Ar
AChE
4-Tolyl
0.75 ± 0.04
0.59 ± 0.04
0.81 ± 0.05
3.27 ± 0.19
0.31 ± 0.02
>10
7a
2-Fluorophenyl
7b
7c
4- Fluorophenyl
3-Nitrophenyl
7d
7e
4-Methoxyphenyl
2-Methoxyphenyl
>100
>100
7f
2,5-Dimethoxyphenyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
2,3-Dichlorophenyl
1-Naphthyl
0.50 ± 0.03
0.19 ± 0.01
2.18 ± 0.13
0.87 ± 0.06
0.32 ± 0.02
0.72 ± 0.04
0.28 ± 0.02
>100
7g
>100
7h
7i
>100
>100
7j
15.0 ± 1.1
16.0 ± 1.1
0.35 ± 0.2
7k
7l
2-Thienyl
-
Tacrine
a Data are expressed as mean ± S.E. of at least three different experiments.

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Fig. 1. The structures of tacrine and some previously reported tacrine-based anti-AChE agents
(A, B and C). Compounds 7a-l were designed in this work, as new tetracyclic tacrine analogs
containing pyrano[2,3-c]pyrazole.
1

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Fig. 2. Inhibition kinetics of compound 7h using Lineweaver-Burk plot with eelAChE.
2

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Fig. 3. The Lineweaver-Burk secondary plot of compound 7h, slope vs. various concentrations
of compound 7h.
3

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Fig. 4. Neuroprotective effect of compound 7h against H2O2-induced cell death in PC12 cells.
###
Data are expressed as mean ± SD (n = 8),
**P <0.05 vs. H2O2 treated only.
P <0.001 vs. control, *** P <0.001 vs. H2O2 and
4

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Fig. 5. The proposed orientation of R (green) and S (magenta) enantiomers of 7h in the AChE
active site.
5

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Fig. 6. Illustration of (R)-7h binding mode in the AChE active site. The hydrogen bond is
presented as red dashed line and rings having π–π stacking are filled with yellow color.
6

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7

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Fig. 7. Illustration of (S)-7h enantiomer binding mode in the AChE active site. The hydrogen
bond is presented as red dashed line and rings having π–π stacking are filled with green color.
Ar
Ultrasonic
CN
CN
Irradiation
O
O
O
HN
+
NH2NH2.H2O
+
+
(S)-proline
H2O/EtOH, r.t.
10-35 min
Ar
H
N
OEt
O
NH2
CN
2
1
5a-l
3
4
O
AlCl3,
ClCH2CH2Cl
6
Ar = phenyl; substituted phenyl;
naphthalen-1-yl;
Ar
4
NH2
thiophen-2-yl
6
5
3
HN
N
O
N
1
9
7a-l
Scheme 1. Synthesis of compounds 7a-l.
8

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Highlights
•
•
A series of tacrine-based compounds 7a-l were synthesized as AChE inhibitors.
Compound 7h bearing a 3,4-dimethoxyphenyl group was the most active derivative.
•
•
•
Compound 7h with IC50 = 0.19 µM was more potent than reference drug tacrine.
Compound 7h could significantly protect neurons against oxidative stress.
Docking study showed that (R)-7h preferably binds to CAS while (S)-7h binds to PAS.

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Supplementary material
New tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: efficient synthesis,
biological assessment and docking simulation study
Mehdi Khoobi, Farzaneh Ghanoni, Hamid Nadri, Alireza Moradi, Morteza Pirali Hamedani, Farshad Homayouni
Moghadam, Saeed Emami, Mohsen Vosooghia, Reza Zadmard, Alireza Foroumadi, Abbas Shafiee*
1H and 13C spectra for selected compounds:
Compound
page
4-(2-Fluorophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (7b)
2,3
4,5
6,7
4-(4-Fluorophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine(7c)
4-(4-Methoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (7e)
4-(2,5-Dimethoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (7g) 8,9
4-(3,4-Dimethoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine(7h)10,11
3-Methyl-4-(naphthalen-1-yl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (7k)
3-Methyl-4-(thiophen-2-yl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (7l)
12,13
14,15
1

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F
NH2
HN
N
O
N
7b
2