Bioorganic & Medicinal Chemistry Letters 28 (2018) 2481–2484

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Bioorganic & Medicinal Chemistry Letters

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A cascade synthesis, in vitro cholinesterases inhibitory activity and docking T

studies of novel Tacrine-pyranopyrazole derivatives
⁎
Chamseddine Derablia, Imen Boualiaa, Ahmed B. Abdelwahabb,c, Raouf Boulcinaa,d, ,
Chawki Bensouicie, Gilbert Kirschb, Abdelmadjid Debachea
a
Laboratory of Synthesis of Molecules with Biological Interest, Frères Mentouri-Constantine University, 25000 Constantine, Algeria
b
SRSMC, Lorraine University, 1 Boulevard Arago, 57070, France
c
Chemistry of Natural Compounds Department, National Research Centrer, El-Behoos St. 33, Dokki, Cairo 12622, Egypt
d
Faculty of Technology, Batna 2 University, 05000 Batna, Algeria
e
Biotechnology Research Center, Constantine, Algeria

A R T I C LE I N FO A B S T R A C T

Keywords: In this work, we describe the preparation of some new Tacrine analogues modified with a pyranopyrazole
Multicomponent reaction moiety. A one-pot multicomponent reaction of 3-methyl-1H-pyrazol-5(4H)-one, aryl(or hetero)aldehydes, mal-
Tacrine-pyranopyrazole derivatives ononitrile and cyclohexanone involving a Friedländer condensation led to the title compounds. The synthesized
Cholinesterase inhibition heterocyclic analogues of this molecule were evaluated in vitro for their AChE and BChE inhibitory activities in
Alzheimer’s disease
search for potent cholinesterase enzyme inhibitors. Most of the synthesized compounds displayed remarkable
Docking study
AChE inhibitory activities with IC50 values ranging from 0.044 to 5.80 µM, wherein compounds 5e and 5j were
found to be most active inhibitors against AChE with IC50 values of 0.058 and 0.044 µM respectively. Molecular
modeling simulation on AChE and BChE receptors, showed good correlation between IC50 values and binding
interaction template of the most active inhibitors docked into the active site of their relevant enzymes.

Alzheimer’s disease (AD) is a progressive neurological disorder by thiophene A, thiazole B, 4-azaisoindole C, or selenophene D het-
leading to impairment in memory, language skills, judgment, and or- erocycles were described.7–12 In an approach for finding multi-targeted
ientation.1 It arises pathologically from acetylcholine (ACh) neuro- AChE inhibitors, a series of 1,8-naphthyridine E derivatives structurally
transmission hindrance by the formation of β-amyloid plaques. Thus related to Tacrine were designed.13 Recently, a series of benzochro-
one of the possible treatment is by inhibition of acetylcholinesterase mene F14,15 tetrahydropyranodiquinolin-8-amines G,16 and (benz)imi-
(AChE) and butyrylcholinesterase (BChE) (to some extent) to maintain dazolopyridino-based H Tacrines17 were developed and evaluated as
the ACh neurotransmission as long as possible. The enhancement of the potent and selective inhibitors of AChE (Fig. 1).
levels of the neurotransmitter ACh could be achieved through the ad- On the other hand, pyrano[2,3-c]pyrazoles are a large class of
ministration of acetylcholinesterase inhibitors. Currently, only four heterocyclic compounds possessing many important biological activ-
agents have been approved for AD therapy: Donepezil, Galantamine ities, such as analgesic, anti-inflammatory, anti-tumor and, insecticidal
and Rivastigmine,2a are very efficient for the inhibition of AChE, hence activities.18 Literature survey reveals a variety of three and four com-
raise the acetylcholine levels in the brain.3 Unfortunately, these AChE ponent MCR’s for the construction of different pyranopyrazoles. Among
inhibitors do not cure and stop the progression of the disease. On the them a three-component reaction between aromatic aldehyde, mal-
other hand, Memantine (Noojerone), was approved by US FDA drug for ononitrile, and substituted pyrazolin-5-ones in the presence of various
AD therapy is considered a low-affinity noncompetitive antagonist of base catalysts. These bases included triethylamine,19 triethanolamine,20
the NMDA subtype of glutamate receptors.2–4 Tacrine (9-amino-1,2,3,4- piperidine,21 N-methylmorpholine,22 D,L-proline,23 MgO,24 silica so-
tetrahydroacridine) (commercial name Cognex®) is an AChE inhibitor dium carbonate,25 and CsCO3 supported on hydroxy apatite-coated
that was proved to have a beneficial effect in patients with mild to Ni0.5 Zn0.5 Fe2O4 magnetic nanoparticles,26 Non-catalytic conditions for
moderate dementia of AD.5 Unfortunately, the administration of Ta- this sort of synthesis was also reported.27
crine is associated with high occurrence of hepatotoxicity.6 Different The first approach to synthesize the Tacrine–Pyranopyrazole deri-
synthetic pathways in which the benzene ring in Tacrine was replaced vatives was developed by Khoobi et al.28 It described the synthesis of

⁎
Corresponding author at: Laboratory of Synthesis of Molecules with Biological Interest, Frères Mentouri-Constantine University, 25000 Constantine, Algeria.
E-mail address: [email protected] (R. Boulcina).

https://doi.org/10.1016/j.bmcl.2018.05.063
Received 24 February 2018; Received in revised form 30 May 2018; Accepted 31 May 2018
Available online 01 June 2018
0960-894X/ © 2018 Elsevier Ltd. All rights reserved.
C. Derabli et al. Bioorganic & Medicinal Chemistry Letters 28 (2018) 2481–2484

specific time. Then, cyclohexanone 4, and aluminum chloride (AlCl3)

were added to the mixture, and stirring was continued for a specific
time to afford the desired product 5a in high yield (Scheme 1). With the
optimized reaction conditions in hand, we set out to explore the scope
and limitation of the protocol by investigating the reaction of various
aryl(or hetero)aldehydes.
All of the reactions proceeded smoothly to produce the expected
Tacrine-Pyranopyrazoles 5a–t with good to excellent yields. The use of
aromatic or heterocyclic aldehydes hasn’t significant impact on the
conversions. All of the products were fully characterized by melting
point, FTIR, 1H NMR, and 13C NMR spectroscopy further confirmed the
structure of products.29
In order to carry on with the investigation of the in vitro antic-
holinesterase potential of new Tacrine-pyranopyrazole derivatives, we
Fig. 1. The structures of reported Tacrine-derived AChE/BChE inhibitors. focused our attention to evaluating the influence of the substituent in
position 4 of pyran ring with aromatic or heteroaromatic moieties to-
ward this activity. From the synthesized Tacrine-pyranopyrazoles, 12
novel compounds were selected for structure-activity relationships
purposes. In fact, the anti-cholinesterase activity of target novel com-
pounds was assessed in vitro against AChE from Electrophorus electricus
(eel-AChE) and horse serum butyrylcholinesterase (eqBChE) according
to the spectrophotometric method of Ellman et al.30 For comparison
Scheme 1. Possible functionalization of the Tacrine–Pyranopyrazoles scaffold.
purpose, Tacrine and Galanthamine were used as reference compounds.
The IC50 values of all test compounds and their selectivity index for
Tacrine analogues from pyrano[2,3-c]pyrazoles and cyclohexanone in BChE over AChE were summarized in Table 1. The obtained IC50 values
the presence of aluminum chloride. revealed that all tested compounds showed significant inhibitory ac-
As continuing of our research on various heterocyclic compound tivity against AChE (IC50 values = 0.044–5.80 µM). The most active
families, we report herein our results concerning the cascade synthesis, compound was biphenyl derivative 5j with an IC50 value of 0.044 µM.
docking studies of new Tacrine–Pyranopyrazole analogues and in vitro Indeed, its anti-AChE activity was superior to that of reference drugs.
anticholinesterase activity and compared to Tacrine and Galantamine. The IC50 of compound 5a was estimated to a moderate value of
The main preparation goals were to reach target compounds 1.23 µM; However, The IC50 values of 4-substituted phenyl analogs 5b,
through convenient, fast, and low cost synthesis steps, with a view to 5c, 5d and 5e revealed that the phenyl and S-methyl groups on para-
identifying more potent and selective anticholinesterase derivatives. position of phenyl ring is more favorable than others substituents. The
Firstly, we planned the prepare Tacrine–Pyranopyrazole 5a by a one- introduction of two methyl or methoxy groups on phenyl ring resulted
pot, overall 4CR integrating Friedländer reaction of cyclohexanone 4 in compounds 5f and 5g respectively with improved anti-AChE activity.
with a 3CR of 3-methyl-1H-pyrazol-5(4H)-one 1, benzaldehyde 2a, However, the insertion of both chloro and nitro groups on phenyl ring
malononitrile 3 in the presence AlCl3 in 1,2-dichloroethane (DCE) diminished the activity, as observed in compounds 5h and 5i. In ad-
under heating. Unfortunately, no desired target product was detected dition, 3-pyridine moiety 5k had potent activity with an IC50 value of
after analysis of the crude reaction mixture. In a second attempt, we 0.33 µM. The activity of compound 5l showed that the bicyclic 2-
adopted a combinatorial synthesis of Tacrine–Pyranopyrazoles using a methoxy-1-naphthyl group is truly tolerated on the pyran part of the
sequential, one-pot, two-step reaction, in which both processes are molecule. In term of anti-BChE activity, all tested compounds showed
carried out in the same flask, without the isolation of pyranopyrazole moderate to good activities. 2-chloro-6-nitro derivative 5i with an IC50
intermediates. The reactions were first carried out by mixing 3-methyl- value of 1.84 µM was the most potent compound against BChE.
1H-pyrazol-5(4H)-one 1, benzaldehyde 2a, and malononitrile 3 in the The data obtained for inhibition of BChE/AChE by target com-
absence of any catalyst in 1,2-dichloroethane (DCE) by heating at re- pounds 5a–l demonstrated that the majority of the compounds had high
flux, and the corresponding pyranopyrazole was produced after a

Table 1
Inhibition of cholinesterases activity and selectivity index of compounds.
Compound R AChE IC50 ± SD (µM)a BChE IC50 ± SD (µM)a Selectivity indexc

5a C6H5 1.23 ± 0.18 36.01 ± 3.97 29.28

5b 4-Cl-C6H4 1.66 ± 0.27 > 68.15 > 41.05
5c 4-Br-C6H4 1.80 ± 0.27 11.64 ± 0.36 6.47
5d 4-(NO2)C6H4 5.80 ± 1.11 2.73 ± 0.16 0.47
5e 4-(MeS)-C6H4 0.058 ± 0.005 > 66.05 > 1138.8
5f 2,4-(Me)2-C6H3 0.29 ± 0.03 39.03 ± 0.17 134.59
5g 2,4-(MeO)2-C6H3 0.26 ± 0.05 31.11 ± 0.15 119.65
5h 2-(Cl)-5-(NO2)-C6H3 1.04 ± 0.48 2.50 ± 0.56 2.40
5i 2-(Cl)-6-(NO2)-C6H3 1.77 ± 0.19 1.84 ± 0.12 1.04
5j C6H5-C6H4 0.044 ± 0.002 > 61.20 > 1390.9
5k 3-Pyridinyl 0.33 ± 0.03 4.26 ± 0.54 12.91
5l 2-(MeO)-1-naphthyl 0.13 ± 0.05 11.35 ± 0.58 87.31
b
Galantamine – 21.82 ± 4.00 40.72 ± 2.85 1.87
b
Tacrine34 – 0.26 ± 0.008 0.05 ± 0.003 0.20

a
IC50 values represent the means ± SD of three parallel measurements (p < 0.05).
b
Reference compound.
c
Selectivity index = (IC50 of BChE)/(IC50 of AChE).

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C. Derabli et al. Bioorganic & Medicinal Chemistry Letters 28 (2018) 2481–2484

Fig. 2. Binding modes of R-5j (white stick) (on the left) and S-5j (white and pink stick) (on the right) in AChE, the hydrogen bonds are drawn in red dashes.

Fig. 3. Binding orientation of R-5i (white and blue stick) (on the left) and S-5i (white and yellow stick) (on the right) in BChE.

selectivity for AChE over BChE. In fact, both compounds 5e and 5j a way to be oriented the biphenyl group inside the pocket. In this po-
showed the highest selectivity towards AChE in the whole series of the sition, the product formed two hydrogen bonds with Ser286 and one
novel compounds. It was noted also that compounds 5f, 5g, and 5l bond with Tyr70. The S configuration was rotated in the opposite way
displayed 134, 119, and 87-fold higher affinities to AChE. According to to direct the biphenyl ring in the same direction as the R isomer. Since it
Taylor et al.31, compounds which gives a good inhibitory activity of was inversely oriented, it formed 3 hydrogen bonds with different
acetylcholinesterase (AChE) are more beneficial for human health. amino acid residues, Arg289 (two hydrogen bonds) and Tyr334 (one
Thanks to the availability of the 3D structures of AChE and BChE, hydrogen bond). The pyrazole ring and the amino group seemed to act
executing the process of molecular docking was approachable. The as the pharmacophores for these interactions. For BChE and 5i inter-
protein structures were downloaded from the Protein Data Bank action, the nitro group which is attached to the phenyl group performed
(PDB).32 The 3D structures of the compounds were introduced in the a role in stabilization of the R enantiomer inside the pocket. It directed
energy-minimized form by MOPAC algorithm. We took in our account by the nitro group to accept two hydrogen bonds from His438. In case
the center of asymmetry which is characteristic for our synthesized li- of S configuration, one proton from the pyrazole ring and the amino
brary, so all the examples were designed in S and R configurations. group protons formed three hydrogen bonds with Asp70, Pro285, and
AutoDock Vina33 was utilized for docking; the method was validated by Ser287 respectively. There was no great difference between the energy
extraction and re-docking of the co-crystallized native ligand. The si- of interaction of the selected poses for the isomers of the 5i and 5j. This
mulated poses of the native ligands were quite similar to the real or- may describe the inhibitory activity of these compounds in spite of the
ientations. expected presence of both isomers in the tested solution inequal per-
The energy of interaction which calculated during the automated centages.
docking of the native ligands was used as a reference to determine the In summary, we have developed a simple, efficient, and practical
stability of the complexes originated from the docking of synthesized one-pot, tandem, sequential, four-component reaction for the synthesis
compounds. The most active hits from the biological study against the of a wide range of Tacrine-pyranopyrazole derivatives via Friedländer
enzymes were picked up to run the modeling study for comprehension condensation of cyclohexanone followed by a 3CR with 3-methyl-1H-
of the mode of interaction between the compounds and the proteins. pyrazol-5(4H)-one, malononitrile, and aryl(or hetero)aldehydes
The most promising inhibitor candidates from the biological analysis without isolation and/or purification of intermediates. This approach
were 5j and 5i toward AChE and BChE respectively (Figs. 2 and 3). allows a diverse range of compounds. These compounds were evaluated
By investigation the poses in which the product was oriented, it was on their in vitro anticholinesterase activity, permitting to identify two
found that 5j was more likely to be fitted on the entrance of the gorge in molecules (5e and 5j) with high potential (respective IC50 values 58

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C. Derabli et al. Bioorganic & Medicinal Chemistry Letters 28 (2018) 2481–2484

and 44 nM) in comparison to Galantamine as reference drug. The

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Niazimbetova ZI, Evans DH. Org Lett. 2002;4:423.
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methyl-1H-pyrazol-5(4H)-one (1, 1 mmol), aryl(or hetero)aldehydes (2, 1 mmol), and
1. Palmer AM. Trends Pharmacol Sci. 2011;32:141. malononitrile (3, 1.1 mmol) was added into adequate volume of dry 1,2-dichloroethane
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6. Watkins PB, Zimmerman HJ, Knapp MJ, Gracon SI, Lewis KW. JAMA. 1994;271:992. mp > 250 °C; IR (KBr) ν max: 3398 and 3240 (NH2), 1582 (C]N) cm−1. 1H NMR (250
7. Thomae D, Kirsch G, Seck P. Synthesis. 2007;1027. MHz, DMSO-d6) δ (ppm) 12.05 (br s, 1H), 8.18 (s, 1H), 7.82 (d, 1H, J = 9.1 Hz),
8. Thomae D, Perspicace E, Hesse S, Kirsch G, Seck P. Tetrahedron. 2008;64:9309. 7.40–7.32 (m, 2H), 5.61 (s, 1H), 5.02 (br s, 2H), 3.84 (s, 2H), 2.39–2.22 (s, 2H), 2.00 (s,
9. Seck P, Thomae D, Kirsch G. J Heterocyclic Chem. 2008;45:853. 3H), 1.74 (s, 4H).13C NMR (62.5 MHz, DMSO-d6) δ (ppm) 158.2, 153.4, 152.2, 142.5,
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 Supplementary Material

A cascade synthesis, in vitro cholinesterases inhibitory activity and docking studies of

novel Tacrine-pyranopyrazole derivatives

Chamseddine Derabli a, Imen Boualia a, Ahmed B. Abdelwahab b,c, Raouf Boulcina a,d*, Chawki Bensouicie,
Gilbert Kirschb, and Abdelmadjid Debachea

a
Laboratory of Synthesis of Molecules with Biological Interest, Frères Mentouri-Constantine University, 25000 Constantine,
Algeria
b
SRSMC, Lorraine University, 1 Boulevard Arago, 57070, France
c
Chemistry of Natural Compounds Department, National Research Centre, El-Behoos St. 33, Dokki, Cairo, 12622, Egypt
d
Faculty of Technology, Batna 2 University, 05000 Batna, Algeria
e
Center for Research in Biotechnology, Constantine, Algeria

Table of Contents:

1. Instruments and materials………………………............................................................................. SI-2

2. Anticholinesterase activity…………………................................................................................... SI-2

3. Statistical analysis………………………........................................................................................ SI-3

4. Molecular modeling………………………..................................................................................... SI-3

5. General procedure for the preparation of compounds 5a-t………………....................................... SI-3

1
6. H and 13 C NMR spectra of new compounds……………………….............................................. SI-8

SI-1
 1. Instruments and materials

Melting points were determined on an Electrothermal capillary fine control apparatus. IR spectra were
recorded on a Shimadzu FT-IR 8201 spectrometer, only significant absorption band frequencies are cited. 1H
and 13C NMR spectra were recorded on a Bruker Avance 400 instrument at 400 and 100 MHz respectively
and Brüker advance DPX 250 (250 MHz for the 1H, 62.5 for the 13
C), in CDCl3 or DMSO-d6. Chemical
shifts (δ) are given in part per million downfield from TMS as an internal standard and J values in Hz. The
chemicals were used as obtained commercially. High Resolution Mass spectra were recorded with a
MicroTof-Q 98.
The measurements and calculations of the activity results were evaluated by using bioactivity measurements
were carried out on a 96-well microplate reader, Perkin Elmer Multimode Plate Reader EnSpire at National
Center of biotechnology Research. Acetylcholinesterase from electric eel (AChE, Type-VI-S, EC 3.1.1.7,
827,84 U/mg, Sigma), butyrylcholinesterase from horseserum (BChE, EC 3.1.1.8, 7,8 U/mg, Sigma),
acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB),
Galantamine were obtained from Sigma Chemical Co.(Sigma-Aldrich GmbH, Stern-heim, Germany),All
other chemicals and solvents were of analytical grade.

2. Anticholinesterase activity

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity was measured, by the
spectrophotometric method developed by Ellman, G.L., and al.[1] Briefly, 150 µl of 100 mM sodium
phosphate buffer (pH 8.0), 10 µl of sample solution dissolved in methanol atdifferent concentrations and 20
µl AChE (5.32 x10-3 U) or BChE (6.85 x10-3U) solution were mixed and incubated for 15 min at 25ºC, and
10 µl of 0.5 mM DTNB[5,5´-dithio-bis(2-nitrobenzoic) acid] were added. The reaction was then initiated by
the addition of 10 µl of acetylthiocholine iodide (0.71 mM) or butyrylthiocholine chloride (0.2 mM). The
hydrolysis of these substrates were monitored spectrophotometrically by the formation of yellow 5-thio-2-
nitrobenzoate anion, as the result of the reaction of DTNB with thiocholine, released by the enzymatic
hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride, respectively, at a wavelength of 412
nm, every 5 min for 15 min, utilising a 96-well microplate reader (Perkin Elmer Multimode Plate Reader
EnSpire, USA) in triplicate experiments. Galanthamine was used as reference compound. The results were
given as 50% inhibition concentration (IC50) and the percentage of inhibition of AChE or BChE was
determined by comparison of reaction rates of samples relative to blank sample (methanol in phosphate
buffer, pH 8) using the formula

E−S
Inhibition of AChE or BChE % = × 100
E

Where E is the activity of enzyme without test sample, and S is the activity of enzyme with test sample.
SI-2
 3. Statistical analysis

All data on anticholinesterase activity tests were the average of triplicate analyses. The data were recorded as mean ±
standard deviation. Analysis of variance was performed by ANOVA procedures. Significant differences between
means were determined by student’s-t test, p values <0.05 were regarded as significant

4. Molecular modeling

The program AutodockVina 1.1.11was used for docking simulations. The compounds were firstly designed
in 2D by ChemBioDraw Ultra 14.0andtransformed to 3D by ChemBio3D Ultra 14.0. The most stable
conformer was obtained by MOPAC algorithm which plugged with VEGA ZZ 3.0.5 software2. This last was
used for the preparation of the crystal structure of acetylcholinesterase (1EVE)3and butyrylcholinesterase
(5DYW)4 which were downloaded from Protein Data Bank (PDB). Docking parameters have been adjusted
to correspond the co-crystallized molecules as the following: for acetylcholinesterase (1EVE)grid box size:
25 *25 *25 Å, the center of box: x = 2.5, y = 63.2, z = 67.0, for butyrylcholinesterase (5DYW) grid box size:
20 *20 *20 Å, the center of box: x = -3.9, y = 9.7, z = -13.2. The visualization of the protein/compounds
interaction was performed by Pymol software.5All hydrogen bonds which were shown by the visualization
software and not fitting the stable hydrogen bonds criteria was excluded.

5. General procedure for the preparation of compounds 5a-t

In a 50 mL round bottom flask, a mixture of 3-methyl-1H-pyrazol-5(4H)-one (1, 1 mmol), aryl(or

hetero)aldehydes (2, 1 mmol), and malononitrile (3, 1.1 mmol) was added into adequate volume of dry 1,2-
dichloroethane (DCE) and stirred at reflux for 3-4 hours until completion of the reaction (monitored by
TLC). Then, Aluminum chloride (1.5 mmol) and cyclohexanone (4, 1.5 mmol) were added into the reaction.
The mixture was continuously stirred for 24 hours (monitored by TLC) at constant temperature. After
completion, water was added and the mixture was basified with 10% sodium hydroxide solution to pH = 8-
9. After stirring for 30 min, the precipitate was filtered and washed with water. Purification by column
chromatography on silica gel (ethyl acetate/methanol = 9:1, v/v) afforded the title compounds 5a-t.

Table1. Synthesis of Tacrine–Pyranopyrazole derivatives

Compound R Yielda (%)

5a C6H5 92
5b 4-Cl-C6H4 92
5c 4-Br-C6H4 91
5d 4-(NO2)-C6H4 80
5e 4-(MeS)-C6H4 94
5f 2,4-(Me)2-C6H3 91
5g 2,4-(MeO)2-C6H3 90
5h 2-(Cl)-5-(NO2)-C6H3 92
5i 2-(Cl)-6-(NO2)-C6H3 80
5j C6H5-C6H4 94
SI-3
 5k 3-Pyridinyl 93
5l 2-(MeO)-1-naphthyl 75
5m 4-F-C6H4 90
5n 3-(NO2)-C6H4 80
5o 4-(Me)2NC6H4 75
5p 4-Me-C6H4 93
5q 4-C2H5-C6H4 91
5r 2-(MeO)-C6H4 90
5s 4-(MeO)-C6H4 93
5t 2-(Cl)-6-(OMe)-3-quinolyl 70
a
Isolated yields.

3-Methyl-4-phenylpyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (5a):
Yield 92%;light brown solid; mp > 250 °C; IR (KBr) ν max: 3405 and 3285 (NH2), 1575 (C=N)cm-1.1H NMR
(250 MHz, DMSO-d6) δ (ppm) 11.90 (br s, 1H), 7.34-7.13 (m, 5H), 5.35 (br s, 2H), 5.19 (s, 1H), 2.60-2.52
13
(s, 2H), 2.26 (s, 2H), 2.01 (s, 3H), 1.72 (s, 4H). C NMR (62.5 MHz, DMSO-d6) δ (ppm) 155.8, 152.5,
152.2, 145.2, 135.0, 128.5, 127.4, 126.4, 118.1, 112.1, 99.9, 98.6, 34.3, 32.1, 23.0, 22.4, 22.2, 9.9. HRMS
(ESI+) m/z 333.1723 [M+H]+., calcd for C20H21N4O: 333.1715.

4-(4-Chlorophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5b):
Yield 92%; white solid; mp > 250 °C;IR (KBr) ν max: 3402 and 3232 (NH2), 1585 (C=N)cm-1.1H NMR (250
MHz, DMSO-d6) δ (ppm) 11.83 (br s, 1H), 7.30 (m, 4H), 5.21 (s, 1H), 5.13 (br s, 2H), 2.65 (s, 2H), 2.40-
13
2.18 (s, 2H), 2.01 (s, 3H), 1.77 (s, 4H). C NMR (62.5 MHz, DMSO-d6) δ (ppm) 155.4, 152.4, 152.0,
143.4, 135.0, 131.1, 128.9, 128.1, 112.1, 98.9, 97.9, 34.1, 31.9, 30.5, 22.7, 22.2, 22.0, 9.8. HRMS (ESI+)
m/z 367.1320 [M+H]+., calcd for C20H20ClN4O: 367.1326.

4-(4-Bromophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5c)
Yield 91%; brown solid;mp 238-240 °C; IR (KBr) ν max:3391 and 3225 (NH2), 1581(C=N) cm-1.1H NMR
(250 MHz, DMSO-d6) δ (ppm) 12.0 (br s, 1H), 7.44 (d, 2H, J = 8.4 Hz), 7.21 (d, 2H, J = 8.4 Hz), 5.44 (br s,
2H), 5.25 (s, 1H), 2.58 (s, 2H), 2.35-2.21 (m, 2H), 2.00 (s, 3H),1.66 (s, 4H).13C NMR (62.5 MHz, DMSO-
d6) δ (ppm) 155.7, 152.6, 152.1, 144.6, 131.3, 129.6, 119.3, 112.1, 99.4, 98.2, 33.5, 32.1, 27.4, 23.0, 22.4,
22.2, 9.9. HRMS (ESI+) m/z 411.0821 [M+H]+., calcd for C20H20BrN4O: 411.0820.

4-(4-Nitrophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5d)

SI-4
Yield 80%;white solid;mp 242-244 °C; IR (KBr) ν max: 3422 and 3365 (NH2), 1585 (C=N) cm-1.1H NMR
(250 MHz, DMSO-d6) δ (ppm) 11.94 (br s, 1H),8.07 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 5.39 (br s,
2H), 5.29 (s, 1H), 2.61 (s, 2H), 2.36-2.10 (m, 2H), 2.01 (s, 3H), 1.72 (s, 4H). 13C NMR (62.5 MHz, DMSO-
d6) δ (ppm) 154.1, 151.4, 151.0, 150.6, 144.4, 133.8, 127.0, 121.9, 118.2, 110.7, 97.0, 96.0, 32.8, 30.6, 21.4,
20.8, 20.7, 8.4. HRMS (ESI+) m/z 378.1583 [M+H]+., calcd for C20H20N5O3: 378.1566.

3-Methyl-4-(4-(methylthio)phenyl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5e)
Yield 94%;white solid; mp > 250 °C; IR (KBr) ν max:3481 and 3405 (NH2), 1572 (C=N) cm-1.1H NMR (400
MHz, DMSO-d6) δ (ppm) 11.88 (br s, 1H), 7.19 (dd, J=8.4, 2.0 Hz, 2H), 7.13 (dd, J=8.4, 2.0 Hz, 2H), 5.33
(br s, 2H), 5.17 (s, 1H), 2.58 (s, 2H), 2.41 (s, 3H), 2.30 (d, J=16.4 Hz, 2H), 2.09 (d, J=16.4 Hz, 2H), 2.01 (s,
3H), 1.71 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 155.6, 152.3, 152.0, 141.8, 135.7, 135.6, 128.2,
128.0, 127.9, 126.1, 112.0, 99.6, 98.4, 33.6, 32.0, 22.9, 22.3, 22.1, 9.8. HRMS (ESI+) m/z 379.1601
[M+H]+., calcd for C21H23N4OS: 379.1593.

4-(2,4-Dimethylphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5f)
Yield 91%;yellow solid; mp 216-218 °C; IR (KBr) ν max: 3471 and 3292 (NH2), 1580(C=N) cm-1.1H NMR
(400 MHz, DMSO-d6) δ (ppm) 11.85 (br s, 1H), 7.16 (d, J=7.2 Hz, 1H), 6.93 (d, J=8.0 Hz, 1H), 6.91 (s,
1H), 5.19 (s, 1H), 4.91 (br s, 2H), 2.58 (s, 2H), 2.33-2.23 (m, 4H), 2.28 (s, 6H), 1.86 (s, 3H), 1.72 (s, 4H).
13
C NMR (100 MHz, DMSO-d6) δ (ppm) 155.4, 152.3, 152.1, 138.6, 135.6, 134.5, 131.9, 129.4, 127.0,
112.2, 98.1, 59.7, 33.6, 31.9, 22.7, 22.3, 22.1, 20.4, 18.6, 14.1, 9.9. HRMS (ESI+) m/z 361.2026 [M+H]+.,
calcd for C22H25N4O: 361.2028.

4-(2,4-Dimethoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5g)
Yield 90%;light brown solid; mp 210-212 °C; IR (KBr) ν max:3480 and 3275 (NH2), 1578(C=N) cm-1.1H
NMR (400 MHz, DMSO-d6) δ (ppm) 11.84 (br s, 1H), 6.70 (d, J=8.4 Hz, 1H), 6.58 (d, J=2.4 Hz, 1H), 6.70
(dd, J=8.4, 2.4 Hz, 1H), 5.22 (s, 1H), 5.13 (br s, 2H), 3.90 (s, 3H), 3.70 (s, 3H), 2.56 (s, 2H), 2.32-2.21 (m,
4H), 1.88 (s, 3H), 1.70 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 155.7, 153.1, 152.1, 146.6, 142.7,
138.2, 135.5, 131.5, 125.4, 123.1, 112.5, 96.8, 96.6, 48.5, 33.1, 31.9, 22.8, 22.2, 22.0, 9.8. HRMS (ESI+)
m/z 393.1935 [M+H]+., calcd for C22H25N4O3: 393.1927.

4-(2-Chloro-5-nitrophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5h)

SI-5
Yield 92%;light brown solid; mp 240-242 °C; IR (KBr) ν max:3451 and 3370 (NH2), 1583 (C=N) cm-1.1H
NMR (400 MHz, DMSO-d6) δ (ppm) 12.01 (br s, 1H), 8.31 (s, 1H), 8.05 (dd, J=8.8, 2.8 Hz, 1H), 7.68 (d,
J=8.8 Hz, 1H), 5.69 (s, 1H), 5.23 (br s, 2H), 2.59 (s, 2H), 2.33 (d, J=16.4 Hz, 2H), 2.21 (d, J=16.4 Hz, 2H),
1.99 (s, 3H), 1.68 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 155.7, 153.1, 152.1, 146.6, 142.7, 138.2,
135.5, 131.5, 125.4, 123.1, 112.5, 96.7, 96.6, 48.5, 33.1, 31.9, 22.8, 22.2, 22.0, 9.8. HRMS (ESI+) m/z
434.1014 [M+Na]+., calcd for C20H18ClN5NaO3: 434.0996.

4-(2-Chloro-6-nitrophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5i)
Yield 80%;brown solid; mp 222-224 °C; IR (KBr) ν max: 3481 and 3395 (NH2), 1590 (C=N) cm-1.1H NMR
(400 MHz, DMSO-d6) δ (ppm) 12.05 (br s, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.56 (t,
J=8.0 Hz, 1H), 5.59 (s, 1H), 5.55 (br s, 2H), 2.68 (s, 2H), 2.31 (d, J=16.4 Hz, 2H), 2.19 (d, J=16.4 Hz, 2H),
1.83 (s, 3H), 1.71 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 155.6, 152.9, 151.6, 146.4, 142.5, 137.9,
136.2, 131.2, 129.8, 122.9, 112.2, 94.1, 60.7, 31.9, 30.3, 29.2, 22.8, 22.2, 22.0, 9.4. HRMS (ESI+) m/z
434.0984 [M+Na]+., calcd for C20H18ClN5O3: 434.0996.

4-(4-Biphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (5j)
Yield 94%;yellow solid; mp 228-230 °C; IR (KBr) ν max:3491 and 3402 (NH2), 1584(C=N) cm-1.1H NMR
(250 MHz, DMSO-d6) δ (ppm) 11.94 (br s, 1H), 7.63-7.54 (m, 4H), 7.47-7.33 (m, 5H) 5.33 (br s, 2H), 5.27
(s, 1H), 2.51 (s, 2H), 2.34-2.27 (m, 2H), 2.08 (s, 3H), 1.75 (s, 4H).13C NMR (62.5 MHz, DMSO-d6) δ (ppm)
156.0, 152.8, 152.2, 144.5, 140.1, 138.6, 135.3, 129.0, 128.0, 127.4, 127.0, 126.7, 112.3, 99.8, 98.7, 34.2,
32.2, 23.1, 22.5, 22.3, 9.9. HRMS (ESI+) m/z 409.2023 [M+H]+., calcd for C26H25N4O: 409,2028.

3-Methyl-4-(pyridin-3-yl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (5k)
Yield 93%;light brown solid; mp > 250 °C; IR (KBr) ν max: 3421 and 3365 (NH2), 1585 (C=N) cm-1.1H
NMR (400 MHz, DMSO-d6) δ (ppm) 12.01 (br s, 1H), 8.59 (d, J=2.0 Hz, 1H), 8.33 (dd, J=4.8, 1.6 Hz, 1H),
7.48 (dd, J=8.0, 2.0 Hz, 1H), 7.24 (dd, J=8.0, 4.8 Hz, 1H), 5.51 (br s, 2H), 5.32 (s, 1H), 2.58 (s, 2H), 2.34
(d, J=16.4 Hz, 2H), 2.18 (d, J=16.4 Hz, 2H), 2.01 (s, 3H), 1.71-1.69 (s, 4H). 13C NMR (100 MHz, DMSO-
d6) δ (ppm) 155.6, 152.6, 151.9, 140.4, 134.6, 123.7, 112.1, 99.1, 97.7, 79.2, 32.0, 31.4, 22.9, 22.3, 22.0, 9.7.
HRMS (ESI+) m/z 356.1504 [M+Na]+., calcd for C19H19N5NaO: 356.1487.

4-(2-Methoxynaphthalen-1-yl)-3-methyl-2,4,6,7,8,9hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-
5-amine (5l)
Yield 75%;light brown solid; mp > 250 °C; IR (KBr) ν max: 3331 and 3275 (NH2), 1587 (C=N) cm-1.1H
NMR (400 MHz, DMSO-d6) δ (ppm) 11.83 (br s, 1H), 7.91 (d, J=9.2 Hz, 1H), 7.82-7.80 (m, 1H), 7.64-7.61
(m, 1H), 7.59 (d, J=9.2 Hz, 1H), 7.22-7.19 (m, 2H), 6.09 (s, 1H), 5.18 (br s, 2H), 4.16 (s, 3H), 2.57 (s, 2H),
SI-6
2.22-2.11 (m, 4H), 1.66 (s, 3H), 1.64 (s, 4H).13C NMR (100 MHz, DMSO-d6) δ (ppm) 155.5, 153.1, 152.2,
152.1, 141.7, 137.2, 135.8, 131.4, 130.0, 129.7, 128.9, 126.1, 123.1, 121.5, 112.8, 111.9, 98.2, 98.0, 56.9,
31.8, 25.8, 22.6, 22.2, 22.0, 9.2. HRMS (ESI+) m/z 413.1953 [M+H]+, calcd for C25H24N4O2: 413.1978.

4-(4-Fluorophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5m):
Yield 90%;white solid; mp > 250 °C (lit.: > 250°C)6; IR (KBr) ν max: 3510 and 3395 (NH2), 1588 (C=N) cm-
11
. H NMR (400 MHz, DMSO-d6) δ (ppm) 11.55 (br s, 1H), 7.20-7.17 (m, 2H), 6.91-6.86 (m, 2H), 4.92 (s,
1H), 438 (br s, 2H), 2.66 (s, 2H), 2.26 (d, J=16.0 Hz, 2H), 2.19 (d, J=16.0 Hz, 2H), 1.94 (s, 3H), 1.73 (s,
4H). 13C NMR (100 MHz, DMSO-d6) δ 155.9, 155.4, 153.0, 151.6, 139.9, 139.8, 135.4, 128.7, 128.6, 115.2,
115.0, 112.2, 98.6, 98.0, 35.1, 32.0, 22.5, 22.1, 22.0, 9.9. HRMS (ESI+) m/z 351.1629 [M+H]+., calcd for
C20H20FN4O: 351.1621.

3-Methyl-4-(3-nitrophenyl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5n)
Yield 80%;yellow solid; mp > 250 °C (lit.: > 250°C)6;IR (KBr) ν max:3420 and 3370 (NH2), 1583 (C=N) cm-
11
. H NMR (400 MHz, DMSO-d6) δ (ppm) 12.01 (br s, 1H),8.11 (s, 1H),7.96 (dd, J=8.0, 1.2 Hz, 1H), 7.00
(d, J=8.0 Hz, 1H), 7.39 (t, J=8.0 Hz, 1H), 5.17 (s, 1H), 4.69 (br s, 2H), 2.50 (s, 2H), 2.27 (d, J=16.0 Hz, 2H),
13
2.17 (d, J=16.0 Hz, 2H), 1.96 (s, 3H), 1.73 (s, 4H). C NMR (100 MHz, DMSO-d6) δ (ppm) 154.2, 151.4,
150.7, 146.2, 133.7, 128.0, 126.0, 120.5, 119.7, 110.8, 97.4, 96.2, 32.5, 30.7, 29.6, 21.4, 20.9, 20.7, 8.4.
HRMS (ESI+) m/z 378.1561 [M+H]+., calcd for C20H20N5O3: 378.1566.

4-(4-Aminodimethylphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5o)
Yield 75%; green solid; mp > 250 °C; IR (KBr) ν max: 3402 and 3320 (NH2), 1587 (C=N) cm-1.1H NMR
(250 MHz, DMSO-d6) δ (ppm) 11.60 (br s, 1H), 7.02 (d, J = 8.4 Hz, 2H), 6.54 (d, J = 8.4 Hz, 2H), 4.79 (s,
1H), 4.60 (br s, 2H),2.81 (s, 6H), 2.63 (s, 2H), 2.22 (s, 2H), 1.96 (s, 3H), 1.71 (s, 4H). 13C NMR (62.5 MHz,
DMSO-d6) δ (ppm) 154.7, 151.8, 151.3, 148.2, 134.8, 130.7, 127.08, 119.0, 111.6, 111.4, 98.7, 97.9, 34.2,
31.1, 21.8, 21.5, 21.3, 9.3. HRMS (ESI+) m/z376.2153[M+H]+., calcd for C22H26N5O3: 376.2137.

3-Methyl-4-(p-tolyl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (5p)
Yield 93%; white solid; mp > 250 °C (lit.: > 250°C)6; IR (KBr) ν max:3391 and 3195 (NH2), 1582 (C=N)cm-
11
. H NMR (400 MHz, DMSO-d6) δ (ppm) 11.61 (br s, 1H),7.08 (d, J=5.6 Hz, 2H), 7.00 (d, J=5.6 Hz, 2H),
5.07 (br s, 2H), 4.95 (s, 1H), 2.64 (s, 2H), 2.21 (s, 3H), 2.26- 2.20 (m, 2H),1.96 (s, 3H), 1.73 (s, 4H).13C
NMR (100 MHz, DMSO-d6) δ (ppm) 154.6, 153.0, 152.1, 140.6, 135.7, 134.9, 129.0, 127.0, 112.2, 99.0,

SI-7
98.3, 34.8, 30.8, 22.4, 21.8, 21.7, 20.5, 9.9. HRMS (ESI+) m/z 347.1888 [M+H]+., calcd for C21H23N4O:
347.1872.

4-(4-Ethylphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5q)
Yield 91%;white solid; mp > 250 °C; IR (KBr) ν max:3423 and 3268 (NH2), 1579 (C=N) cm-1.1H NMR (250
MHz, DMSO-d6) δ (ppm) 11.59 (br s, 1H), 7.12-7.03 (m, 4H), 5.40 (s, 2H), 4.49 (s, 1H), 2.68 (s, 2H), 2.54
(s, 2H) 2.27-2.23 (m, 2H), 1.95 (s, 3H),1.74 (s, 4H), 1.12 (t, 3H). 13C NMR (62.5 MHz, DMSO-d6) δ (ppm)
155.4, 152.4, 152.0, 142.6, 139.7, 131.1, 127.9, 127.0, 112.5, 98.4, 97.9, 27.8, 22.1, 21.4, 21.2, 14.9, 9.9.
HRMS (ESI+) m/z 361.2023 [M+H]+., calcd for C22H25N4O: 361.2028.

4-(2-Methoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5r)
Yield 90%;white solid;mp > 250 °C (lit.: > 250°C)6; IR (KBr) ν max: 3441 and 3376 (NH2), 1585(C=N) cm-
11
. H NMR (400 MHz, DMSO-d6) δ (ppm) 11.65 (br s, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.91 (d, J = 8.0 Hz,
1H), 6.83 (d, J = 7.6 Hz, 1H), 6.75 (t, J = 7.6 Hz, 1H), 5.33 (s, 1H), 4.96 (br s, 2H), 3.92 (s, 3H), 2.60 (s,
13
2H), 2.28 (d, J=16.0 Hz, 2H), 2.19 (d, J=16.0 Hz, 2H), 1.89 (s, 3H), 1.71 (s, 4H). C NMR (100 MHz,
DMSO-d6) δ (ppm) 156.5, 155.4, 154.6, 151.8, 134.9, 132.1, 129.4, 127.4, 121.3, 111.7, 110.1, 99.3, 98.7,
55.5, 31.7, 26.6, 22.6, 22.2, 22.0, 9.3. HRMS (ESI+) m/z 363.1829 [M+H]+., calcd for C21H22N4O2:
363.1821.

4-(4-Methoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine
(5s)
Yield 93%; white solid; mp > 250 °C (lit.: > 250°C)6; IR (KBr) ν max :3490 and 3405 (NH2), 1575 (C=N) cm-
11
. H NMR (250 MHz, DMSO-d6) δ (ppm) 11.90 (br s, 1H), 7.07 (d, J = 8.0 Hz, 2H), 6.74 (d, J = 8.0 Hz,
2H), 5.35 (br s, 2H), 5.13 (s, 1H), 3.36 (s, 3H), 2.58 (s, 2H), 2.25 (d, J=16.0 Hz, 2H), 2.12 (d, J=16.0 Hz,
2H), 1.99 (s, 3H), 1.62 (s, 4H).13C NMR (62.5 MHz, DMSO-d6) δ (ppm) 157.6, 156.4, 155.7, 152.3, 137.2,
134.9, 128.4, 119.4, 113.8, 112.0, 100.1, 98.7, 33.4, 32.1, 30.8, 23.0, 22.4, 22.2, 9.9. HRMS (ESI+) m/z
363.1834 [M+H]+., calcd for C21H23N4O2: 363.1821.

4-(6-Methoxy-2-chloroquinoline)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (5t)
Yield 70%;light brown solid; mp > 250 °C; IR (KBr) ν max: 3398 and 3240 (NH2), 1582 (C=N) cm-1.1H
NMR (250 MHz, DMSO-d6) δ (ppm) 12.05 (br s, 1H), 8.18 (s, 1H), 7.82 (d, 1H, J = 9.1 Hz), 7.40-7.32 (m,
2H), 5.61 (s, 1H), 5.02 (br s, 2H), 3.84 (s, 2H), 2.39-2.22 (s, 2H), 2.00 (s, 3H), 1.74 (s, 4H).13C NMR (62.5

SI-8
MHz, DMSO-d6) δ (ppm) 158.2, 153.4, 152.2, 142.5, 138.4, 136.0, 117.9, 129.1, 123.5, 119.8, 112.8, 105.3,
55.6, 32.2, 23.0, 22.2, 22.5, 9.9. HRMS (ESI+) m/z 448.1552 [M+H]+., calcd for C24H22ClN5O2: 448.1540.

1
6. H and 13 C NMR spectra of new compounds

3-Methyl-4-phenylpyrazolo[4’,3’:5,6]pyrano[2,3-b]quinolin-5-amine (5a)

SI-9
4-(4-Chlorophenyl)-3-méthyl-2,4,6,7,8,9-hexahydropyrazolo [4’,3’:5,6]pyrano[2,3-b]quinolin-5-amine
(5c)

SI-10
4-(4-Bromophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4’,3’:5,6]pyrano[2,3-b]quinolin-5-amine
(5d)

SI-11
4-(4-Nitrophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo [4’,3’:5,6]pyrano[2,3-b]quinolin-5-amine
(5f)

SI-12
4-(4-Aminodimethylphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo [4’,3’:5,6]- pyrano[2,3-
b]quinolin-5-amine (5g)

SI-13
4-(4-Ethylphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo [4’,3’:5,6]pyrano[2,3-b]quinolin-5-amine
(5i)
SI-14
SI-15
3-Methyl-4-(4-(methylthio)phenyl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5l)

SI-16
4-(2,4-Dimethylphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5m)

SI-17
4-(2,4-Dimethoxyphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5n)

SI-18
4-(2-Chloro-5-nitrophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5o)

SI-19
4-(2-Chloro-6-nitrophenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-
amine (5p)

SI-20
4-(4-Biphenyl)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4’,3’:5,6]pyrano[2,3-b]quinolin-5-amine (5q)

SI-21
3-Methyl-4-(pyridin-3-yl)-2,4,6,7,8,9-hexahydropyrazolo[4',3':5,6]pyrano[2,3-b]quinolin-5-amine (5r)

SI-22
4-(2-Methoxynaphthalen-1-yl)-3-methyl-2,4,6,7,8,9hexahydropyrazolo[4',3':5,6]pyrano[2,3-
b]quinolin-5-amine (5s)

SI-23
4-(6-Methoxy-2-chloroquinoline)-3-methyl-2,4,6,7,8,9-hexahydropyrazolo[4’,3’:5,6]pyrano[2,3-
b]quinolin-5-amine (5t)

SI-24
 Table 2. Acetyl cholinesterase inhibitory activity

Extracts Acetylcholinesterase inhibitory activity

0.391 µg 0.781 µg 1.563 µg 3.125 µg 6.25 µg 12.5 µg 25 µg IC50µg/mL

5o 46.46±1.33 62.19±3.82 81.15±1.30 90.00±2.05 91.15±1.00 94.90±0.36 96.88±1.90 0.43±0.20

5p 30.10±0.88 59.17±0.95 77.08±2.43 79.38±2.65 88.13±0.94 95.31±0.54 96.98±0.72 0.73±0.08

5a 48.27±0.36 71.46±0.56 83.60±0.38 87.42±1.14 93.20±1.59 96.48±1.32 98.61±0.33 0.41±0.06

5f 30.95±0.63 35.18±1.19 45.56±0.41 55.64±1.04 74.66±2.91 77.18±3.16 87.78±2.13 2.19±0.42

5d 28.03±1.08 53.57±0.69 68.16±0.39 79.79±0.51 82.23±2.02 91.06±2.03 97.79±0.63 0.74±0.11

5c 32.68±1.10 60.06±1.43 70.83±0.17 81.63±4.67 87.30±0.56 92.78±1.19 97.08±1.91 0.61±0.10

Galantamineb 35,9±2,28 43,77±0.00 68,50±0,31 80,69 ±0,41 85,78 ±1,63 91,80 ±0,20 94,77 ±0,34 6.27±1.15

Extracts Acetylcholinesterase inhibitory activity

0.006 µg 0.012 µg 0.024 µg 0.049 µg 0.098 µg 0.195 µg 0.391 µg IC50µg/mL

5s 12.56±0.36 16.05±0.69 33.03±0.38 46.62±0.014 60.55±0.54 75.40±0.99 88.51±0.87 0.055 ±0.02

5r 6.61±1.32 13.14±0.82 15.49±0.56 29.05±0.72 44.82±0.36 62.39±0.30 66.76±0.49 0.111 ±0.01

5q 34.37±0.59 43.00±0.88 59.09±0.81 71.19±0.06 81.97±0.63 87.22±0.80 89.67±0.54 0.018 ±0.001

5l 30.36±0.43 36.90±0.61 59.67±0.48 67.64±0.44 80.91±1.04 86.80±1.16 91.30±0.44 0.022 ±0.002

Extracts Acetylcholinesterase inhibitory activity

0.024 µg 0.049 µg 0.098 µg 0.195 µg 0.391 µg 0.781 µg 1.563 µg IC50 µg/mL

5m 28.92±0.71 41.05±0.37 47.18±0.10 64.76±0.42 82.19±0.57 85.28±0.81 92.08±0.72 0.106±0.01

5n 24.16±0.51 32.36±0.57 48.96±0.66 62.89±0.27 70.69±0.52 84.08±0.71 89.22±0.68 0.102±0.02

Galantamineb 35,9±2,28 43,77±0.00 68,50±0,31 80,69 ±0,41 85,78 ±1,63 91,80 ±0,20 94,77 ±0,34 6.27±1.15

SI-25
 Table 3. Butyrylcholinesterase inhibitory activity

Extracts Butyrylcholinesterase inhibitory activity

0.391 µg 0.781 µg 1.563 µg 3.125 µg 6.25 µg 12.5 µg 25 µg IC50 µg/mL

5o 5.53±1.91 10.19±2.36 13.88±1.85 21.55±1.08 35.58±1.82 54.24±0.86 71.93±1.43 12.21±0.23

5m NA NA NA 13.33±0.37 44.75±0.43 49.71±0.67 51.85±0.21 14.07±0.06

5p 37.64±0.78 52.09±0.80 66.21±4.23 85.34±1.35 86.50±1.46 94.27±2.86 98.71±1.54 0.76 ±0.05

5n NA 17.77±1.50 37.09±3.64 43.03±0.90 55.77±0.98 62.82±1.97 80.78±1.75 5.41±0.28

5s 2.86±0.84 7.31±0.12 25.10±0.07 47.89±0.55 51.86±0.46 62.98±0.92 69.97±0.32 4.68±0.24

5r 18.21±0.82 41.59±0.57 53.90±0.56 57.13±0.81 72.05±0.71 74.25±0.87 84.78±1.03 1.42±0.18

5q NA NA NA NA 11.75±0.12 14.05±0.84 20.65±0.11 >25

5l 0.11±0.71 10.05±0.18 23.69±0.40 30.96±0.23 36.60±0.44 44.46±0.10 46.68±0.55 >25

5a 5.38±0.94 10.73±0.90 13.55±1.72 23.86±2.42 30.70±0.60 52.46±0.40 72.88±3.60 11.97 ±1.32

5f 24.89±1.58 37.90±1.12 66.85±1.23 75.14±0.62 90.27±3.54 92.58±2.26 93.21±1.65 1.03 ±0.06

5d 13.09±1.79 17.35±1.22 37.88±0.74 43.06±0.92 58.50±0.53 60.46±1.07 69.90±0.49 4.79 ±0.15

5c 20.75±0.66 22.99±2.56 23.45±1.87 27.94±3.72 31.68±2.07 45.84±3.98 46.88±1.28 >25

Galantamineb 11,13±0,01 21,02±0,58 55,22±1,75 63,87±3,04 78,85±0,87 85,71±2,04 96,98± 2,38 11.70±0.82

References:

[1] Ellman, G.L., Courtney, K.D., Andres, V., Featherston, R.M., 1961. A new and rapidcolorimetric
determination of acetylcholinesterase activity.Biochem.Pharmacol. 7, 88–95.

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