Design, Synthesis and Bioevaluation of Two Series of 3-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]quinazolin-4(3H)-ones and N-(1-Benzylpiperidin-4-yl)quinazolin-4-amines

Article abstract of DOI:10.1002/cbdv.202000290

Two series of 3-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]quinazolin-4(3H)-ones and N-(1-benzylpiperidin-4-yl)quinazolin-4-amines were designed initially as potential acetylcholine esterase inhibitors. Biological evaluation demonstrated that N-(1-benzylpiperidin-4-yl)quinazolin-4-amines significantly inhibited AChE activity. Especially, two compounds of them were found to be the most potent with relative AChE inhibition percentages of 87 percent in comparison to donepezil. The docking studies with AChE showed similar interactions between donepezil and four derivatives. N-(1-Benzylpiperidin-4-yl)quinazolin-4-amines also exhibited significant DPPH scavenging effects. The two series of compound also exerted moderate to good cytotoxicity against three human cancer cell lines, including SW620 (human colon cancer), PC-3 (prostate cancer), and NCI?H23 (lung cancer), with 3-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]quinazolin-4(3H)-one being the most cytotoxic agent. 3-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]quinazolin-4(3H)-one significantly induced early apoptosis and arrested the SW620 cells at G2/M phase. From this study, two compounds of N-(1-benzylpiperidin-4-yl)quinazolin-4-amines could serve as new leads for further design and AChE inhibitors, while 3-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]quinazolin-4(3H)-one could serve as a new lead for the design and development of more potent anticancer agents.

Full text of DOI:10.1002/cbdv.202000290

Title: Design, Synthesis and Bioevaluation of Two Series of 3-((1-
Benzyl-1H-1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-ones and N-
(1-benzylpiperidin-4-yl)quinazolin-4-amines
Authors: Ta Thu Lan, Duong Tien Anh, Hai Pham-The, Do Thi Mai
Dung, Eun Jae Park, Sun Dong Jang, Joo Hee Kwon, Jong
Soon Kang, Nguyen Thi Thuan, Sang-Bae Han, and Nguyen-
Hai Nam
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.202000290
Link to VoR: https://doi.org/10.1002/cbdv.202000290

10.1002/cbdv.202000290
Chemistry & Biodiversity
Chem. Biodiversity
Design, Synthesis and Bioevaluation of Two Series of 3-((1-Benzyl-1H-
1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-ones and N-(1-
benzylpiperidin-4-yl)quinazolin-4-amines
Ta Thu Lan,a Duong Tien Anh,aHai Pham-The,aDo Thi Mai Dung,a Eun Jae Park,bSun Dong Jang,bJoo
Hee Kwon,cJong Soon Kang,cNguyen Thi Thuan,*,a Sang-Bae Han,*,b Nguyen-Hai Nam*,a
aHanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, 10000, Vietnam
cCollege of Pharmacy, Chungbuk National University, 194-31, Osongsaengmyung-1, Heungdeok, Cheongju, Chungbuk, 28160, Republic of
Korea
dBio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Chungbuk, 28116, Republic of Korea
*Corresponding authors: Tel.: +84-4-39330531; Fax: +84-4-39332332; Emails: namnh@hup.edu.vn (N.H. Nam).
Two series of 3-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-ones (4a-j) and N-(1-benzylpiperidin-4-yl)quinazolin-4-amines
(6a-j) were designed initially as potential acetylcholine esterase inhibitors. Biological evaluation demonstrated that series 6a-j
significantly inhibited AChE activity. Especially compounds 6cand 6e were found to be the most potent with relative AChE inhibition
percentages of 87% in comparison to donepezil. The docking studies with AChE showed similar interactions between Donepezil and these
four derivatives. Compounds 6a-j also exhibited significant DPPH scavenging effects. Two compound series also exerted moderate to
good cytotoxicity againsts three human cancer cell lines, including SW620 (human colon cancer), PC-3 (prostate cancer), and NCI-H23
(lung cancer), with compound 4a being the most cytotoxic agent. Compound 4a significantly induced early apoptosis and arrested the
SW620 cells at G2/M phase. From this study, compounds 6c and 6e could serve as new leads for further design and AChE inhibitors, while
compound 4a could serve as a new lead for the design and development of more potent anticancer agents.
Keywords: Acetylcholine esterase inhibitors • quinazolin-4(3H)-one, quinazolin-4-amine • cytotoxicity • docking simulation
Introduction
Alzheimer’s disease (AD) is one the most common form of dementia.[1] It is an age-related neurodegenerative disorder and accounts for
approximately 80% of dementia patients worldwide. Clinically, the disease is characterized by a progressive loss of memory and
impairment of cognitive functions.[1, 2] Currently, the disease is estimated to affect nearly 30 million individuals of people over the age of
65.
Figure 1. Structures of some AChE inhibitors.
Despite enormous efforts of scientists devoted in studying the AD’s pathology, the primary causes of the disease has not yet been
completely understood. It is, therefore, AD remains one of the incurable neurological disorders to date and the treatment of the disease
remains symptomatic treatment.[1] Pathologically, a number of researches have indicated that several factors, such as abnormal
posttranslational modifications of tau protein resulting in neurofibrillary tangles, deposition of amyloid β-protein (Aβ) plagues, and a
1
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decrease in the level of the neurotransmitter acetylcholine, are closely associated with AD’s initiation and progression.[3,4] Therapeutically,
acetylcholine esterase inhibitors are still the only class of drugs approved for treatment of AD hitherto. Donepezil, tacrine, galanthamine
and rivastigmine (Figure 1) are some acetylcholine esterase inhibitors which have been shown to induce significant improvements in
memory and cognitive functions.[5]
Acetylcholine esterase (AChE; EC 3.1.1.7) belongs to the serine hydrolase enzyme class. These enzymes catalyze the hydrolysis of
acetylcholine, a neurotransmitter, to liberate choline and acetic acid, leading to the termination of cholinergic neurotransmission.[6]
Inhibition of acetylcholine esterase, therefore, prevents the breakdown of acetylcholine and boosts cholinergic neurotransmission,
resulting in clinical benefits. Due to its therapeutic importance, medicinal chemists worldwide have devoted great efforts in developing
novel AChE inhibitors. As a result, numerous AChE inhibitors have been described in the past decades, both naturally and synthetic.[7] In
our research program to develop novel AChE inhibitors we have used fragment-hopping and structural similarity approach coupled with
docking probe to design a series of donepezil analogues (Figure 2). This paper describes the synthesis and evaluation of AchE inhibitory
activity of this compounds series. The free radical scavenging effects of these compounds were also evaluated. In addition, since the
compounds possess quinazoline ring which is present in many anticancer agents, the cytotoxicity of the compounds against some human
cancer cell lines was also reported.
Figure 2. Design of novel quinazoline derivatives as potential AChE inhibitors.
Results and Discussion
Chemistry
The target 3-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-ones (4a-j) were synthesized via a three-step pathway, as illustrated
in Scheme 1. Condensation of the anthranilic acids (1a-j) with formamide under high temperature gave quinazolin-4(3H)-ones(2a-j) in
good yields (80-95%). Nucleophilic substitution reaction of compounds 2a-j with propargyl bromide under basic conditions (K2CO3) in
acetone with a catalytic amount of KI gave exclusively the 3-propargylquinazoline-4(3H)-ones (3) in quantitative yields (85-94%). Finally, a
Click reaction of the intermediates 3a-j with benzyl azide was cleanly proceeded using CuI and ascorbate in acetonitrile to give 3-((1-
benzyl-1H-1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-ones (4a-j)in moderate to good yields (65-87%).
Compounds 6a-j were synthesized via a three-step pathway, also from anthranilic acids (1a-j), as illustrated in Scheme 2. The quinazoline-
4(3H)-ones (2a-j) were first chlorinated at position 4 to give 4-chlorinated intermediates 5a-j in good yields (75-84%). The intermediates
5a-j were then reacted with 1-benzylpiperidin-4-amine in 2-propanol under basic conditions to afford target compounds 6a-j in moderate
yields (60-63%).
The structures of the synthesized compounds 4a-j and 6a-jwere determined straightforwardly based on analysis of spectroscopic data,
including IR, MS, 1H and 13C NMR.
2
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Scheme 1.Synthesis of 4-oxoquinazoline derivatives 4a-j
Scheme 2.Synthesis of quinazoline derivatives 6a-j.
Bioactivity
The AChE inhibitory effects of the synthesized compounds 4a-j and 6a-j were screened at 50 M. The AChE inhibitor screening kit
(colorimetric, BioVision (catalog #K197-100)) was used for the evaluation. The absorbance was kinetically measured at 412 nm for 40 min.
The results expressed as the enzyme relative inhibition percentages in comparison to donepezil, a positive control, at the final point are
shown in Figure 3. All compounds in series 4a-j showed no or only negligible effects on the AChE activity. Interestingly, compounds in
series 6a-j exhibited very noted inhibitory activity with relative inhibition percentages up to 87% in comparison to donepezil. Compounds
6c (with 7-CH3 substituent) and compound 6e (with 6,7-(OCH3)2 substituent) were two most potent AChE inhibitors in series 6a-j (relative
inhibition percentages up to 87%). Compound 6b (with 6-CH3 substituent) also displayed strong AChE inhibition (relative inhibition
percentage, 80%). The two fluorinated compounds 6f and 6g were the next most potent AChE inhibitors in series 6a-j (relative inhibition
percentages were 67 and 77%, respectively). Thus, it is clear that series 6a-j, especially compounds 6b, 6c, 6e, and 6g, were promising
AChE inhibitors. It could be easily noted that compounds 6a-j possess structurally features similar to donepezil, which are likely
attributable for their potent AChE inhibitory effects, while compounds in series 4a-j did not show any significant activity.
Since antioxidant activity would be an added value for AChE inhibitors to treat Alzheimer, we decided to evaluate compounds 6a-j for
their DPPH scavenging activity using a method previously described by Ellman et al.[8] The results illustrated in Figure 4 demonstrate that
compounds 6a-j also exhibited moderate DPPH scavenging activity. Among the compounds in the series, compound 6e was the most
potent DPPH scavenger with an inhibition percentage of 64.4%. It could be observed that compounds bearing electrong donating groups
on the quinazoline ring (6b-e) displayed stronger DPPH scavenging activity in comparison to compounds with electrong withdrawing
substituents (6f-j). Thus, electron density would be a factor important for the DPPH scavenging activity of the compounds in series 6a-j.
Figure 3. Relative inhibition of AChE by the compounds synthesized in comparison to donepezil. The enzyme was treated with samples at 50 µM and
donepezil at 20 µM.
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Figure 4. DPPH scavenging activity of compounds 6a-j. The compounds were assayed at 50 µg/mL. Quercetin (50 µg/mL) was used as a positive control.
Structurally, compounds 4a-j and 6a-j possess a 4-oxoquinazoline or quinazoline scaffolds, which are known to be present in various
anticancer agents.[8] Therefore, in addition to AChE inhibitory and DPPH scavenging activities, we also screened these compounds for
their cytotoxicity against three human cancer cell lines, including SW620 (colon cancer), PC-3 (prostate cancer), and NCI-H23 (lung cancer).
SRB method was used. The results are summarized in Table 1. It was found that compounds in series 4a-j were generally more cytotoxic
than compounds in series 6a-j. In series 4a-j, only two compounds 4a and 4e showed IC50 values higher than 10 M in three human cancer
cell lines tested. Eight other compounds (4a-c, 4f-j) displayed lower IC50 values (1.56-8.64 M). These compounds were found to be
stronger in term of cytotoxicity in comparison to 5-FU, a current widely used anticancer agent. Within two series 4a-j and 6a-j, three
compounds 4a, 4h, and 4i, especially compound 4a, emerged as the most cytotoxic compound and this compound could serve as a new
lead to further design more potent anticancer agents.
Table 1. Cytotoxicity of the synthesized compounds in some human cancer cell lines
Cp
Cytotoxicity (IC50
,
[b]M)/Cell lines[c]
d
R
MW
LogP[a]
NCI-H23
SW620
PC3
4a
4b
4c
4d
4e
4f
H
6-CH3
7-CH3
7-OCH3
6,7-(OCH3)2
6-F
317.35
331.38
331.38
347.38
337.40
335.34
335.34
351.79
396.25
443.25
318.42
332.45
332.45
348.45
378.48
2.21
2.76
2.76
2.29
1.85
2.41
2.41
2.85
3.10
3.38
3.96
4.50
4.50
4.04
3.60
2.21±0.011
4.80±0.044
5.67±0.094
11.12±0.481
10.42±0.321
5.55±0.094
8.64±0.173
3.07±0.203
3.45±0.098
5.57±0.282
14.34±1.014
18.34±1.936
15.21±1.012
21.35±2.011
>30
1.56±0.103
3.22±0.240
5.65±0.216
2.01±0.213
2.69±0.268
6.05±0.408
12.34±0.921
11.33±0.862
5.05±0.466
5.67±0.152
3.61±0.054
2.14±0.001
4.86±0.380
12.23±1.341
14.23±1.081
11.23±0.902
20.47±1.121
>30
10.23±0.871
11.32±0.723
3.34±0.148
7.03±0.428
2.72±0.272
3.16±0.092
4.10±0.219
13.21±1.341
13.76±1.213
9.23±0.903
18.56±1.761
>30
4g
4h
4i
7-F
6-Cl
6-Br
4j
6-I
6a
6b
6c
6d
6e
H
6-CH3
7-CH3
7-OCH3
6,7-(OCH3)2
4
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6f
6g
6-F
7-F
336.41
336.41
352.87
396.32
444.32
130.08
543.5
4.16
4.16
4.60
4.85
5.12
-0.81
1.27
13.23±0.911
15.32±0.912
16.21±1.021
17.56±1.311
20.76±1.901
8.84±1.92
18.31±1.012
17.21±1.901
20.54±1.705
22.54±0.901
15.21±0.121
13.61±0.46
1.16±0.28
18.27±0.971
15.31±1.071
24.23±2.010
19.49±1.971
27.32±2.012
13.45±3.92
1.29±0.12
6h
6i
6-Cl
6-Br
6-I
6j
5-FU[d]
ADR[e]
1.12±0.14
SAHA]f]
1.12±0.10
1.82±0.09
1.44±0.17
264.32
1.44
[a]Calculated by KowWin v.167 program; [b]The concentration (M) of compounds that produces a 50% reduction in enzyme activity or cell growth, data represent
means ± standard errors of the mean (SEM)of triplicate experiments (n = 3);[c] Cell lines: SW620, colon cancer; PC3, prostate cancer; NCI-H23, lung cancer; [d]5-FU:
5-Fluorouracil, a positive control. [e]5-ADR: Adriamycin, a positive controle. [f]SAHA: vorinostat, an anticancer agent acting by inhibition of hisone deacetylase, used
as a positive control.
With the results from the SRB assays, we selected compound 4afor flow cytometry analysis to see whether the compound affect the cell
cycle. SW620 cells were used for this experiment. The cells were initially pre-incubated for 24 h. Then, the cells were treated with
compound 4a (10 µM) or SAHA (1 µM) for 24 h. After that, the cells were harvested and the DNA contents were analyzed. It was found
that compound 4a killed a substantial population of cells at G0/G1 phase (27.40%). Among the viable cells, compounds caused a large
polulation of cells at G2/M phase (29.52%, vs. 5.31% of the VH) (Figure 5). Thus, it was clear that compound 4a arrested cell cycle of
SW620 cells at G2/M phase. This was in contrast to the effects of SAHA, which caused SW620 cells arrest at G0/G1 phase (Figure 5).
Next, the Annexin V-FITC/PI dual staining assay was used to determine whether compound 4a induced apoptosis in SW620 cells. SW620
human colon cancer cells were plated in 6-well culture plates and allowed to grow for 24 h. The cells were treated with compound 4a at 30
µM for 24 h, then harvested. The harvested cells were washed twice with ice-cold PBS and incubated in the dark at room temperature in
100 ml of 1× binding buffer containing 1 µl Annexin V-FITC and 12.5 mL PI . The results clearly demonstrated that compound 4a
significantly induced early apopotosis (stained red with Annexin V) (Figure 6).
In good consistence with the effects of the compound 4a on the cell cycle and apoptosis, the morphology of SW620 colon cancer cells was
substantially changed similar to that caused by SAHA, a positive control (Figure 7).
Figure 5. Cell cycle analysis of representative compounds 4a. SW620 cells (human colon cancer) (2x105 cells) were treated with compound (30 M) or SAHA (a
positive control) (1 M) for 24 h. The harvested cells were stained with propidium iodide (PI) in the presence of RNase and then were analyzed for DNA
content. UN: untreated, VH: vehicle (DMSO. 0.05%). Data was represented as histograms (left) and bar graphs (right).
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Figure 6. Apoptosis (Annexin V/PI) analysis of representative compounds 4a. SW620 cells (human colon cancer) (2x105 cells) were treated with compound (30
M) or SAHA (a positive control) (1 M) for 24 h. The harvested cells were stained with propidium iodide (PI) in the presence of RNase and then were analyzed
for DNA content. UN: untreated, VH: vehicle (DMSO. 0.05%). Area 1 = PI positive population, Area 2: Annexin V-postivie population. Data was represented as
histograms (left) and bar graphs (right).
Figure 7. Morphology changes of cells treated with representative compound 4a. SW620 (human colon cancer) cells (2 x 105 cells/well in 6-well plate, pre-
incubated for 2 h) were treated with compounds (30 M) or SAHA (a positive control)(1 M) for 24 h. Then, the cells were photographed using an Imaging
Device: Biostation with 20X lens. Scale bar: 50 µm.
Molecular docking studies
In this study several 3-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-ones and N-(1-benzylpiperidin-4-yl)quinazolin-4-amines
were synthesized and evaluated inhibitory activity against AChE enzyme. Some of them (6b, 6c, 6e and 6g) show good relative inhibition
(77-87%) against AChE in comparison with donepezil. To better understand the difference between tested compounds and donepezil, as
well as to investigate the structure-activity relationships of AChE inhibitors, semi-flexible docking simulations were performed using ICM
Pro 3.8 software.[9] A crystal complex of AChE-Donepezil reported by Cheung et al. was used for this study.[10] There are controversial
literatures related to the role of water molecule in forming tight hydrogen bonds between the heterocyclic amine like piperidine ring of
Donepezil toward key residues such as Tyr341 and Tyr337 of AChE. We then adopted the docking protocol reported by Roberts and
Mancera,[11] with a slight modification by removing water molecules in the binding site for docking assays.[12]
In order to demonstrate the validity of docking protocol, donepezil was firstly redocked into the active site of AChE. As the results, the
binding orientations of the redocked and co-crystal structures were highly overlapped (RMSD = 0.397Å). As can be seen in Figure 8A, the
key hydrogen bonding interaction between Donepezil with Phe295 of AChE was conserved. This interaction is important to accomodate
indanone ring at the peripheral anionic site (PAS) of AChE.[13] In addition, the phenyl moiety of donepezil could interact with the choline-
binding site and forms multiple π–π stacking interactions with Trp86 aromatic side chain. The affinity score calculated for donepezil was -
27.67 kCal/mol. The results obtained are suitable and the applied protocol could be utilized for further docking studies.
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Figure 8. Superposition between: (A) redocked donepezil (yellow carbon), and (B) synthesized compounds (6b, 6c, 6e, 6g represent as colored molecules)
and co-crystal Donepezil (green carbon).
The next step involved into docking four derivatives (6b, 6c, 6e and 6g) into the binding site of AChE following the protocols described
previously. Being illustrated in the Figure 8B, all four compounds showed similar interactions as donepezil. Compound 6e exhibited the
highest affinity with AChE compared to the other derivatives. The docking scores determined for four 6b, 6c, 6e and 6g were -26.50, -
26.91, -27.38, and -25.73 kCal/mol, respectively, slightly lower than that of donepezil. The docking scores agreed well with the ranking
order of in vitro values. For all the compounds, the quinazoline moiety formed multiple stacking interactions with Trp286 and Tyr341.
These interactions were similar to indanone ring of donepezil as shown in Figure 8A and played important role for stabilizing the ligand on
the entrance of the PAS cavity. Figure 9 showed an interaction comparison of two most active compounds (6c and 6e). Compound 6e
formed two hydrogen bonds with Tyr124 and Phe295, and the remaining interactions were almost identical. Meanwhile for 6c, the
distance between the quinazoline ring and NH backbone of Phe295 was pushed as far as 4.26 Å and it could not form hydrogen bond with
that residue. The lack of that hydrogen bond may affect the affinity of compound 6c in comparison with 6e. In addition, the 6,7-
dimethoxy substituents clearly improved the interactions of quinazoline rings with the periphery site of AChE enzyme.
Figure 9. Binding pose of compound 6c (A) and 6e (B)in the active binding site of AChE
Conclusions
In this study, we have designed and synthesized twonovel series of 3-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-ones (4a-j)
and N-(1-benzylpiperidin-4-yl)quinazolin-4-amines(6a-j). Series 6a-j were found to significantly inhibit AChE activity, especially
compounds 6cand 6e were the most potent with relative AChE inhibition percentages of 87% in comparison to Donepezil. We performed
docking studies for these four derivatives and the results showed similar interaction to donepezil. Among them, compound 6e exhibited
the highest affinity with a score of -27.38 kCal/mol. Compounds 6a-j also exhibited significant DPPH scavenging effects, which would be
an added value for these compounds as potential agents to treat AD. Two compound series also exerted moderate to good cytotoxicity
against three human cancer cell lines, including SW620 (human colon cancer), PC-3 (prostate cancer), and NCI-H23 (lung cancer), with
compound 4a being the most cytotoxic agent. Compound 4a significantly induced early apoptosis and arrested the SW620 cells at G2/M
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phase. Thus, from this study, compounds 6c and 6e could serve as new leads for further design and AChE inhibitors, while compound 4a
could serve as a new lead for the design and development of more potent anticancer agents.
Experimental Section
Chemistry
Thin layer chromatography which was performed using Whatman® 250 m Silica Gel GF Uniplates and visualized under UV light at 254 nm,
was used to check the progress of reactions and preliminary evaluation of compounds’ homogeneity. Melting points were measured using
a Gallenkamp Melting Point Apparatus (LabMerchant, London, United Kingdom) and are uncorrected. Purification of compounds was
carried out using crystallization methods and/or open silica gel column flash chromatography employing Merck silica gel 60 (240 to 400
mesh) as stationary phase. Nuclear magnetic resonance spectra (1H NMR) were recorded on a Bruker 500 MHz spectrometer with DMSO-
d6 as solvent unless otherwise indicated. Tetramethylsilane was used as an internal standard. Chemical shifts are reported in parts per
million (ppm), downfield from tetramethylsilane. Mass spectra with different ionization modes including electron ionization (EI),
Electrospray ionization (ESI), were recorded using PE Biosystems API2000 (Perkin Elmer, Palo Alto, CA, USA) and Mariner® (Azco Biotech,
Inc.Oceanside, CA, USA) mass spectrometers, respectively. The elemental (C, H, N) analyses were performed on a Perkin Elmer model
2400 elemental analyzer. All reagents and solvents were purchased from Aldrich or Fluka Chemical Corp. (Milwaukee, WI, USA) or Merck
unless noted otherwise. Solvents were used directly as purchased unless otherwise indicated.
The synthesis of novel compounds 4a-j and 6a-jwas carried out as illustrated in Schemes 1 and 2. Details are described below.
General procedures for the synthesis of compounds 4a-j
A solution of a respective 2-aminobenzoic acid (1a-j) (2 mmol) in excess amount of formamide (4 mL) was stirred at 120 C for 6 hours.
After completion of the reaction, the resulting mixture was cooled and poured into ice-cold water (10 mL). A solution of NaHCO3 5% was
gradually added to adjust pH to 7, which led to the formation of white solids. The solids were filtered and dried to give the corresponding
quinazolinone derivative 2, which was used for the next step without further purification.
To a respective solution of compounds 2a-j (1 mmol) in DMF (3 mL) were added K2CO3 (165.5 mg, 1.2 mmol). The mixtures were stirred at
80°C for 1 hour, then then a catalytic amount of KI (8.3 mg, 0.05 mmol) was added. After stirring for further 15 minutes, 0.15 ml of a
solution of propargyl bromide 80% in toluene was dropped slowly into the mixtures. The reaction mixtures were again stirred at 60°C for 3
hours. The reaction was checked by TLC. After the reaction completed, the resulting mixtures were cooled, poured into ice-cold water and
acidified to pH~4. The white solids formed were filtered and dried to give the propargylated compounds 3, which were used for the next
step without further purification.
A respective solution of compounds 3a-j and (azidomethyl)benzene (0.5 mmol) in acetonitrile (10 mL) were stirred at room temperature
for 10 minutes, then CuI (10 mg, 0.05 mmol) was added. The mixture was stirred at 60°C until the reaction completed (12-24 hours). The
corresponding resulting mixtures were evaporated under reduced pressure to give the residues, which were re-dissolved in 50 ml of DCM.
The mixtures were filtered and the DCM layers were evaporated under reduced pressure to give the targeting compounds 4a-j.
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)quinazolin-4(3H)-one (4a)
White solid; Yield: 68%. mp: 169-170 oC. Rf = 0.67 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3138, 3061 (CH, arene); 2941 (CH, CH2); 1676 (C=O);
1609, 1474 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.61 (1H, s, H-2); 8.24 (1H, s, H-6’); 8.15 (1H, d, J = 8.00 Hz, H-5); 7.84 (1H, t, J = 7.50
Hz, H-7); 7.77 (1H, d, J = 7.50 Hz, H-8); 7.56 (1H, t, J = 7.50 Hz, H-6); 7.40-7.31 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 5.57 (2H, s, H-1”a, H-1”b);
5.30 (2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 160.30, 148.67, 148.15, 136.21, 134.96, 129.23, 129.19, 128.91, 128.68,
128.63, 128.53, 127.74, 126.56, 124.66, 122.03, 54.07, 53.44, 41.62, 31.16. ESI-MS m/z: 318.00 [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-methylquinazolin-4(3H)-one (4b)
White solid; Yield: 65%. mp: 175-176 oC. Rf = 0.68 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3113, 3067 (CH, arene); 2986 (CH, CH2); 1657 (C=O);
1603, 1454 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.40 (1H, s, H-2); 8.06 (1H, s, H-6’); 7.85 (1H, s, H-5); 7.57 (1H, dd, J = 8.25 Hz, J’ =
1.75 Hz, H-7); 7.51 (1H, d, J = 8.50 Hz, H-8); 7.29-7.21 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 5.47 (2H, s, H-1”a, H-1”b); 5.16 (2H, s, H-1’a, H-
1’b); 2.35 (3H, s, 6-CH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ 160.28, 147.57, 146.39, 137.43, 136.37, 136.19, 129.24, 129.23, 129.22, 129.21,
129.20, 129.19, 128.63, 128.50, 128.49, 127.60, 125.86, 124.25, 121.84, 53.31, 41.45, 21.28.ESI-MS m/z: 332.10 [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-7-methylquinazolin-4(3H)-one (4c)
8
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10.1002/cbdv.202000290
Chemistry & Biodiversity
Chem. Biodiversity
White solid; Yield: 67%. mp: 176-177 oC. Rf = 0.68 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3136, 3063 (CH, arene); 2955 (CH, CH2); 1661 (C=O);
1603, 1454 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.44 (1H, d, J = 8.00 Hz, H-2); 8.11 (1H, d, J = 9.50 Hz, H-6’); 7.93 (1H, d, J = 8.50 Hz,
H-5); 7.43 (1H, d, J = 5.50 Hz, H-8); 7.29-7.21 (6H, m, H-6, H-3”, H-4”, H-5”, H-6”, H-7”); 5.47 (2H, d, J = 2.50 Hz, H-1”a, H-1”b); 5.17 (2H, d, J =
6.50 Hz, H-1’a, H-1’b); 2.37 (3H, s, 7-CH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ 160.21, 148.59, 145.50, 136.29, 129.21, 129.07, 128.64,
128.51, 127.34, 126.40, 124.47, 119.68, 53.38, 41.43, 21.76. ESI-MS m/z: 332.00 [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-7-methoxyquinazolin-4(3H)-one (4d)
White solid; Yield: 58%. mp: 191-192 oC. Rf = 0.62 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3084 (CH, arene); 2972 (CH, CH2); 1674 (C=O); 1609,
1450 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.50 (1H, s, H-2); 8.15 (1H, s, H-6’); 7.95 (1H, d, J = 9.00 Hz, H-5); 7.29-7.22 (6H, m, H-8, H-
3”, H-4”, H-5”, H-6”, H-7”); 7.05 (1H, d, J = 9.00 Hz, H-6); 5.48 (2H, d, J = 3.00 Hz, H-1”a, H-1”b); 5.17 (2H, s, H-1’a, H-1’b); 3.81 (3H, s, 7-
OCH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ 164.51, 129.23, 129.19, 128.92, 128.69, 128.55, 128.22, 117.45, 109.06, 56.35, 54.07, 53.47.ESI-
MS m/z: 348.00 [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6,7-dimethoxyquinazolin-4(3H)-one (4e)
White solid; Yield: 54%. mp: 211-212 oC. Rf = 0.59 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3061 (CH, arene); 2970 (CH, CH2); 1662 (C=O); 1607,
1499 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.41 (1H, s, H-2); 8.13 (1H, d, J = 5.00 Hz, H-6’); 7.31-7.22 (6H, m, H-5, H-3”, H-4”, H-5”, H-
6”, H-7”); 7.13 (1H, s, H-8); 5.49 (2H, s, H-1”a, H-1”b); 5.18 (2H, s, H-1’a, H-1’b); 3.78 (3H, s, 7-OCH3); 3.76 (3H, s, 6-OCH3). 13C NMR (125 MHz,
DMSO-d6, ppm): δ 159.53, 154.97, 149.33, 147.33, 144.20, 136.24, 129.23, 128.68, 128.56, 124.61, 115.06, 108.50, 105.51, 56.49, 56.18, 53.45,
41.58, 31.16.ESI-MS m/z: 378.10 [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-fluoroquinazolin-4(3H)-one (4f)
White solid; Yield: 63%. mp: 181-182 oC. Rf = 0.65 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3061, 3028 (CH, arene); 2922 (CH, CH2); 1689 (C=O);
1605, 1483 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.47 (1H, s, H-2); 8.12 (1H, s, H-6’); 7.74-7.64 (3H, m, H-5, H-7, H-8); 7.29-7.21 (5H, m,
H-3”, H-4”, H-5”, H-6”, H-7”); 5.48 (2H, s, H-1”a, H-1”b); 5.19 (2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 161.74, 159.78,
159.72, 147.91, 145.28, 136.33, 130.71, 130.64, 129.22, 128.65, 128.51, 127.93, 124.42, 123.52, 123.33, 111.30, 111.11, 53.35, 41.65.ESI-
MS m/z: 336.00 [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-7-fluoroquinazolin-4(3H)-one (4g)
White solid; Yield: 60%. mp: 185-186 oC. Rf = 0.65 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3107, 3061 (CH, arene); 2954 (CH, CH2); 1661 (C=O);
1603, 1477 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.53 (1H, s, H-2); 8.13 (1H, s, H-6’); 8.11-8.10 (1H, m, H-5); 7.43 (1H, d, J = 10.00 Hz,
H-6); 7.35-7.21 (6H, m, H-8, H-3”, H-4”, H-5”, H-6”, H-7”); 5.48 (2H, s, H-1”a, H-1”b); 5.18 (2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6,
ppm): δ 167.08, 165.08, 163.37, 159.69, 150.46, 149.88, 136.29, 129.80, 129.71, 129.22, 128.65, 128.51, 127.93, 124.49, 119.13, 116.38, 116.19,
112.97, 112.80, 53.37, 41.60.ESI-MS m/z: 336.00 [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-chloroquinazolin-4(3H)-one (4h)
White solid; Yield: 67%. mp: 191-192 oC. Rf = 0.64 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3107 (CH, arene); 2999, 2953 (CH, CH2); 1665 (C=O);
1605, 1472 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.50 (1H, s, H-2); 8.11 (1H, s, H-6’); 7.99 (1H, d, J = 2.50 Hz, H-5); 7.78 (1H, dd, J =
9.00 Hz, J’ = 2.50 Hz, H-7); 7.64 (1H, d, J = 9.00 Hz, H-8); 7.28-7.21 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 5.47 (2H, s, H-1”a, H-1”b); 5.18 (2H,
s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.42, 148.89, 147.13, 142.98, 136.34, 135.07, 131.95, 130.05, 129.22, 128.64, 128.50,
125.52, 124.35, 123.32, 53.33, 41.72.ESI-MS m/z: 352.10 (35Cl), 354.00 (37Cl) [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-bromoquinazolin-4(3H)-one (4i)
White solid; Yield: 63%. mp: 223-124 oC. Rf = 0.66 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3127, 3063 (CH, arene); 2945 (CH, CH2); 1676 (C=O);
1615, 1460 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.51 (1H, s, H-2); 8.12 (1H, d, J = 2.00 Hz, H-5); 8.11 (1H, s, H-6’); 7.89 (1H, dd, J =
8.50 Hz, J’ = 2.50 Hz, H-7); 7.56 (1H, d, J = 8.50 Hz, H-8); 7.29-7.21 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 5.48 (2H, s, H-1”a, H-1”b); 5.18 (2H,
s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.29, 148.97, 147.40, 142.89, 137.77, 136.36, 130.16, 129.22, 128.64, 128.49, 124.29,
123.66, 120.12, 53.33, 41.73.ESI-MS m/z: 396.00 (79Br), 398.00 (80Br) [M+H]+ .
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-iodoquinazolin-4(3H)-one (4j)
White solid; Yield: 65%. mp: 231-232 oC. Rf = 0.63 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3132, 3059 (CH, arene); 2986, 2930 (CH, CH2); 1665
(C=O); 1605, 1458 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.50 (1H, s, H-2); 8.32 (1H, d, J = 2.00 Hz, H-5); 8.11 (1H, s, H-6’); 8.04 (1H, dd,
J = 8.50 Hz, J’ = 2.00 Hz, H-7); 7.41 (1H, d, J = 8.50 Hz, H-8); 7.29-7.21 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 5.47 (2H, s, H-1”a, H-1”b); 5.17
(2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.10, 149.04, 147.68, 143.30, 136.35, 134.81, 129.93, 129.23, 128.65, 128.50,
124.34, 123.85, 92.81, 53.33, 41.74.ESI-MS m/z: 444.00 [M+H]+ .
General procedures for the synthesis of compounds 6a-j
Compounds 6a-j were synthesized via a three-step pathway as illustrated in scheme 2. The first step was similar to that described for
compounds 4a-j, followed by chlorination with thionyl chloride resulting in 4-chloro-quinazoline derivatives 5a-j. A mixture of compounds
2 andthionyl chloride was stirred at 120 C for 5 hours. Then, the resulting mixtures were evaporated under reduced pressure to give the
9
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10.1002/cbdv.202000290
Chemistry & Biodiversity
Chem. Biodiversity
residues, which were re-dissolved in 50 ml of water. After that, the mixtures were neutralised with a solution of NaHCO3 5% and extracted
with DCM (3 × 15 mL). The organic layers were combined and filtered with anhydrous Na2SO4. The solvent was removed under reduced
pressure to give the intermediates 5a-j.
The 4-chloro-quinazolines were reacted with 1-benzylpiperidin-4-amine in the presence of K2CO3. A respective solution of the
intermediates 5a-j and 1-benzylpiperidin-4-amine (1 mmol) in 2-propanol (10 mL) were stirred at room temperature for 10 minutes, then
K2CO3 (165.5 mg, 1.2 mmol) was added. The mixture was stirred at 70°C until the reaction completed (5 hours). The resulting mixtures
were evaporated under reduced pressure to give the residues, which were re-dissolved in 50 ml of DCM. The mixtures were filtered and
the DCM layers were evaporated under reduced pressure to give the white solids. The crude products were further purified by column
chromatography (DCM/methanol = 100 : 5) to give the target compounds 6a-j.
N-(1-Benzylpiperidin-4-yl)quinazolin-4-amine (6a)
White solid; Yield: 69%. mp: 171-172 oC. Rf = 0.46 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3063 (CH, arene); 2951 (CH, CH2); 1580 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.45 (1H, s, H-2); 8.31 (1H, d, J = 8.00 Hz, NH); 7.91 (1H, d, J = 7.50 Hz, H-5); 7.75 (1H, t, J = 8.00 Hz, H-7);
7.66 (1H, d, J = 8.00 Hz, H-8); 7.50 (1H, t, J = 7.25 Hz, H-6); 7.33-7.25 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 4.23-4.17 (1H, m, H-1’); 3.50 (2H, s,
H-1”a, H-1”b); 2.89-2.87 (2H, m, H-3’a, H-5’a); 2.10-2.06 (2H, m, H-3’b, H-5’b); 1.93-1.90 (2H, m, H-2’a, H-6’a); 1.70-1.67 (2H, m, H-2’b, H-
6’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.20, 155.57, 149.68, 139.17, 132.91, 129.17, 128.64, 127.91, 127.32, 125.83, 123.34, 115.37,
62.61, 52.84, 48.39, 31.64.ESI-MS m/z: 319.20 [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-6-methylquinazolin-4-amine (6b)
White solid; Yield: 65%. mp: 185-186 oC. Rf = 0.48 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3026 (CH, arene); 2934 (CH, CH2); 1580 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.40 (1H, s, H-2); 8.12 (1H, s, H-5); 7.89 (1H, d, J = 7.50 Hz, H-7); 7.60-7..57 (1H, m, H-8); 7.36-7.25 (5H, m,
H-3”, H-4”, H-5”, H-6”, H-7”); 4.21-4.15 (1H, m, H-1’); 3.50 (2H, s, H-1”a, H-1”b); 2.88-2.86 (2H, m, H-3’a, H-5’a); 2.47 (3H, s, 6-CH3); 2.09-
2.05 (2H, m, H-3’b, H-5’b); 1.92-1.90 (2H, m, H-2’a, H-6’a); 1.71-1.63 (2H, m, H-2’b, H-6’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 158.82,
154.81, 147.93, 139.18, 135.37, 134.54, 129.17, 128.64, 127.73, 127.32, 122.27, 115.19, 62.60, 52.85, 48.34, 31.70, 21.59.ESI-MS m/z: 333.20
[M+H]+ .
N-(1-Benzylpiperidin-4-yl)-7-methylquinazolin-4-amine (6c)
White solid; Yield: 67%. mp: 188-189 oC. Rf = 0.49 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3026 (CH, arene); 2938 (CH, CH2); 1574 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.40 (1H, s, H-2); 8.20 (1H, d, J = 8.50 Hz, H-5); 7.82 (1H, d, J = 7.50 Hz, H-6); 7.46 (1H, s, H-8); 7.36-7.25
(5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 4.20-4.14 (1H, m, H-1’); 3.50 (2H, s, H-1”a, H-1”b); 2.88-2.86 (2H, m, H-3’a, H-5’a); 2.09 (3H, s, 7-CH3);
2.20-2.05 (2H, m, H-3’b, H-5’b); 1.92-1.89 (2H, m, H-2’a, H-6’a); 1.71-1.63 (2H, m, H-2’b, H-6’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ
159.07, 155.65, 149.89, 145.89, 143.01, 139.15, 129.19, 128.64, 127.56, 127.33, 127.11, 126.17, 123.15, 113.22, 62.60, 52.84, 48.31, 31.68,
21.74.ESI-MS m/z: 333.20 [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-7-methoxyquinazolin-4-amine (6d)
White solid; Yield: 54%. mp: 201-202 oC. Rf = 0.45 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3071 (CH, arene); 2947 (CH, CH2); 1582, 1566
(C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.47 (1H, s, H-2); 8.33 (1H, d, J = 9.50 Hz, H-5); 8.01 (1H, d, J = 7.50 Hz, H-8); 7.45 (1H, t, J = 9.50
Hz, H-6); 7.36-7.25 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 4.21-4.18 (1H, m, H-1’); 4.02 (3H, s, 7-OCH3); 3.50 (2H, s, H-1”a, H-1”b); 2.89-2.86
(2H, m, H-3’a, H-5’a); 2.09-2.05 (2H, m, H-3’b, H-5’b); 1.92-1.89 (2H, m, H-2’a, H-6’a); 1.70-1.64 (2H, m, H-2’b, H-6’b). 13C NMR (125 MHz,
DMSO-d6, ppm): δ 162.77, 159.13, 158.90, 158.02, 156.72, 156.09, 152.00, 147.48, 139.16, 129.17, 128.64, 127.33, 124.94, 123.42, 116.89,
116.24, 112.18, 110.58, 109.54, 107.29, 62.57, 57.19, 55.93, 52.84, 52.81, 48.61, 48.26, 31.65.ESI-MS m/z: 349.10 [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-6,7-dimethoxyquinazolin-4-amine (6e)
White solid; Yield: 51%. mp: 231-232 oC. Rf = 0.43 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3132 (CH, arene); 2934 (CH, CH2); 1582 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.32 (1H, s, H-2); 7.61 (1H, s, H-5); 7.57 (1H, d, J = 7.50 Hz, NH); 7.36-7.25 (5H, m, H-3”, H-4”, H-5”, H-6”,
H-7”); 7.07 (1H, s, H-8); 4.20-4.17 (1H, m, H-1’); 3.99 (2H, s, H-1”a, H-1”b); 3.90 (3H, s, 7-OCH3); 3.89 (3H, s, 6-OCH3); 2.90-2.88 (2H, m, H-3’a,
H-5’a); 2.10-2.06 (2H, m, H-3’b, H-5’b); 1.94-1.92 (2H, m, H-2’a, H-6’a); 1.71-1.64 (2H, m, H-2’b, H-6’b). 13C NMR (125 MHz, DMSO-d6, ppm):
δ 158.06, 154.16, 153.99, 148.71, 146.61, 139.24, 129.12, 128.65, 127.32, 108.89, 107.51, 102.54, 62.56, 56.61, 56.11, 52.95, 48.22, 32.03.ESI-
MS m/z: 379.20 [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-6-fluoroquinazolin-4-amine (6f)
White solid; Yield: 61%. mp: 197-198 oC. Rf = 0.54 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3136, 3069 (CH, arene); 2941 (CH, CH2); 1584
(C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.45 (1H, s, H-2); 8.20 (1H, dd, J = 10.25 Hz, J’ = 2.75 Hz, H-5); 7.87 (1H, d, J = 7.00 Hz, NH); 7.76-
7.73 (1H, m, H-7); 7.68-7.64 (1H, m, H-8); 7.35-7.25 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 4.18-4.15 (1H, m, H-1’); 3.49 (2H, s, H-1”a, H-1”b);
2.88-2.86 (2H, m, H-3’a, H-5’a); 2.09-2.04 (2H, m, H-3’b, H-5’b); 1.93-1.91 (2H, m, H-2’a, H-6’a); 1.69-1.62 (2H, m, H-2’b, H-6’b). 13C NMR
(125 MHz, DMSO-d6, ppm): δ 160.49, 158.97, 158.94, 158.56, 155.09, 155.08, 146.79, 139.13, 130.76, 130.69, 129.17, 128.63, 127.32, 122.33,
122.13, 115.82, 115.75, 107.84, 107.66, 62.59, 52.75, 48.54, 31.60, 31.14.ESI-MS m/z: 337.20 [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-7-fluoroquinazolin-4-amine (6g)
10
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10.1002/cbdv.202000290
Chemistry & Biodiversity
Chem. Biodiversity
White solid; Yield: 63%. mp: 187-188 oC. Rf = 0.53 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3022 (CH, arene); 2926 (CH, CH2); 1591 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.45 (1H, s, H-2); 8.43-8.40 (1H, m, H-5); 8.01 (1H, d, J = 7.50 Hz, H-6); 7.42-7.23 (6H, m, H-8, H-3”, H-4”,
H-5”, H-6”, H-7”); 4.20-4.16 (1H, m, H-1’); 3.48 (2H, s, H-1”a, H-1”b); 2.87-2.84 (2H, m, H-3’a, H-5’a); 2.09-2.03 (2H, m, H-3’b, H-5’b); 1.91-
1.89 (2H, m, H-2’a, H-6’a); 1.70-1.63 (2H, m, H-2’b, H-6’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 165.75, 163.76, 159.00, 156.71, 151.80,
151.70, 139.13, 129.15, 128.62, 127.30, 126.67, 126.59, 115.17, 114.98, 112.48, 111.86, 111.70, 62.58, 52.77, 48.48, 40.50, 40.26, 40.09,
31.61.ESI-MS m/z: 337.20 [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-6-chloroquinazolin-4-amine (6h)
White solid; Yield: 64%. mp: 194-195 oC. Rf = 0.54 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3086 (CH, arene); 2941 (CH, CH2); 1580 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.50 (1H, d, J = 2.00 Hz, H-2); 8.47 (1H, s, H-5); 8.03 (1H, d, J = 7.50 Hz, NH); 7.76 (1H, dd, J = 8.75 Hz, J’ =
2.75 Hz, H-7); 7.68 (1H, d, J = 8.50 Hz, H-8); 7.35-7.25 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 4.19-4.13 (1H, m, H-1’); 3.49 (2H, s, H-1”a, H-
1”b); 2.88-2.85 (2H, m, H-3’a, H-5’a); 2.09-2.04 (2H, m, H-3’b, H-5’b); 1.92-1.90 (2H, m, H-2’a, H-6’a); 1.67-1.64 (2H, m, H-2’b, H-6’b). 13C
NMR (125 MHz, DMSO-d6, ppm): δ 158.46, 155.95, 148.40, 139.11, 133.30, 130.12, 130.04, 129.17, 128.63, 127.32, 122.68, 116.19, 62.57, 52.74,
48.62, 31.54, 31.14.ESI-MS m/z: 353.10 (35Cl), 355.10 (37Cl) [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-6-bromoquinazolin-4-amine (6i)
White solid; Yield: 66%. mp: 211-212 oC. Rf = 0.55 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3061 (CH, arene); 2940 (CH, CH2); 1574 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.64 (1H, d, J = 2.00 Hz, H-2); 8.48 (1H, s, H-5); 8.06 (1H, d, J = 7.50 Hz, NH); 7.87 (1H, dd, J = 9.00 Hz, J’ =
2.00 Hz, H-7); 7.61 (1H, d, J = 9.00 Hz, H-8); 7.35-7.25 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 4.17-4.14 (1H, m, H-1’); 3.48 (2H, d, J = 2.00 Hz,
H-1”a, H-1”b); 2.87-2.85 (2H, m, H-3’a, H-5’a); 2.09-2.04 (2H, m, H-3’b, H-5’b); 1.92-1.90 (2H, m, H-2’a, H-6’a); 1.69-1.64 (2H, m, H-2’b, H-
6’b). 13C NMR (125 MHz, DMSO-d6, ppm): δ 158.30, 156.00, 148.62, 139.13, 135.92, 130.24, 129.16, 128.63, 127.31, 125.81, 118.28, 116.71,
62.57, 52.75, 48.63, 31.55.ESI-MS m/z: 397.10 (79Br), 399.10 (80Br) [M+H]+ .
N-(1-Benzylpiperidin-4-yl)-6-iodoquinazolin-4-amine (6j)
White solid; Yield: 62%. mp: 234-235 oC. Rf = 0.57 (DCM : MeOH = 14 : 1).IR (KBr, cm-1): 3059 (CH, arene); 2945 (CH, CH2); 1572 (C=C).1H-
NMR (500 MHz, DMSO-d6, ppm): δ 8.77 (1H, d, J = 2.00 Hz, H-2); 8.47 (1H, s, H-5); 8.06 (1H, d, J = 7.50 Hz, NH); 8.01 (1H, dd, J = 8.75 Hz, J’ =
1.75 Hz, H-7); 7.45 (1H, d, J = 9.00 Hz, H-8); 7.36-7.24 (5H, m, H-3”, H-4”, H-5”, H-6”, H-7”); 4.18-4.15 (1H, m, H-1’); 3.50 (2H, s, H-1”a, H-1”b);
2.88-2.86 (2H, m, H-3’a, H-5’a); 2.09-2.04 (2H, m, H-3’b, H-5’b); 1.92-1.90 (2H, m, H-2’a, H-6’a); 1.70-1.62 (2H, m, H-2’b, H-6’b). 13C NMR
(125 MHz, DMSO-d6, ppm): δ 157.97, 155.96, 148.88, 141.28, 139.15, 131.84, 130.03, 129.15, 128.64, 127.32, 117.21, 90.79, 62.55, 52.77, 48.60,
31.57.ESI-MS m/z: 445.10 [M+H]+.
Bioactivity
AChE inhibition assay
AChE inhibitory activity was measure using the Ellman’s method as previously reported[14] with slight modifications. Briefly, to 60 μL of 50
mM NaHPO4 buffer ( pH 7.7) were added 10 μL of respective assayed sample (at stock solution of 0.2 mM). Then, 10 μL of enzyme (0.005
unit enzyme per well) was added. The resulting contents were mixed and pre-read at 405 nm, then the contents were pre-incubated for 10
min at 37 °C. The reaction in each well was initiated by the addition of 10 μL of 0.5 mM substrate (acetylthiocholine iodide or
°
butyrylthiocholine bromide) to each well, followed by the addition of 10 μL DTNB (0.5 mM per well). The wells were incubated for at 37 C,
then the absorbance of each well was measured at 412 nm using 96-well plate reader Synergy HT, Biotek, USA. All experiments were
carried out in triplicate. Donepezil (0.2 mM stock solution) was used as a positive control. The inhibition percentages were calculated by
the following formula:
퐶표푛푡푟표푙퐴푏푠 − 푆푎푚푝푙푒퐴푏푠
퐼푛ℎꢀ푏ꢀ푡푡ꢀ표푛푝푒푟푐푒푛푡푎ꢁ푒 =
푥 100
퐶표푛푡푟표푙퐴푏푠
IC50 values (in case measured) were calculated using EZ–Fit Enzyme kinetics software (Perrella Scientific Inc. Amherst, USA).
DPPH radical scavenging assay
1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity was measured according to a described method.[15] Briefly, 5 µl of each
sample dissolved in MeOH were added to 195 µl of 150 µM methanolic DPPH in 96-well plates. The solution was mixed for 1 min and
incubated at room temperature in a dark place. After 30 min, the absorbance of the reaction mixture was measured at 520 nm on a
microplate reader. The scavenging activity was expressed as the degree of radical reduction of a test group, in comparison to that of the
control.
Cytotoxicity assay
11
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10.1002/cbdv.202000290
Chemistry & Biodiversity
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The cytotoxicity of the synthesized compounds was evaluated against three human cancer cell lines, including SW620 (colon cancer), PC3
(prostate cancer), and NCI-H23 (lung cancer). The cell lines were purchased from a Cancer Cell Bank at the Korea Research Institute of
Bioscience and Biotechnology (KRIBB). The media, sera and other reagents that were used for cell culture in this assay were obtained
from GIBCO Co. Ltd. (Grand Island, New York, USA). The cells were culture in DMEM (Dulbecco’s Modified Eagle Medium) until
confluence. The cells were then trypsinized and suspended at 3 × 104 cells/mL of cell culture medium. On day 0, each well of the 96-well
plates was seeded with 180 µL of cell suspension. The plates were then incubated in a 5% CO2 incubator at 37 oC for 24 h. Compounds
were initially dissolved in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations by culture medium. Then 20 µL of each
compounds’ samples, which were prepared as described above, were added to each well of the 96-well plates, which had been seeded
with cell suspension and incubated for 24-h, at various concentrations. The plates were further incubated for 48 h. Cytotoxicity of the
compounds was measured by the colorimetric method, as described previously[16] with slight modifications.[17-20] The IC50 values were
calculated using a Probits method[21] and were averages of three independent determinations (SD ≤ 10%).
Cell cycle analysis
SW620 human colon cancer cells (2 × 105/ml per well) were plated in 6-well culture plates and allowed to grow for 24 h. The cells were
treated with compounds at appropriate concentrations for 24 h, respectively, and then harvested. The harvested cells were washed twice
with ice-cold PBS, fixed in 75% ice-cold ethanol, and stained with propidium iodide (PI) in the presence of RNase at room temperature for
30 min. The stained cells were analyzed for DNA content using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the
data were processed using Cell Quest Pro software (BD Biosciences).
Apoptosis assay
The Annexin V-FITC/PI dual staining assay was used to determine the percentage of apoptotic cells. SW620 human colon cancer cells (2 ×
105/ml per well) were plated in 6-well culture plates and allowed to grow for 24 h. The cells were treated with compounds at appropriate
concentrations for 24 h, respectively, and then harvested. The harvested cells were washed twice with ice-cold PBS and incubated in the
dark at room temperature in 100 ml of 1× binding buffer containing 1 µl Annexin V-FITC and 12.5 mL PI. After 15 min incubation, cells were
analyzed for percentage undergoing apoptosis using a FACScalibur flow cytometer (BD Biosciences). The data were processed using Cell
Quest Pro software (BD Biosciences).
Molecular docking studies
The X-ray crystal structure of AChE-donepezil complex was retrieved from Protein Data bank (PDB ID: 4EY7) [10]. This protein structure is
then prepared by retaining chain A and removing water molecules, correcting protein structure errors, protonating the structure, setting
tethers of different strengths on the receptors, fixing atoms beyond a 8 distance-distance from the active site and finally optimizing the
fixed structure by minimizing the structure to an RMS gradient of 0.1 kcal/mol/Å.[12, 22] The AMBER99 force field is added and the partial
charge is recalculated for all atoms. All protein preparations were performed using MOE 2015.10 software.[12, 17, 23] The 2D structures of
new compounds were built by using Marvin Sketch software and optimized by MOE energy minimizing mode. Molecular docking was
performed using ICM-Pro 3.8-3 software (http://www.molsoft.com/icmpro/).[9,24] Before performing docking studies, protocol docking was
validated by re-docking the co-crystal ligands Donepezil. The suitability of the docking procedures was assessed via calculating RMSD
between redocked and co-crystal ligand. The docking poses were selected based on the ICM scores and interaction profiles.
Supplementary Material
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number.
Acknowledgements
We acknowledge the principal financial supports from the National Foundation for Science and Technology of Vietnam (NAFOSTED,
Grant number 104.01-2018.301). The work was also partly supported by a grant funded by the Korean Government (NRF, Grant number
2017R1A5A2015541).
Author Contribution Statement
12
This article is protected by copyright. All rights reserved.

10.1002/cbdv.202000290
Chemistry & Biodiversity
Chem. Biodiversity
N. T. T., N-H. N and S-B. H. proposed the work. N-H. N, T. T. L.., and D. T. A. mainly developed the synthesis studies, S-B. H, E. J. P., J. S.
K., J. H. K, andS. D. J. performed the biological testing assays.P-T. H., andD. T. M. D. performed the docking simulations and
physicochemical computations.All authors read and approved the final manuscript.
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