Design, synthesis, docking study, α-glucosidase inhibition, and cytotoxic activities of acridine linked to thioacetamides as novel agents in treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bioorg.2018.06.035

A novel series of acridine linked to thioacetamides 9a–o were synthesized and evaluated for their α-glucosidase inhibitory and cytotoxic activities. All the synthesized compounds exhibited excellent α-glucosidase inhibitory activity in the range of IC₅₀ = 80.0 ± 2.0–383.1 ± 2.0 μM against yeast α-glucosidase, when compared to the standard drug acarbose (IC₅₀ = 750.0 ± 1.5 μM). Among the synthesized compounds, 2-((6-chloro-2-methoxyacridin-9-yl)thio)-N-(p-tolyl) acetamide 9b displayed the highest α-glucosidase inhibitory activity (IC₅₀ = 80.0 ± 2.0 μM). The in vitro cytotoxic assay of compounds 9a–o against MCF-7 cell line revealed that only the compounds 9d, 9c, and 9n exhibited cytotoxic activity. Cytotoxic compounds 9d, 9c, and 9n did not show cytotoxic activity against the normal human cell lines HDF. Kinetic study revealed that the most potent compound 9b is a competitive inhibitor with a K_i of 85 μM. Furthermore, the interaction modes of the most potent compounds 9b and 9f with α-glucosidase were evaluated through the molecular docking studies.

Full text of DOI:10.1016/j.bioorg.2018.06.035

Accepted Manuscript
Design, synthesis, docking study, α-glucosidase inhibition, and cytotoxic ac-
tivities of acridine linked to thioacetamides as novel agents in treatment of type
2
diabetes
Maryam Mohammadi-Khanaposhtani, Sepideh Rezaei, Reza Khalifeh, Somaye
Imanparast, Mohammad Ali Faramarzi, Saeed Bahadorikhalili, Malihe Safavi,
Fatemeh Bandarian, Ensieh Nasli Esfahani, Mohammad Mahdavi, Bagher
Larijani
PII:
S0045-2068(18)30251-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.035
YBIOO 2415
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 March 2018
17 June 2018
29 June 2018
Please cite this article as: M. Mohammadi-Khanaposhtani, S. Rezaei, R. Khalifeh, S. Imanparast, M. Ali Faramarzi,
S. Bahadorikhalili, M. Safavi, F. Bandarian, E. Nasli Esfahani, M. Mahdavi, B. Larijani, Design, synthesis, docking
study, α-glucosidase inhibition, and cytotoxic activities of acridine linked to thioacetamides as novel agents in
treatment of type 2 diabetes, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.035
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Design, synthesis, docking study, α-glucosidase inhibition, and cytotoxic activities of
acridine linked to thioacetamides as novel agents in treatment of type 2 diabetes
a
b
b
c
Maryam Mohammadi-Khanaposhtani , Sepideh Rezaei , Reza Khalifeh , Somaye Imanparast ,
c
d
e
f
Mohammad Ali Faramarzi , Saeed Bahadorikhalili , Malihe Safavi , Fatemeh Bandarian ,
f
f,
f,
Ensieh Nasli Esfahani , Mohammad Mahdavi *, Bagher Larijani *
a
Cellular and Molecular Biology Research Center, Health Research Institute, Babol University
of Medical Sciences, Babol, Iran
b
Department of Chemistry, Shiraz University of Technology, Shiraz, Iran
c
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology
Research Center, Tehran University of Medical Sciences, Iran
d
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
e
Department of Biotechnology, Iranian Research Organization for Science and Technology
(IROST), Tehran, Iran
f
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
Abstract:
A novel series of acridine linked to thioacetamides 9a–o were synthesized and evaluated for their
α-glucosidase inhibitory and cytotoxic activities. All the synthesized compounds exhibited
excellent α-glucosidase inhibitory activity in the range of IC50 = 80.0 ± 2.0–383.1 ± 2.0 µM
against yeast α-glucosidase, when compared to the standard drug acarbose (IC50 = 750.0 ± 1.5
µM). Among the synthesized compounds, 2-((6-chloro-2-methoxyacridin-9-yl)thio)-N-(p-tolyl)
acetamide 9b displayed the highest α-glucosidase inhibitory activity (IC50 = 80.0 ± 2.0 μM). The
in vitro cytotoxic assay of compounds 9a–o against MCF-7 cell line revealed that only the
compounds 9d, 9c, and 9n exhibited cytotoxic activity. Cytotoxic compounds 9d, 9c, and 9n did
not show cytotoxic activity against the normal human cell lines HDF. Kinetic study revealed that

the most potent compound 9b is a competitive inhibitor with a K of 85 μM. Furthermore, the
i
interaction modes of the most potent compounds 9b and 9f with α-glucosidase were evaluated
through the molecular docking studies.
Key words: α-Glucosidase; Type 2 diabetes; Acridine; Thioacetamide; Docking study
1
. Introduction
α-Glucosidase leads to the breakdown of oligo- and disaccharides from dietary complex
carbohydrates. Inhibition of this enzyme reduced the absorption of monosaccharides and control
of postprandial glucose level [1]. Several α-glucosidase inhibitors such as acarbose, voglibose,
and miglitol are approved for the treatment of type 2 diabetes [2-4]. In addition, certain
evidences suggest that α-glucosidase inhibitors can be used to treat other carbohydrate mediated
diseases including cancer, viral infections, and hepatitis [5-7]. Thus, the design and development
of new α-glucosidase inhibitors is a noteworthy goal for pharmacologists [8-12].
Nitrogen-containing aromatic tricyclic scaffolds such as acridine, acridone, and carbazole reveal
a
wide variety of biological activities including anticancer, antimicrobial,
antiacetylcholinesterase, and anticonvulsant activities [13-24]. Furthermore, several natural and
synthetic derivatives of the mentioned scaffolds have been reported as α-glucosidase inhibitors
[
25-27].
On the other hand, Wang et al. recently reported the synthesis and anti-α-glucosidase activity of
a series of thioacetamide derivatives [28].
Hence, in continuation of our efforts for developing new α-glucosidase inhibitors, herein we
report for the first time the synthesis of a novel series of acridine linked to thioacetamides
derivatives 9a–o, and evaluated their α-glucosidase inhibitory activity [29]. Furthermore, kinetic

study and molecular docking of synthesized compounds were also performed. Considering the
numerous reports of cytotoxic effects of acridine derivatives, all synthesized compounds were
evaluated by the MTT assay against breast cancer cell line MCF-7.
2
. Chemistry
The pathway for the synthesis of acridine linked to thioacetamides 9a–o has been depicted in
Scheme 1. This pathway begins from the Ullmann condensation reaction of 4-methoxyaniline 1
and 2,4-dichloro-benzoic acid 2 in the presence of potassium carbonate and copper in DMF at
reflux to yield 4-chloro-2-((4-methoxyphenyl)amino)benzoic acid 3. Compound 3 easily
participated in the cyclization reaction in the presence of POCl at reflux and yeilded 6,9-
3
dichloro-2-methoxyacridine 4. 6,9-Dichloro-2-methoxyacridine 4 reacted with thiourea to
produce 6-chloro-2-methoxyacridine-9-thiol 5 in ethanol at room temperature. On the other
hand, N-phenyl (or benzyl)-2-chloroacetamides 8 were prepared by the reaction between amines
6
and chloroacetyl chloride 7 in DMF at room temperature. Finally, the 6-chloro-2
methoxyacridine-9-thiol 5 reacted with acetamides 8 in the presence of potassium carbonate in
ethanol at reflux to provide the corresponding products 9 in good yields (80–95%).
3
. Results and discussion
3
.1. In vitro α-glucosidase inhibitory activity
The synthesized compounds 9a–o were evaluated for their in vitro α-glucosidase inhibition
potential in comparison with the marketed α-glucosidase inhibitor acarbose as a standard drug.
The IC50 values of the two series of compounds N-phenylacetamide and N-benzylacetamide
derivatives (9a–m and 9n–o, respectively) are summarized in Table 1. Results revealed that all
of the synthesized compounds are more potent than the standard against yeast α-glucosidase

enzyme. The most active compounds were N-phenylacetamide derivatives 9b, 9f, 9a, and 9l with
IC50 values 80.0 ± 2.0, 92.2 ± 2.0, 95.0 ± 1.9, and 96.6 ± 2.0 µM, respectively. Furthermore, the
remaining compounds exhibited high α-glucosidase inhibitory activities around 2–7 folds more
than acarbose with the IC50 values ranging from 102.0 ± 1.3 to 383.1 ± 2.0 µM.
3
.2. Structure–activity relationships
To study the structure–activity relationships and to optimize the anti-α-glucosidase activity of the
designed scaffold, we used aniline, substituted anilines, benzyl amine, and 4-florobenzylamine to
synthesize compounds 9a–o.
In N-phenylacetamide derivatives series, compound 9b with 4-methyl group on N-phenyl ring
was found to be the most active compound among all of the synthesized compounds 9a–o.
Removal of 4-methyl group of compound 9b, slightly reduced the inhibitory activity as observed
in the third most potent compound 9a, whereas replacement of this group with ethyl or fluorine
substituents (respectively compounds 9c and 9d), resulted in a significant decrease in the
inhibitory potential.
The inhibitory activity of the halogenated N-phenylacetamide derivatives against α-glucosidase
demonstrated that 3-chloroN-phenylacetamide derivative 9f (the second most potent) showed the
most potent activity, whereas 4-fluoro derivative 9d was the less active halo-substituted
compound. The observed IC50 values of the chloroderivatives 9e–g revealed that the trend of
activity was 3-Cl > 3,4-dichloro > 4-Cl > 2-Cl. In addition, 4-bromo derivative 9j exhibited more
activity than its 2-bromo analog (compound 9i). Interestingly, trend activity in the methoxy
derivatives 9k–m is similar to that in the chloro derivatives 9e–g in the order of 3-OCH
3
> 4-
OCH > 2-OCH . These findings indicated that the interaction of synthesized compounds with α-
3
3

glucosidase enzyme, in addition to the type of substituents, is dependent on their position on the
phenyl ring of N-phenylacetamide moiety.
Inhibitory activities of N-benzylacetamide derivatives 9n and 9o do not reveal significant
difference with their corresponding N-phenylacetamide analogs 9a and 9d, respectively.
3
.3. Cytotoxicity studies
Cytotoxicity of all synthesized compounds 9a–o was evaluated against the breast cancer cell line
by using MTT assay [30]. As can be seen in Table 2, the synthesized compounds did not indicate
any cytotoxic activity against MCF-7 (IC50 > 100 μM) with the exception of compounds 9d, 9c,
and 9n. Compound 9d with IC50 value of 13.42 ± 0.6 μM was almost as potent as the standard
drug etoposide (IC50 = 12.4 ± 4.7 μM). To further assess of the cytotoxic effects of synthesized
compounds, compounds 9d, 9c, and 9n (cytotoxic compounds against MCF-7) were evaluated
against the normal human cell lines HDF. Results revealed that these compounds were
noncytotoxic at 100-μM concentrations to the studied normal cells.
3
.4. Kinetic study
To study the mechanism of action of the synthesized compounds on α-glucosidase, a kinetic
study was performed on the most potent compound 9b. The mode of inhibition and value of K
were determined by Lineweaver–Burk plots and secondary re-plot of these plots, respectively.
i
Fig. 1A shows that in the presence of various concentrations of compound 9b the Vmax was not
affected, while the Km increased (Fig. 1A), which indicated a competitive mode of inhibition.
Also, the K
i
value of compound 9b was 85 µM (Fig. 1B).
3
.5. Docking study

In order to clarify the interactions of the synthesized compounds in the active site of α-
glucosidase and explain the related inhibitory activities, molecular modeling studies were
performed using Autodock Tools (version 1.5.6). Since, any crystallographic structure of
S.cerevisiae α-glucosidase (used in in vitro assay section) do not report up-to yet, we used of the
crystal structure of S. cerevisiae isomaltase (PDB ID: 3A4A) with 71% identity and 84%
similarity toward the S. cerevisiae α-glucosidase for docking purpose [31]. Superimposition
structure of acarbose (standard inhibitor) and most potent compound 9b in the active site of
isomaltase is presented in Figure 2.
As observed in Figure 3, acarbose interacted with the active site of isomaltase via six hydrogen
bonds and one hydrophobic interaction with residues Asp69, Arg213, Glu277, Arg315, Asp352,
Arg442, and Phe303.
The most active compound 9b interacted with residues Arg315, Asp352, Arg442, Phe303,
Thr306, Tyr72, Phe178, and Asp215 (Fig. 4). Phe303 and Arg315 demonstrated, respectively, π–
π and π–anion interactions with acridine ring. Arg315 also made a weak hydrophobic interaction
with 6-Cl substituent of acridine ring and Thr303 formed a hydrogen bond with 2-methoxy
substituent of this ring. A hydrogen bond between Asp352 and NH group of compound 9b
observed. Finally, Tyr72 and Phe178, through two π–π interactions, and Asp215 and Arg442,
through two π–anion interactions, interacted with the phenyl ring of the N-(4-
methyphenyl)acetamide moiety.
In the second most active compound 9f, interactions with the active site residues Glu277,
Arg315, Arg442, Phe303, Tyr72, His112, Tyr158, and Phe178 were observed (Fig. 5). The
acridine moiety in this compound, only through the acridine ring, interacted with active site
(
Phe303 and Arg315), whereas in the most potent compound, 9b, additional interactions were

observed between the substituents of the acridine ring and the residues in the active site. In the
compound 9f, the sulfur atom showed a weak hydrophobic interaction with Tyr158. Important
residues, Glu277 and Arg442, made π-anion interactions with the phenyl ring of the N-(3-
chlorophenyl)acetamide moiety. In addition, Tyr72 formed a π–π interaction with the
aforementioned ring. Furthermore, the weak hydrophobic interactions between 3-chloro
substituent of the phenyl ring and the active site residues Tyr72, His112, and Phe178 were also
observed.
4
. Conclusion
In conclusion, the novel series of acridine linked to thioacetamide derivatives 9a–o were
designed, synthesized, and evaluated for their α-glucosidase inhibitory and cytotoxic activities.
All of the target compounds revealed high α-glucosidase inhibitory activity against S. cerevisiae
α-glucosidase (IC50s < 383.1 ± 2.0 µM), when compared to acarbose as the standard drug (IC50
50.0 ± 1.5 µM). Among all the synthesized compounds, only 9d, 9c, and 9n exhibited cytotoxic
activity against MCF-7 cell line. In addition, kinetic analysis revealed that the most potent
compound 9b behaves as a competitive inhibitor with a K of 85 µM. The binding interaction of
=
7
i
the active compounds 9b and 9f with the active site of enzyme was confirmed via molecular
docking. This study introduced a new class of compound containing acridine moiety for the
development of novel α-glucosidase inhibitors.
5
. Experimental
All commercially available reagents were purchased from Merck. TLC was performed on silica
gel 250 μ, F254 plates. Melting points were measured using a Kofler hot stage apparatus and are
uncorrected. The IR spectra were recorded using Nicolet FT-IR Magna 550 spectrographs (KBr
1
1
disks). H and CNMR spectra were recorded on an NMR instrument Bruker FT-500 MHz.

Elemental analyses were carried out by an Elementar Analysen system GmbH VarioEL CHN
mode.
5
.1. General procedure for the synthesis of 4-chloro-2-((4-methoxyphenyl)amino)benzoic acid 3
A solution of 4-methoxyaniline 1(5 mmol), 2,4-dichloro-benzoic acid 2 (3 mmol), copper
powder (0.2 mmol), and potassium carbonate (1.5 mmol) in DMF (30 ml) was heated under
reflux for 7 h. The reaction was monitored by TLC and the product, after cooling at room
temperature, was poured into hot water (1 l) and boiled in the presence of activated charcoal for
1
0 min. Thereafter, the obtained mixture was filtered through celite and the filtrate was acidified
with HCl to obtain the precipitated product. The residue was purified using recrystallization from
ethanol to obtain pure products.
5
.2. General procedure for the synthesis of 6,9-dichloro-2-methoxyacridine 4
A mixture of 4-chloro-2-((4-methoxyphenyl)amino)benzoic acid 3 (1 mmol) and POCl
3
(4 mmol)
was heated under reflux for 3 h. After completion of reaction (checked by TLC), the reaction
mixture was cooled down at room temperature, poured into crushed ice and was adjusted to
alkaline pH by liquor ammonia. Thereafter, the mixture was extracted using chloroform (3 × 10
ml), washed with water, and dried over Na
2
SO . The solvent was evaporated under reduced
4
pressure and the residue was purified by chromatography on silica gel eluting with petroleum
ether/ethyl acetate (4:1) to obtain pure products.
5
.3. General procedure for the synthesis of 6-chloro-2-methoxyacridine-9-thiol 5
Thiourea (2 mmol) was added to a stirring solution of compound 4 (1 mmol) in ethanol (10 ml)
and the reaction mixture was stirred at room temperature for 3 h. At the end of the reaction
(
checked by TLC), the obtained precipitate was filtered. The residue was washed with 10 ml of

water, diluted sodium bicarbonate, and diluted sodium hydroxide and was dried at 60 °C to
obtain pure 6-chloro-2-methoxyacridine-9-thiol 5.
5
.4. General procedure for the synthesis of N-phenyl (or benzyl)-2-chloroacetamide 8
Mixture of amines 6 and chloroacetyl chloride 7 in DMF were stirred at room temperature for 30
min. At the end of the reaction (checked by TLC), the reaction mixture was diluted with water,
poured into ice, and the obtained white precipitate was filtered off. The residue was washed with
water to obtain pure N-phenyl (or benzyl)-2-chloroacetamide 8.
5
.5. General procedure for the synthesis of acridine linked to thioacetamide 9
A mixture of 6-chloro-2-methoxyacridine-9-thiol 5 (1 mmol), N-phenyl (or benzyl)-2-
chloroacetamide 8 and potassium carbonate (0. 6 mmol) in acetone (10 ml) was heated under
reflux for 3 h. Thereafter, the mixture was poured in cold water and was extracted using
dichloromethane (4 × 10 ml), and the organic layer was dried by using Na
2
SO . Moreover, the
4
solvent was evaporated under reduced pressure and the residue was purified using
recrystallization from ethanol to obtain the pure products (80–95%).
5
.5.1. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-phenylacetamide (9a)
Pale yellow crystal; yield: 91%, Mp: 218-220 °C. IR (KBr): 3109, 2922, 1630, 1419, 1215, 811
-
1 1
cm . H NMR (500 MHz, DMSO-d
6
): 9.84 (s, , 1H, NH), 8.675 (d, J =15 Hz, 1H, H8), 8.14 (s,
H, H5), 8.035 (d, J =15 Hz, 1H, H7), 7.88 (s, 1H, H1), 7.62 (dd, J = 12.5, 5 Hz, 1H, H4), 7.48-
.51 (dd, J = 12.5, 5 Hz, 1H, H3), 7.16-7.21 (m, 4H, H2', H3', H5', H6'), 6.93-6.97 (m, 1H, H4'),
1
7
3
1
1
13
.84 (s, 3H, OCH
46.91, 146.54, 139.03, 138.69, 134.43, 132.03, 130.68, 129.11, 128.87, 128.41, 127.79, 126.63,
23.99, 119.58, 102.69, 55.92. Anal. Calcd for C22 S: C, 64.62; H, 4.19; N, 6.85.
3
), 3.68 (s, 2H, CH
2
S). C NMR (100 MHz, DMSO-d ): 166.97, 158.55,
6
H17ClN
2
O
2
Fund: C, 64.51; H, 4.31; N, 6.68.

5
.5.2. 2-(6-chloro-2-methoxy acridine -9-ylthio)-N-p-tolylacetanide (9b)
Pale yellow crystal; yield: 82%, Mp: 226-228 °C. IR (KBr): 3136, 2924, 1629, 1558, 1213, 816
-1 1
cm . H NMR (500 MHz, DMSO-d6): 9.74 (s, , 1H, NH), 8.68 (d, J =10 Hz, 1H, H8), 8.12 (s,
H, H5), 8.05 (d, J = 10 Hz, 1H, H7), 7.89 (s, 1H, H1), 7.59 (dd, J = 12.5, 5 Hz, 1H, H4), 7.49
dd, J = 12.5, 5 Hz, 1H, H3), 7.08 (d, J = 10 Hz, 2H, H2', H6'), 6.95 (d, J = 10 Hz, 2H, H3', H5'),
1
(
1
3
3
1
1
.85 (s, 3H, OCH3), 3.65 (s, 2H, CH
66.67, 158.53, 146.92, 146.56, 138.77, 136.51, 134.43, 132.89, 132.01, 129.42, 128.85, 128.41,
28.32, 126.57, 119.63, 102.71, 55.92, 20.96. Anal.Calcd for C23 S: C, 65.32; H, 4.53;
2
S), 2.17 (s, 3H, CH3). C NMR (100 MHz, DMSO-d6):
H11ClN
2
O
2
N, 6.62. Fund: C, 65.21; H, 4.69; N, 6.46.
.5.3. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(4-ethylphenyl)acetamide (9c)
Pale yellow crystal; yield: 86%, Mp: 211-215 °C. IR (KBr): 3062, 2928, 1629, 1417, 1214, 816
5
-1 1
cm . H NMR (500 MHz, DMSO-d
6
): 9.77 (s, , 1H, NH), 8.68 (d, J = 15 Hz, 1H, H8), 8.15 (d, J
5 Hz, 1H, H5), 8.04 (d, J =15 Hz, 1H, H7), 7.89 (d, J = 5 Hz, 1H, H1), 7.61-7.64 (dd, J = 12.5,
Hz, 1H, H4), 7.50-7.53 (dd, J = 12.5, 5 Hz, 1H, H3), 7.10 (d, J = 10 Hz, 2H, H2', H6'), 6.99 (d,
), 3.68 (s, 2H, CH S), 2.41 (m, 2H, CH ), 1.08 (t, J =
). C NMR (100 MHz, DMSO-d ): 166.74, 158.57, 146.92, 146.56, 139.42,
38.80, 136.72, 134.45, 132.06, 128.92, 128.42, 128.30, 127.81, 126.66, 119.69, 102.75, 55.96,
=
5
J = 10 Hz, 2H, H3', H5'), 3.85 (s, 3H, OCH
3
2
2
1
3
1
1
2
4
2.5 Hz, 3H, CH
3
6
8.09, 16.20. Anal.Calcd for C24
.66; N, 6.59.
H21ClN
2
2
O S: C, 65.97; H, 4.84; N, 6.41. Fund: C, 66.11; H,
5
.5.4. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(4-fluorphenyl)acetamide (9d)
Pale yellow crystal; yield: 91%, Mp: 200-203 °C. IR (KBr): 3064, 2103, 1630, 1517, 1215, 816
-1 1
cm . H NMR (500 MHz, DMSO-d ): 8.66 (d, J =5 Hz, 1H, NH), 8.67 (dd, J = 10, 5 Hz, 1H,
6

H8), 8.15-8.13 (m, 1H, H5), 8.01 (dd, J = 10, 5 Hz,1H, H7), 7.86-7.87 (m, 1H, H1), 7.63-7.59
(
m, 1H, H4), 7.51-7.48 (m, 1H, H3), 7.19-7.16 (m, , 2H, H2', H6'), 7.02-6.96 (m, 2H, H3', H5'),
1
3
3
1
1
6
.84 (s, 3H, OCH
3
), 3.65 (s, 2H, CH
2
S). C NMR (100 MHz, DMSO-d ): 166.91, 158.56,
6
46.91, 146.54, 138.58, 135.39, 134.43, 132.04, 130.69, 128.85, 128.42, 127.81, 126.61, 121.33,
21.25, 115.80, 115.58, 102.66, 55.944. Anal.Calcd for C22
.56. Fund: C, 61.78; H, 3.54; N, 6.73.
H16ClFN
2
2
O S: C, 61.90; H, 3.78; N,
5
.5.5. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(2-chlorophenyl)acetamide (9e)
Pale yellow crystal; yield: 84%, Mp: 200-202 °C. IR (KBr): 3060, 2925, 1629, 1427, 1216, 816
-
1 1
cm . H NMR (500 MHz, DMSO-d
5 Hz, 1H, H5), 8.05 (d, J =10 Hz, 1H, H7), 7.88 (d, J = 5 Hz, 1H, H1), 7.62-7.64 (dd, J = 10, 5
Hz, 1H, H4), 7.52-7.55 (dd, J = 10, 5 Hz, 1H, H3), 7.34-7.29 (m, 2H, H3', H6'), 7.14 (m, 1H,
6
): 9.43 (s, , 1H, NH), 8.69 (d, J =10 Hz, 1H, H8), 8.15 (d, J
=
1
3
H5'), 7.07 (m, 1H, H4'), 3.87 (s, 3H, OCH
3
), 3.83 (s, 2H, CH
): 167.61, 158.59, 156.32, 146.94, 146.60, 138.62, 134.80, 134.45, 132.12, 129.86, 129.00,
28.40, 127.85, 127.71, 126.84, 126.62, 126.41, 125.97, 102.81, 56.01. Anal.Calcd for
S: C, 59.60; H, 3.64; N, 6.32. Fund C, 59.46; H, 3.83; N, 6.56.
2
S). C NMR (100 MHz, DMSO-
d
1
6
C
22
H
16Cl
2
N
2
O
2
5
.5.6. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(3-chlorophenyl)acetamide (9f)
Pale yellow crystal; yield: 92%, Mp: 172-178 °C. IR (KBr): 3123, 2925, 1632, 1513, 1216, 815
-
1 1
cm . H NMR (500 MHz, DMSO-d
H, H5), 8.01 (d, J =10 Hz, 1H, H7), 7.85 (d, J = 5 Hz, 1H, H1), 7.57-7.60 (dd, J = 10, 5 Hz, 1H,
H4), 7.48 (dd, J = 10, 5 Hz, 1H, H3), 7.31 (s, 1H, H2'), 7.18-7.14 (m, 1H, H6'), 7.00-6.98 (m,
6
): 9.97 (s, , 1H, NH), 8.65 (d, J =10 Hz, 1H, H8), 8.13 (s,
1
1
3
2
1
H, H4', H5'), 3.85 (s, 3H, OCH
3
), , 3.65 (s, 2H, CH
2
S). C NMR (100 MHz, DMSO-d ):
6
67.32, 158.57, 146.92, 146.55, 140.35, 138.32, 134.43, 133.45, 132.01, 130.70, 128.77, 128.43,

1
5
27.82, 126.54, 123.67, 119.11, 117.91, 102.56, 55.87. Anal.Calcd for C22
9.30; H, 3.64; N, 6.32. Fund: C, 59.41; H, 3.83; N, 6.19.
H
16Cl
2
N
2
2
O S: C,
5
.5.7. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(4-Chlorophenyl)acetamide (9g)
Pale yellow crystal; yield: 80%, Mp: 224-226 °C. IR (KBr): 2923, 2853, 1628, 1466, 1213, 815
-1 1
cm . H NMR (500 MHz, DMSO-d
6
): 9.96 (s, , 1H, NH), 8.65 (dd, J =10, 5 Hz, 1H, H8), 8.14
(
s, 1H, H5), 8.02 (dd, J =10, 5 Hz, 1H7), 7.86 (s, 1H, H1), 7.59-7.62 (m, 1H, H4), 7.51-7.48 (m,
1
2
1
H, H3), 7.34-7.325(m, 2H, H2', H6' ), 7.16-7.14 (m, 2H, H3', H5' ), 3.85 (s, 3H, OCH
3
), 3.67 (s,
): 167.17, 158.57, 146.91, 146.54, 138.49, 138.39,
34.43, 132.07, 131.94, 130.66, 128.82, 128.43, 127.80, 126.61, 121.40, 115.59, 102.63, 55.96.
S: C, 59.60; H, 3.64; N, 6.32; . Fund: C, 59.77; H, 3.81; N, 6.19.
.5.8. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(3,4-dichlorophenyl)acetamide (9h)
Pale yellow crystal; yield: 83%, Mp: 220-223 °C. IR (KBr): 3120, 2922, 1629, 1473, 1215, 812
13
H, CH
2
S). C NMR (100 MHz, DMSO-d
6
Anal.Calcd for C22
H
16Cl
2
N
2
O
2
5
-1 1
cm . H NMR (500 MHz, DMSO-d
6
): 10.03 (d, J = 5 Hz, 1H, NH), 8.65-8.62 (m, 1H, H8), 8.14-
8
7
.13 (m, 1H, H5), 8.02-8.00 (m, 1H, H7), 7.85-7.83 (m, 1H, H1), 7.60-7.58 (m, 1H, H4), 7.50-
1
3
.38 (m, 3H, H3, H2', H6'), 7.03-6.99 (m, 1H, H5'), 3.85 (s, 3H, OCH
): 167.50, 158.60, 146.92, 146.54, 138.97, 134.44, 132.06, 131.33,
31.03, 130.73, 128.77, 128.43, 127.85, 126.54, 120.73, 119.53, 102.55, 55.93. . Anal.Calcd for
S: C, 55.30; H, 3.16; N, 5.86. Fund: C, 55.47; H, 2.98; N, 5.93.
3
), 3.64 (s, 2H, CH
2
S). C
NMR (100 MHz, DMSO-d
6
1
C
22
H
15Cl
3
N
2
O
2
5
.5.9. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(2-bromophenyl)acetamide (9i)
Pale yellow crystal; yield: 86%, Mp: 203-207 °C. IR (KBr): 3131, 2957, 1629, 1511, 1215, 817
-1 1
cm . H NMR (500 MHz, DMSO-d
6
): 9.41 (s , 1H, NH), 8.73 (d, J =10 Hz, 1H, H8), 8.18 (d, J
=5 Hz 1H, H5), 8.07 (d, J =10 Hz, 1H, H7), 7.92 (d, J = 5 Hz, 1H, H1), 7.66 (dd, J = 10, 5 Hz,

1
H, H4), 7.56 (dd, J = 10, 5 Hz, 1H, H3), 7.50 (d, J =10 Hz, 1H, H6'), 7.23-7.17 (m, 1H, H3',
13
H5'), 7.04-7.00 (m, 1H, H4'), 3.90 (s, 3H, OCH
3
), 3.84 (s, 2H, CH S). C NMR (100 MHz,
2
DMSO-d
32.17, 130.59, 129.52, 129.08, 128.43, 127.89, 126.67, 102.82, 56.10. Anal.Calcd for
S: C, 54.17; H, 3.31; N, 5.74. Fund: C, 54.28; H, 3.52; N, 5.59.
.5.10. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(4-bromophenyl)acetamide (9j)
Pale yellow crystal; yield: 83%, Mp: 226-228 °C. IR (KBr): 2922, 2852, 1627, 1469, 1218, 808
6
): 167.49, 158.64, 147.00, 146.64, 144.48, 138.71, 136.15, 134.10, 133.10, 132.89,
1
C
22
H
16BrClN
2
O
2
5
-
1 1
cm . H NMR (500 MHz, DMSO-d
5 Hz, 1H, H5), 8.01 (d, J =10 Hz, 1H, H7), 7.86 (d, J = 5 Hz, 1H, H1), 7.57 (dd, J = 10, 5 Hz,
H, H4), 7.48 (dd, J = 10, 5 Hz, 1H, H3), 7.17-7.22 (m, 4H, H2', H3', H5', H6'), 3.85 (s, 3H,
6
): 9.94 (s, , 1H, NH), 8.65 (d, J =10 Hz, 1H, H8), 8.11 (d, J
=
1
1
3
OCH
3
), 3.65 (s, 2H, CH
2
S). C NMR (100 MHz, DMSO-d ): 167.08, 158.54, 146.88, 146.51,
6
1
1
3
38.52, 137.94, 134.44, 131.99, 130.68, 128.94, 128.77, 128.39, 127.80, 127.59, 126.54, 121.01,
02.60, 55.92. . Anal.Calcd for C22
.48; N, 5.39.
H
16BrClN
2
2
O S: C, 54.17; H, 3.31; N,5.74. Fund: C, 54.25; H,
5
.5.11. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(2-methoxyphenyl)acetamide (9k)
Pale yellow crystal; yield: 95%, Mp: 190-191 °C. IR (KBr):3119, 2924, 1628, 1468, 1215, 814
-
1 1
cm . H NMR (500 MHz, DMSO-d
5 Hz 1H, H5), 8.02 (d, J =10 Hz, 1H, H7), 7.85 (d, J = 5 Hz, 1H, H1), 7.60 (dd, J = 10, 5 Hz,
H, H4), 7.59 (dd, J = 10, 5 Hz, 1H, H3),7.50 (dd, J = 10, 5 Hz , 1H , H6'), 7.23 (s, 1H, H3'),
6
): 8.99(s, , 1H, NH), 8.65 (d, J =10 Hz, 1H, H8), 8.13 (d, J
=
1
6
1
3
.74 (m, 2H, H4', H5'), 3.85 (s, 3H, OCH
3
), 3.78 (s, 2H, CH
2
S), 3.54 (s, 3H, OCH ). C NMR
3
(
100 MHz, DMSO-d
6
): 167.16, 158.48, 147.86, 146.90, 146.54, 134.38, 131.99, 129.16, 129.04,

1
28.35, 128.26, 126.53, 125.20, 122.90, 111.19, 102.83, 55.92, 55.88. Anal.Calcd for
S: C, 62.94; H, 4.36; N, 6.38. Fund: C, 62.81; H, 4.21; N, 6.49.
.5.12. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(3-methoxyphenyl)acetamide (9l)
Pale yellow crystal; yield: 86%, Mp: 220-224 °C. IR (KBr): 3119, 2923, 1628, 1518, 1214,
C
23
H19ClN
2
O
3
5
-
1 1
8
8
7
1
12cm . H NMR (500 MHz, DMSO-d ): 9.80 (s, , 1H, NH), 8.67 (d, J =10 Hz, 1H, H8), 8.12-
6
.11 (d, J =5 Hz 1H, H5), 8.01 (d, J =10 Hz, 1H, H7), 7.89 (s, 1H, H1), 7.60-7.56 (m, 1H, H4),
.49-7.45 (m, 1H, H3), 7.06-7.02 (m, 1H, H5'), 6.85 (d, J = 5 Hz, 1H, H2'), 6.73 (d, J = 10 Hz,
H, H6'), 6.53-6.50 (m, 1H, H3'), 3.85 (s, 3H, OCH
3
), 3.78 (s, 2H, CH
): 166.93, 159.83, 158.53, 146.91, 146.55, 140.16, 138.64,
34.44, 131.96, 130.71, 129.78, 128.40, 128.31, 127.80, 126.55, 55.88, 55.32. Anal.Calcd for
S: C, 62.94; H, 4.36; N, 6.38. Fund: C, 62.81; H, 4.21; N, 6.52.
.5.13. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(4-methoxyphenyl)acetamide (9m)
Pale yellow crystal; yield: 91%, Mp: 216-218 °C. IR (KBr): 2923, 2853, 1627, 1467, 1214, 815
2
S), 3.54 (s, 3H, OCH ).
3
1
3
C NMR (100 MHz, DMSO-d
6
1
C
23
H
19ClN
2
O
3
5
-
1 1
cm . H NMR (500 MHz, DMSO-d
5 Hz, 1H, H5), 8.00 (d, J =10 Hz, 1H, H7), 7.90 (d, J = 5 Hz, 1H, H1), 7.56 (dd, J = 10, 5 Hz,
H, H4), 7.47 (dd, J = 10, 5 Hz, 1H, H3), 7.51-7.48 (m, 1H, H3), 7.08 (d, J = 10 Hz, 2H, H2',
H6'), 6.69 (d, J = 10 Hz, 2H, H3', H5'), 3.86 (s, 3H, OCH ), 3.64 (s, 3H, OCH ), 3.62 (s, 2H,
): 166.39, 165.23, 158.51, 155.83, 146.91, 146.55,
38.80, 134.44, 132.10, 131.93, 128.81, 128.38, 128.23, 126.50, 121.16, 114.06, 102.70, 55.89,
6
): 9.65 (s, , 1H, NH), 8.68 (d, J =10 Hz, 1H, H8), 8.10 (d, J
=
1
3
3
1
3
CH
2
S). C NMR (100 MHz, DMSO-d
6
1
5
4
5.54, 40.66. Anal.Calcd for C23
.51; N, 6.21.
H
19ClN
2
3
O S: C, 62.94; H, 4.36; N, 6.36. Fund: C, 63.13; H,
5
.5.14. 2-(6-chloro-2-methoxyacridin-9-ylthio)-N-(benzyl)acetamide (9n)

Pale yellow crystal; yield: 88%, Mp: 199-203 °C. IR (KBr): 3111, 2923, 1628, 1417, 1213, 819
-
1 1
cm . H NMR (500 MHz, DMSO-d
6
): 8.60 (d, J =15 Hz, 1H, H8), 8.20 (t, J = 10 Hz, 1H, NH),
.08 (d, J = 5 Hz, 1H, H5), 8.01 (d, J =10 Hz, 1H, H7), 7.85 (d, J = 5 Hz, 1H, H1), 7.57 (dd, J =
0, 5 Hz, 1H, H4), 7.51 (dd, J = 12.5, 5 Hz, 1H, H3), 7.13-7.05 (m, 3H, H3', H4', H5'), 6.70-6.72
8
1
1
3
(
m, 2H, H2', H3'), 3.98 (d, 2H, J = 5 Hz, CH
2
N), 3.93 (s, 3H, OCH
3
), 3.57 (s, 2H, CH S). C
2
NMR (100 MHz, DMSO-d
6
): 168.12, 158.45, 146.64, 146.45, 138.97, 138.93, 134.39, 131.98,
1
3
6
30.38, 129.00, 128.59, 128.30, 128.18, 127.71, 127.45, 127.25, 126.52, 102.87, 56.09, 42.82,
9.52. Anal.Calcd for C23
.83.
H19ClN
2
2
O S: C, 65.32; H, 4.53; N, 6.62. Fund C, 65.51; H, 4.39; N,
5
.5.15. N-(4-fluorobenzyl)-2-(6-chloro-2-methoxyacridin-9-ylthio)acetaide (9o)
Pale yellow crystal; yield: 93%, Mp: 201-203 °C. IR (KBr):3116, 2924, 1629, 1468, 1217, 807
-
1 1
cm . H NMR (500 MHz, DMSO-d
.12 (d, J = 5 Hz, 1H, H5), 8.035 (d, J =15 Hz, 1H, H7), 7.87 (d, J = 5 Hz, 1H, H1), 7.59-7.61
dd, J = 10, 5 Hz, 1H, H4), 7.52-7.55 (dd, J = 10, 5 Hz, 1H, H3), 6.87-6.93 (m, , 2H, H2', H6'),
6
): 8.62 (d, J =10 Hz, 1H, H1), 8.23 (t, J =10 Hz, 1H, NH),
8
(
1
3
6
1
1
.78-6.75 (m, 2H, H3', H5'), 3.95-3.94 (m, 5H, OCH
3
, CH
2
S). C NMR (100 MHz, DMSO-d ):
6
68.12, 161.35 (d, JC-F = 301.25 Hz), 158.49, 146.88, 146.49, 134.44, 132.03, 129.44, 129.36,
29.01, 128.33, 128.26, 126.59, 115.38, 115.17, 102.88, 56.12, 42.04. Anal.Calcd for
C
23
H18ClFN
2
O
2
S: C 62.65; H, 4.11; N, 6.35. Fund C 62.76; H, 4.28; N, 6.18.
5
.6. α-Glucosidase inhibition assay
Saccharomyces cerevisiae α-glucosidase (EC3.2.1.20, 20 U/mg) and substrate (p-nitrophenyl
glucopyranoside) were purchased from Sigma-Aldrich. Enzyme was prepared in potassium

phosphate buffer (pH 6.8, 50 mM), and the synthesized compounds 9a–o were dissolved in
DMSO (10 % final concentration).
The various concentrations of compounds 9a-o (20 µL), enzyme solution (20 µL), and potassium
phosphate buffer (135 µL), were added in the 96-well plate and incubated at 37 °C for 10 min.
Thereafter, the substrate (25 µL, 4 mM) was added to the mixture and allowed to incubate at 37
°C for 20 min. Eventually, the change in absorbance was measured at 405 nm by using
spectrophotometer (Gen5, Power wave xs2, BioTek, America). DMSO as control (10 % final
concentration) and acarbose as the standard drug were used. The percentage of inhibition for
each entry was calculated by using the following formula:
%
Inhibition = [(Abs control _ Abs sample)/Abs control] ×100
IC50 values were obtained from the nonlinear regression curve using the Logit method.
.7. In vitro cytotoxicity assay
5
Evaluation of cytotoxic effects of the synthesized compounds 9a–o was performed exactly based
on our previous report [10].
5
.8. Kinetic study
To evaluate the inhibition mode of the synthesized compound, kinetic studies were performed.
The 20 µl of enzyme solution (1 U/mL) was incubated with 20 µl of various concentrations (0,
6
0, 80, and 100 µM) of the most potent compound 9b for 15 min at 30 °C. The reaction was then
initiated by adding different concentrations of p-nitrophenyl glucopyranoside as substrate (1–4
mM), and the change in absorbance was determined for 20 min at 405 nm by using a
spectrophotometer (Gen5, Power wave xs2, BioTek, America).

5
.9. Molecular docking study
Docking studies were performed using the AUTODOCK 4.2 program and the pdb structure of
3
A4A was taken from the Brookhaven protein database (http://www.rcsb.org). Subsequently, the
additional molecules (water molecules and the original inhibitors) were removed from the
protein structure. The 3D structure of the most active compounds 9b and 9f were provided using
MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com). After the preparation of
the autodock format of protein and selected compounds, the obtained enzyme structure was used
as an input for the AUTOGRID program. AUTOGRID for each atom type in the ligand
performed a precalculated atomic affinity grid maps, plus an electrostatics map and a separate
desolation map presented in the ligand. All maps were calculated with 0.375 Å spacing between
the grid points. The center of the grid box was placed in coordinates x = 22.6225, y = −8.069, z =
2
4.158. The dimensions of the active site box for all docks that were set at 50 × 50 × 50, centered
at the geometrical center of co-crystallized ligand, were used. Flexible ligand dockings were
accomplished for the selected ligands. Each docked system was carried out by 50 runs of the
AUTODOCK search by the Lamarckian genetic algorithm (LGA). The best pose of each ligand
was selected for analyzing the interactions between isomaltase and the inhibitor. The results were
visualized using Discovery Studio 4.0 Client (Figs. 2–5).
Acknowledgment
The authors gratefully acknowledge the Research Council of Tehran University of Medical
Sciences and Iran National Science Foundation (INSF).
Support information
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Captions:
Fig. 1. Kinetics of α-gluosidase inhibition by 9b. (a) The Lineweaver– Burk plot in the absence
and presence of different concentrations of 9b (µM); (b) the secondary plot between K
m
and
various concentrations of 9b.
Fig. 2. Superimposition of the most potent compound 9b (pink) and acarbose (cyan) in the active
site of enzyme.
Fig. 3. Docking pose of acarbose in the active site of enzyme.
Fig. 4. Docking pose of the compound 9b in the active site of enzyme.
Fig. 5. Docking pose of the compound 9f in the active site of enzyme.
Scheme 1. Synthesis of compounds 9. Reagents and conditions:: (a) K
7h, (b) POCl , reflux, 3 h, (c) Thiourea, EtOH, r.t, 1 h, (d ) r.t, 30 min, and (e) EtOH, K
reflux, 2h.
2
CO
3
, Cu, DMF, reflux
,
3
2
CO ,
3

Table 1
In vitro α-glucosidase inhibitory activity of compounds 9a–o.
a
Compound
n
0
R
H
IC50 (µM)
9
9
a
95.0 ± 1.9
80.0 ± 2.0
129.5 ± 2.0
241.0 ± 1.7
195.0 ± 1.2
92.2 ± 2.0
144.2 ± 1.1
102.0 ± 1.3
173.4 ± 1.6
118.2 ± 2.0
383.1 ± 2.0
96.6 ± 2.0
147.0 ± 1.5
118.3 ± 1.7
220.0 ± 1.5
750.0 ± 1.5
b
0
0
0
0
0
0
0
0
0
0
0
0
1
1
-
4-CH3
4-CH2CH3
4-F
9
c
9
d
9
e
2-Cl
9
f
3-Cl
9
9
g
4-Cl
h
3,4-dichloro
2-Br
9
i
9
j
4-Br
9
k
2-OCH3
3-OCH3
4-OCH3
H
9
l
9
m
9
n
o
9
F
Acarbose
-
a
Data are expressed as mean ± S.E. of at least three different experiments.

Table 2
Cytotoxicity evaluation of compounds 9a-o
a
a
Compound
MCF-7 (IC50 μM)
>100
Compound
MCF-7 (IC50 μM)
>100
9
9
a
9i
b
>100
9j
>100
9
c
26.59 ± 1.3
13.42 ± 0.6
>100
9k
>100
9
d
9l
9m
>100
9
e
>100
9
f
>100
9n
55.23 ± 0.8
>100
9
9
g
>100
9o
h
>100
Etoposide
12.4 ± 4.7
a
Data are expressed as mean ± S.E. of at least three different experiments.

a)
2
2
1
1
5
0
5
0
5
0
5
Concentration of Inhibitor
0
6
8
1
µM
0 µM
0 µM
00 µM
-
1.0
-0.5
0.0
0.5
1.0
1.5
-
1/(S, mM)
-
10
b)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-100
-50
0
50
100
150
Inhibitor (µM)
Fig. 1. Kinetics of α-gluosidase inhibition by 9b. (a) The Lineweaver– Burk plot in the absence
and presence of different concentrations of 9b (µM); (b) the secondary plot between K
m
and
various concentrations of 9b.

Fig. 2. Superimposition of the most potent compound 9b (pink) and acarbose (cyan) in the active
site of enzyme.

Fig. 3. Docking pose of acarbose in the active site of enzyme.

Fig. 4. Docking pose of the compound 9b in the active site of enzyme.