Design, synthesis, biological evaluation, and docking study of new acridine-9-carboxamide linked to 1,2,3-triazole derivatives as antidiabetic agents targeting α-glucosidase

Article abstract of DOI:10.1002/jhet.4142

A new series of acridine-9-carboxamide-1,2,3-triazole derivatives 7a-m were designed, synthesized, and evaluated as novel α-glucosidase inhibitors. Acridine-9-carboxamide-1,2,3-triazole scaffold has been designed by combination of effective moieties from potent α-glucosidase inhibitors. Most of the synthesized compounds were more potent than standard inhibitor acarbose. Among the title compounds, the most potent compounds were compounds 7j, 7k, and 7a with IC₅₀ values of 120.2 ± 1.0, 151.1 ± 1.4, and 157.6 ± 1.6 μM, respectively (IC₅₀ value of acarbose = 750.0 ± 10.0 μM). Docking study of the most potent compounds demonstrated that these compounds formed stable complexes with α-glucosidase active site. Anti-α-amylase assay of compounds 7j, 7k, and 7a was performed and no activity was observed. in vitro cytotoxicity assay of the latter compounds revealed that these compounds were not cytotoxic toward human normal (HDF) and cancer (MCF-7) cell lines. ADME and toxicity prediction of compounds 7j, 7k, and 7a were also performed.

Full text of DOI:10.1002/jhet.4142

Design, synthesis, biological evaluation, and docking study of new acridine-9-carboxamide
linked to 1,2,3-triazole derivatives as antidiabetic agents targeting α-glucosidase
a
b
c
Mohammad Sadegh Asgari, Behnam Tahmasebi, Somayeh Mojtabavi, Mohammad Ali
c
d
a
b
b
Faramarzi, Rahmatollah Rahimi, Parviz Rashidi Ranjbar, Mahmood Biglar, Bagher Larijani,
e
f
b
Hossein Rastegar, Maryam Mohammadi-Khanaposhtani, * Mohammad Mahdavi, *
a
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
b
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
*
E-mail: momahdavi@tums.ac.ir
c
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
d
Department of Chemistry, Iran University of Science and Technology, Tehran, Iran
e
Food and Drug Research Institute, Food and Drug Administration, MOHE, Tehran, Iran
f
Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of
Medical Sciences, Babol, Iran
*
E-mail: maryammoha@gmail.com
A new series of acridine-9-carboxamide-1,2,3-triazole derivatives 7a-m were designed,
synthesized, and evaluated as novel α-glucosidase inhibitors. Acridine-9-carboxamide-1,2,3-
triazole scaffold has been designed by combination of effective moieties from potent α-
glucosidase inhibitors. Most of the synthesized compounds were more potent than standard
inhibitor acarbose. Among the title compounds, the most potent compounds were compounds 7j,
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which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/jhet.4142
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7
k, and 7a with IC values of 120.2 ± 1.0, 151.1 ± 1.4, and 157.6 ± 1.6 μM, respectively (IC
50 50
value of acarbose = 750.0 ± 10.0 μM). Docking study of the most potent compounds
demonstrated that these compounds formed stable complexes with α-glucosidase active site.
Anti-α-amylase assay of compounds 7j, 7k, and 7a was performed and no activity was observed.
In vitro cytotoxicity assay of the latter compounds revealed that these compounds were not
cytotoxic toward human normal (HDF) and cancer (MCF-7) cell lines. ADME and toxicity
prediction of compounds 7j, 7k, and 7a were also performed.
Keyword: Acridine-9-carboxamide; 1,2,3-Triazole; α-Glucosidase; Docking; ADME
INTRODUCTION
Diabetes mellitus is a prevalent metabolic disorder which led to defective homeostasis of glucose
in the blood due to inadequate insulin secretion (Type 1) and/or defects in insulin action (Type 2)
[
1]. Based on the type of diabetes, various medications are used for regulation of blood glucose;
commonly, insulin is used for type 1 diabetes and biguanidus, sulfonylureas, thiazolidinedones,
and α-glucosidase inhibitors are used for type 2 diabetes [2-4]. α-Glucosidase is an intestinal
enzyme that plays a major role in the degradation of carbohydrates to glucose so inhibiting it can
reduce the release of glucose into the bloodstream [5]. α-Glucosidase inhibitors like acarbose,
miglitol, and voglibose are used effectively for treatment of type 2 diabetes [6]. However these
drugs created side effects such as hepatic disorders, flatulence, gastrointestinal diseases,
improper absorption, and diarrhea [7, 8]. Therefore, during the last two decades, pharmaceutical
chemists reported various α-glucosidase inhibitors with different scaffolds [9, 10]. Furthermore,
there are evidences that shows α-glucosidase inhibitors can be useful in the treatment of other
carbohydrate related diseases [11].
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Aromatic tricyclic heterocycles such as acridine, xanthone, and carbazole exhibited various
pharmacological properties such as anti-cancer, anticonvulsant, anti-HIV, antioxidant, anti-
inflammatory, and anticholinesterase activities [12-14]. These scaffolds are also found in the
potent α-glucosidase inhibitors such as compounds A-C (Fig. 1) [15-17]. Another important
pharmacophore in the design of α-glucosidase inhibitors is 1,2,3-triazole ring. In addition to
compounds B and C, this ring is also found in several strong α-glucosidase inhibitors such as
compounds D [18]. Thus, in continuation of our attempts for design and synthesis of new
heterocyclic compounds as potent α-glucosidase inhibitors, herein, we designed a new series of
acridine-9-carboxamide-1,2,3-triazoles 7a-m and synthesized them with simple reactions. Then,
these compounds evaluated against yeast α-glucosidase by in vitro and in silico assays [19-21].
Since acridine is found in numerous anti-tumor agents, cytotoxic effects of title compounds were
evaluated against cancer and normal human cells by MTT assay [22].
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Figure 1. Design strategy for new α-glucosidase inhibitors 7a-m with acridine-9-carboxamide
,2,3-triazole scaffold.
1
RESULTS AND DISCUSSION
Chemistry. Synthetic procedure for the preparation of the acridine-9-carboxamide-1,2,3-triazole
derivatives 7a-m is depicted in Scheme 1. This procedure was started with reaction between acridine-9-
carboxylic acid 1 and propargylamine 2 in the presence of HOBt/EDCI in dry acetonitrile for produce N-
(prop-2-yn-1-yl)acridine-9-carboxamide 3. On the other hand, azide derivatives 6a-m were prepared
according to our previous work [18]. In the final step, N-(prop-2-yn-1-yl)acridine-9-carboxamide 3,
catalytic amount of sodium ascorbate, and CuSO were added to the freshly prepared azides 6a-m to
4
provide desired compounds 7a-m.
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Scheme 1. Synthesis of compounds 7a-m. Reagents and conditions: (a) HOBT/EDCI, dry
CH CN, R.T., 24 h; (b) NEt , H O/t-BuOH, R.T., 1 h; (c) CuSO ·5H O, sodium ascorbate, R.T.,
3
3
2
4
2
1
6–24 h.
In vitro α-glucosidase inhibition assay. The in vitro α-glucosidase inhibition of the synthesized
compounds 7a-m was evaluated against yeast form of this enzyme (Saccharomyces cerevisiae).
The IC values of compounds 7a-m in comparison to acarbose as standard drug are presented in
5
0
Table 1. All the synthesized compounds (IC = 120.2 ± 1.0-624.4 ± 9.3 µM), with the exception
5
0
of 7d and 7f (IC > 750 µM), exhibited higher inhibitory activity respect to acarbose (IC =
5
0
50
1
20.2 ± 1.0-624.4 ± 9.3 µM). Among the synthesized compounds, compounds 7j, 7k, and 7a
with inhibitory activities 6-4 folds more than standard inhibitor were the most potent compounds.
As can be seen in Table 1, compound 7a with un-substituted pendant phenyl ring was the third
most potent compound (IC = 157.6 ± 1.6 µM). The introduction of electron-donating
50
substituents 2-metyl or 3-methoxy on pendant phenyl ring led to a significant decrease in
inhibitory activity as observed in compounds 7b (IC = 624.4 ± 9.3 µM) and 7c (IC = 616.3 ±
5
0
50
9
.0 µM), while inhibitory activity of remaining derivatives with electron withdrawing
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substituents depend on the type and position of the substituent. In this regards, 2-fluoro
derivative 7d and 4-fluoro derivative 7f were inactive against α-glucosidase while their chloro
analogs (2-chloro derivative 7g and 4-chloro derivative 7h) exhibited high anti-α-glucosidase
activity in comparison to standard inhibitor. Moreover, 3-fluoro derivative 7e was a moderate
inhibitor for α-glucosidase. Among the synthesized compounds, 3-bromo derivative 7j (IC =
5
0
1
20.2 ± 1.0 µM) showed the most potent activity. Changing the position of bromo substituent in
the pendant phenyl ring from C-3 to C-4, as in compound 7k (second potent compound; IC =
5
0
1
51.1 ± 1.4 µM) slightly diminished the activity, while this changing from C-3 to C-2
dramatically decreased inhibitory activity as observed in compound 7i (IC = 303.7 ± 4.3 µM).
5
0
Similar to fluoro and bromo substituents, nitro substituent showed a better effect when placed in
-position (compound 7l vs 7m).
3
Table 1
α-Glucosidase inhibitory activities of the synthesized compounds 7a-m.
a
a
Compound R
IC (µM)
Compound R
IC (µM)
50
5
0
7
a
H
157.6 ± 1.6
624.4 ± 9.3
616.3 ± 9.0
> 750
7h
7i
4-Cl
165.2 ± 1.7
303.7 ± 4.3
120.2 ± 1.0
151.1 ± 1.4
7
b
2-CH
3
2-Br
3-Br
4-Br
7
c
3-OCH
3
7j
7
d
2-F
7k
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7
e
3-F
4-F
2-Cl
580.6 ± 7.7
> 750
7l
7m
3-NO
2
2
229.7 ± 2.6
404.2 ± 5.6
750.0 ± 10.0
7
f
4-NO
7
g
216.6 ± 2.5
Acarbose
a
Values are the mean ± SD. All experiments were performed at least three times.
Kinetic study. To give the inhibition mode of the synthesized compounds into α-glucosidase, an
enzyme kinetic assay was performed on the most potent compound 7j. As can be seen in Fig. 2a,
with increase of concentration of inhibitor 7j, V did not change while K increased. So,
m
m
compound 7j is a competitive inhibitor for α-glucosidase. The value of K for compound 7j was
i
1
16 µM (Fig. 2b).
a)
b)
7
6
5
4
3
2
1
0
3
2
1
0
0
0
0
Concentration of Inhibitor
0
4
8
1
µM
0 µM
0 µM
20 µM
-
1.5
-1.0
-0.5
0.0
0.5
1.0
-1
1
/[S](PNPG, mM )
-
10
-150
-100
-50
0
50
100
150
-1
Compound (7j)
Figure 2. Kinetics of α-glucosidase inhibition by compound 7j. (a) The Lineweaver– Burk plot
in the absence and presence of different concentrations of compound 7j; (b) The secondary plot
between K and various concentrations of compound 7j.
m
Docking study in α-glucosidase active site. Interaction poses of acarbose as standard inhibitor
and the most potent compounds 7j, 7k, and 7a were screened in the active site of modeled α-
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glucosidase by molecular docking study [19]. Acarbose in α-glucosidase active site was showed
in Fig. 3a and details of acarbose interaction in the active site was exhibited in 2-dimensional
form in Fig. 3b. As can be seen in the latter Fig., acarbose established hydrogen bonds with
active site residues Arg312, Thr307, Gln322, Thr301, Glu304, Asn241, and Ser308 and a
hydrophobic interaction with His279. Furthermore, acarbose formed weak carbon hydrogen
bonds with Phe157, Val305, Glu304, and His239 and two unfavorable interactions with Thr307
and Arg312. The binding energy of this drug in the active site of modeled α-glucosidase was -4.4
kcal/mol.
(a)
(b)
Figure 3. (a) Acarbose and (b) it interaction pose in the active site of modeled α-glucosidase.
1
,2,3-Triazole ring of the most potent compound 7j formed π-π and π-cation interactions with
residues His239 and His279, respectively (Fig. 4a). This compound also established a π-anion
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interaction with Glu304 via pendant 3-bromophenyl group. The latter group also formed a
hydrophobic interaction with Pro309 via bromo substituent and two hydrophobic interactions
with Pro309 and Arg312 via phenyl ring. Furthermore, acridine ring established two
hydrophobic interactions with Arg312. The binding energy value for this compound was
calculated to be -9.36 kcal/mol, which was lower from acarbose (−4.04 kcal/mol) and hence,
compound 7j can bind to α-glucosidase active site more easily than acarbose.
4
-Bromo substituent of the second potent compound 7k formed two hydrophobic interactions
with Phe300 and Gln350. 1,2,3-triazole ring of this compound in addition two interactions with
Arg312 (hydrophobic interaction) and Phe157 (π-Lone Pair), formed an unfavorable interaction
with Asp408. Compound 7k also established a hydrogen bond with Arg312 via carbonyl unit.
Acridine ring of this compound formed π-anion interactions with Glu304 and a carbon hydrogen
bond with Asn241.
As can be seen in Fig. 4., the interaction modes of the most potent compound 7j and the third
most potent compound 7a are similar and have only two differences: 1) acridine ring of
compound 7a formed an additional π-π interaction with Phe158 and 2) un-substituted phenyl ring
of compound 7a, unlike compound 7j, only formed a hydrophobic interaction with Pro309.
The comparison of binding energies of the most active compound 7j (-9.36 kcal/mol) with the
second potent compound 7k (−9.09 kcal/mol) and third potent compound 7a (-8.85 kcal/mol)
demonstrated that theoretically the compound 7j bound easily than compounds 7k and 7a to
active site of α-glucosidase. This finding is in agreement to biological data (Table 1).
(a)
(b)
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(
c)
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Figure 4. Interaction modes of the most active compounds 7j (a), 7k (b), and 7a (C) in the active
site pocket.
In vitro α-amylase inhibitory activity assay. α-Amylase is a pancreatic carbohydrate-
hydrolyzing enzyme that polysaccharides converted to oligo- and disaccharides [23]. Although
α-amylase inhibition can play a positive role in lowering blood glucose level, it raises the amount
of undigested polysaccharide in the intestine and causes gastrointestinal side effects [24].
Therefore, α-amylase inhibitory activity of the most potent compounds 7j, 7k, and 7a were
evaluated against porcine pancreatic α-amylase [20]. As can be seen Table 2, In vitro inhibition
assay of the compounds 7j, 7k, and 7a (IC s > 200 ) showed that these compounds are inactive
5
0
against α-amylase in comparison to acarbose (IC = 108 ± 0.71 µM).
5
0
Table 2
Anti-α-amylase activity and cytotoxicity of compounds 7j, 7k, and 7a.
Compound
Enzyme/Cell line
MCF-7
> 200
α-Amylase
> 200
HDF
> 200
> 200
> 200
-
7
j
7
7
k
a
> 200
> 200
108 ± 0.71
-
> 200
> 200
Acarbose
Etoposide
-
22.08 ± 0.39
92.7 ± 1.2
In vitro cytotoxicity. In vitro cytotoxicity of the compounds 7j, 7k, and 7a was evaluated against
human normal cell line HDF and human cancer cell line MCF-7 by MTT assay [15, 18].
Obtained results revealed that at 200 µM, studied compounds were non-cytotoxic against two
cell line MCF-7 and HDF when compared with etoposide as an anti-tumor agent (Table 2).
ADME and Toxicity studies. ADME/T properties of the compounds 7j, 7k, and 7a as most
potent α-glucosidase inhibitors among the synthesized compounds and standard inhibitor
acarbose were predicted using online software PreADMET (Table 3) [25]. As can be seen in
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Table 3, new synthesized compounds and acarbose have poor penetration into Caco-2 cell, blood
brain barrier (BBB), and skin. Moreover, compounds 7j, 7k, and 7a have high human oral
absorption (HIA) while acarbose does not show HIA. In term of toxicity, all studied compounds
with the exception of the most potent compound 7j, were mutagenic. In silico toxicity assay also
demonstrated that cardiotoxicity acarbose is ambiguous while compounds 7j, 7k, and 7a
exhibited medium risk in term of cardiotoxicity (hERG inhibition). Furthermore, the latter new
compounds, unlike acarbose, have not carcinogenic effect on mouse. On the other hand, only
compound 7a, like acarbose have not carcinogenic effect on rat.
Table 3. ADME/T Profile of the most potent compounds 7j, 7k, and 7a.
a
ADME/T
Compound
7
j
7k
7a
Acarbose
9.44448
Caco2
HIA
24.4555
97.623813
0.0296523
-2.76734
24.4254
21.2183
97.090144
0.0218381
-2.87797
97.623813
0.0282977
-2.76843
Mutagen
Medium risk
Negative
Positive
0.000000
0.0271005
-5.17615
Mutagen
Ambiguous
Positive
BBB
Skin Permeability
Ames test
Non-mutagen
Medium risk
Negative
Mutagen
hERG inhibition
Carcino Mouse
Carcin Rat
Medium risk
Negative
Positive
Negative
Negative
a
The recommended ranges for Caco2: <25 poor, >500 great, HIA: >80% is high <25% is poor, BBB = –3.0
–
1.2, and Skin_Permeability = –8.0 – –1.0.
CONCLUSION
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In conclusion, new acridine-9-carboxamide-1,2,3-triazole derivatives were designed,
synthesized, and evaluated as potent α-glucosidase inhibitors. Most of the synthesized
compounds were more potent than acarbose and among them, the most potent compounds were
3
-bromo derivative 7j, 4-bromo derivative 7k, and un-substituted derivative 7a with inhibitory
activity around 6-4 fold more than acarbose. Docking study was performed to elucidate the
observed anti-α-glucosidase activity on the most potent compounds using Autodock tools. α-
Amylase inhibitory activity and cytotoxicity of the most potent compounds in order to assess the
possible side effects were screened and no anti-α-amylase activity and no cytotoxicity were
observed. ADME/Toxicity prediction of the most potent compounds and acarbose were also
performed.
EXPERIMENTAL
General chemistry. Melting points of acridine-9-carboxamide-1,2,3-triazoles 7a-m were
1
13
measured on a Kofler hot stage apparatus. The NMR ( H and C) and IR spectra of these
compounds were obtained by using a Bruker FT-500 and Nicolet Magna FTIR 550
spectrophotometer on KBr disks, respectively. Mass spectrum was performed by an Agilent
Technology (HP) mass spectrometer (ionization potential = 70 eV). Elemental analysis was
measured by an Elementar Analysensystem GmbH VarioEL CHN mode. Compounds 6a-m were
obtained according to our previous work [18].
Synthesis of N-(prop-2-yn-1-yl)acridine-9-carboxamide 3. A mixture of acridine-9-carboxylic
acid 1 (1 mmol), hydroxybenzotriazole (HOBt, 0.13 g, 1 mmol), and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI, 0.17 g, 1.1 mmol) was stirred in dry acetonitrile (10
mL) at room temperature for 30 min. After that, propargylamine 2 (0.05 g, 1 mmol) was added to
the latter mixture and the reaction was continued at room temperature. After 24 hours,
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acetonitrile was evaporated under vacuum and the obtained residue was dissolved in
dichloromethane and washed with sodium carbonate 10%. Dichloromethane solution was dried
by anhydrous sodium sulfate (Na SO4) and dichloromethane was evaporated to give pure
2
compound 3.
General procedure for the synthesis of acridine-9-carboxamide-1,2,3-triazole derivatives
7
a–m. A mixture of compound 3 (1 mmol), CuSO4 (7 mol%), and sodium ascorbate was added
to freshly prepared azides 6a-m in H O and t-BuOH (10 mL, 1:1) and obtained mixture was
2
stirred at room temperature for 16-24 h. Then, the reaction mixture was diluted with cold water,
precipitated products 7a-m were filtered off, washed with cold water, and purified by
recrystallization in ethyl acetate.
N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7a. White solid; isolated
-1 1
yield: 89%; mp 166-168 °C; IR (KBr, υ): 3295, 3051, 2926, 1638 cm ; H NMR (500 MHz,
DMSO-d ) δ 9.54 (s, 1H, NH-amide), 8.22 (s, 1H, H-triazole), 8.19 (d, J = 8.7 Hz, 2H, Ar), 7.97
6
(d, J = 8.7 Hz, 2H, Ar), 7.88 (ddd, J = 8.0, 6.6, 1.3 Hz, 2H, Ar), 7.63 – 7.58 (m, 2H, Ar), 7.41 –
1
3
7
.34 (m, 5H, Ar), 5.66 (s, 2H, CH2-Ph), 4.75 (s, 2H, CH -NH); C NMR (126 MHz, DMSO-d )
2 6
δ 166.53, 148.60, 145.07, 142.34, 136.69, 131.06, 129.70, 129.23, 128.60, 128.40, 127.10,
+
1
26.06, 123.70, 122.26, 53.27, 35.16; MS (70 eV, m/z [M ]) = 393; Anal Calcd for C H N O,
2
4
19
5
C, 73.27; H, 4.87; N, 17.80 found: C, 73.28; H, 4.83; N, 17.84.
N-((1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7b. White
-1 1
solid; isolated yield: 83%; mp 162-164 °C; IR (KBr, υ): 3289, 3054, 2922, 1630 cm ; H NMR
(
500 MHz, DMSO-d ) δ 9.54 (s, 1H, NH-amide), 8.19 (d, J = 8.7 Hz, 2H, Ar), 8.09 (s, 1H, H-
6
triazole), 7.97 (d, J = 8.6 Hz, 2H, Ar), 7.93 – 7.81 (m, 2H, Ar), 7.66 – 7.51 (m, 2H, Ar), 7.33 –
1
3
7
.07 (m, 4H, Ar), 5.67 (s, 2H, CH2-Ph), 4.77 (s, 2H, CH -NH), 2.34 (s, 3H, Me); C NMR (126
2
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MHz, DMSO-d ) δ 166.51, 148.61, 144.97, 142.34, 136.83, 134.72, 131.06, 130.92, 129.72,
6
1
29.16, 128.80, 127.10, 126.72, 126.03, 123.69, 122.26, 51.48, 35.18, 19.11; MS (70 eV, m/z
+
[
M ]) = 407; Anal Calcd for C H N O, C, 73.69; H, 5.19; N, 17.19 found: C, 73.74; H, 5.13; N,
2
5
21
5
1
7.14.
N-((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7c. White
-1 1
solid; isolated yield: 88%; mp 169-171 °C; IR (KBr, υ): 3289, 3053, 2929, 1632 cm ; H NMR
500 MHz, DMSO-d ) δ 9.55 (t, J = 5.7 Hz, 1H, NH-amide), 8.22 (s, 1H, H-triazole), 8.19 (d, J =
(
6
8
.8 Hz, 2H, Ar), 7.98 (d, J = 8.6 Hz, 2H, Ar), 7.90 – 7.86 (m, 2H, Ar), 7.63 – 7.58 (m, 2H, Ar),
.32 (t, J = 7.9 Hz, 1H, Ar), 6.96 – 6.91 (m, 3H, Ar), 5.63 (s, 2H, CH2-Ph), 4.77 (d, J = 5.7 Hz,
7
13
2
H, CH -NH), 3.74 (s, 3H, OCH ); C NMR (126 MHz, DMSO-d ) δ 166.54, 159.93, 148.60,
2 3 6
1
45.09, 142.35, 138.12, 131.05, 130.39, 129.71, 127.10, 126.06, 123.71, 122.27, 120.48, 114.30,
+
1
13.81, 55.56, 53.19, 35.16; MS (70 eV, m/z [M ]) = 423; Anal Calcd for C H N O , C, 70.91;
25
21
5
2
H, 5.00; N, 16.54 found: C, 70.92; H, 5.10; N, 16.48.
N-((1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7d. White solid;
-1 1
isolated yield: 87%; mp 180-182 °C; IR (KBr, υ): 3289, 3051, 2924, 1635 cm ; H NMR (500
MHz, DMSO-d ) δ 9.54 (s, 1H, NH-amide), 8.26 – 8.16 (m, 3H, H-triazole& Ar), 7.98 (d, J =
6
8
.6 Hz, 2H, Ar), 7.91 – 7.84 (m, 2H, Ar), 7.65 – 7.58 (m, 2H, Ar), 7.43 (dt, J = 19.7, 7.4 Hz, 2H,
1
3
Ar), 7.32 – 7.22 (m, 2H, Ar), 5.73 (s, 2H, CH2-Ph), 4.76 (s, 2H, CH -NH); C NMR (126 MHz,
2
DMSO-d ) δ 166.54, 148.60, 145.03, 142.33, 131.27, 131.20, 131.07, 129.70, 127.11, 126.05,
6
+
1
25.33, 125.31, 123.78, 122.26, 116.20, 116.04, 49.06, 35.14; MS (70 eV, m/z [M ]) = 411;
Anal Calcd for C H FN O, C, 70.06; H, 4.41; N, 17.02 found: C, 70.09; H, 4.36; N, 17.01.
2
4
18
5
N-((1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7e. White solid;
-1 1
isolated yield: 85%; mp 218-220 °C; IR (KBr, υ): 3297, 3044, 2918, 1638 cm ; H NMR (500
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MHz, DMSO-d ) δ 9.65 – 9.44 (m, 1H, NH-amide), 8.27 (s, 1H, H-triazole), 8.19 (d, J = 8.7 Hz,
6
2
H, Ar), 7.99 (d, J = 9.2 Hz, 2H, Ar), 7.91 – 7.82 (m, 2H, Ar), 7.64 – 7.57 (m, 2H, Ar), 7.49 –
1
3
7
.41 (m, 1H, Ar), 7.20 (t, J = 8.8 Hz, 3H, Ar), 5.70 (s, 2H, CH2-Ph), 4.77 (s, 2H, CH -NH); C
2
NMR (126 MHz, DMSO-d ) δ 166.54, 148.61, 145.15, 142.37, 139.42, 139.36, 131.36, 131.29,
6
1
5
4
31.05, 129.71, 127.08, 126.06, 124.49, 124.47, 123.89, 122.27, 115.54, 115.38, 115.30, 115.13,
2.58, 49.06, 35.16; Anal Calcd for C H FN O, C, 70.06; H, 4.41; N, 17.02 found: C, 70.14; H,
2
4
18
5
.32; N, 17.03.
N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7f. White solid;
-1 1
isolated yield: 83%; mp 173-175 °C; IR (KBr, υ): 3288, 3047, 2928, 1639 cm ; H NMR (500
MHz, DMSO-d ) δ 9.53 (t, J = 5.4, 4.9 Hz, 1H, NH-amide), 8.22 (s, 1H, H-triazole), 8.19 (d, J =
6
8
.8 Hz, 2H, Ar), 7.98 (d, J = 8.7 Hz, 2H, Ar), 7.88 (t, J = 7.2 Hz, 2H, Ar), 7.65 – 7.56 (m, 2H,
Ar), 7.48 – 7.39 (m, 2H, Ar), 7.24 (t, J = 8.8 Hz, 2H, Ar), 5.65 (s, 2H, CH2-Ph), 4.75 (d, J = 5.7
1
3
Hz, 2H, CH -NH); C NMR (126 MHz, DMSO-d ) δ 166.53, 148.60, 145.11, 142.34, 132.93,
2
6
1
31.06, 130.80, 130.73, 129.71, 127.10, 126.07, 123.60, 122.26, 116.15, 115.98, 52.47, 35.16;
Anal Calcd for C H FN O, C, 70.06; H, 4.41; N, 17.02 found: C, 70.01; H, 4.47; N, 17.06.
2
4
18
5
N-((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7g. White
-1 1
solid; isolated yield: 81%; mp 214-216 °C; IR (KBr, υ): 3291, 3056, 2927, 1638 cm ; H NMR
(
500 MHz, DMSO-d ) δ 9.57 (d, J = 5.8 Hz, 1H, NH-amide), 8.20 (s, 1H, H-triazole), 8.18 (d, J
6
=
2.9 Hz, 2H, Ar), 7.99 (d, J = 8.6 Hz, 2H, Ar), 7.91 – 7.86 (m, 2H, Ar), 7.65 – 7.60 (m, 2H, Ar),
7
5
1
.56 (dd, J = 7.7, 1.4 Hz, 1H, Ar), 7.44 – 7.37 (m, 2H, Ar), 7.31 (dd, J = 7.6, 1.6 Hz, 1H, Ar),
13
.78 (s, 2H, CH2-Ph), 4.78 (d, J = 5.6 Hz, 2H, CH -NH); C NMR (126 MHz, DMSO-d ) δ
2
6
66.55, 148.61, 144.98, 142.33, 133.86, 133.16, 131.07, 131.03, 130.76, 130.14, 129.71, 128.20,
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1
27.12, 126.05, 124.01, 122.27, 51.12, 35.16; Anal Calcd for C H ClN O, C, 67.37; H, 4.24;
24 18 5
N, 16.37 found: C, 67.41; H, 4.23; N, 16.40.
N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7h. White
-1 1
solid; isolated yield: 84%; mp 190-192 °C; IR (KBr, υ): 3293, 3052, 2921, 1637 cm ; H NMR
(
500 MHz, DMSO-d ) δ 8.69 (t, J = 5.7 Hz, 1H, NH-amide), 7.38 (s, 1H, H-triazole), 7.34 (d, J =
6
8
.7 Hz, 2H, Ar), 7.14 (d, J = 8.7 Hz, 2H, Ar), 7.03 (ddd, J = 8.1, 6.6, 1.3 Hz, 2H, Ar), 6.79 –
.71 (m, 2H, Ar), 6.62 (d, J = 8.5 Hz, 2H, Ar), 6.55 (d, J = 8.6 Hz, 2H, Ar), 4.82 (s, 2H, CH2-
6
1
3
Ph), 3.92 (d, J = 6.1, 5.6 Hz, 2H, CH -NH); C NMR (126 MHz, DMSO-d ) δ 166.55, 148.61,
2
6
1
45.14, 142.35, 135.68, 133.36, 131.06, 130.41, 129.72, 129.24, 127.10, 126.07, 123.73, 122.27,
2.48, 35.18; Anal Calcd for C H ClN O, C, 67.37; H, 4.24; N, 16.37 found: C, 67.31; H, 4.28;
5
2
4
18
5
N, 16.29.
N-((1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7i. White solid;
-1 1
isolated yield: 81%; mp 157-159 °C; IR (KBr, υ): 3285, 3054, 2920, 1639 cm ; H NMR (500
MHz, DMSO-d ) δ 9.65 – 9.47 (m, 1H, NH-amide), 8.19 (d, J = 8.7 Hz, 3H, H-triazole& Ar),
6
8
7
1
.00 (d, J = 8.6 Hz, 2H, Ar), 7.89 (t, J = 7.6 Hz, 2H, Ar), 7.73 (d, J = 7.9 Hz, 1H, Ar), 7.63 (t, J =
.5 Hz, 2H, Ar), 7.43 (t, J = 7.4 Hz, 1H, Ar), 7.33 (t, J = 7.6 Hz, 1H, Ar), 7.25 (d, J = 7.5 Hz,
1
3
H, Ar), 5.76 (s, 2H, CH2-Ph), 4.79 (d, J = 5.8, 4.6 Hz, 2H, CH -NH); C NMR (126 MHz,
2
DMSO-d ) δ 166.55, 148.60, 145.00, 142.32, 135.46, 133.41, 131.07, 130.96, 130.92, 129.71,
6
1
6
28.75, 127.13, 126.05, 124.01, 123.41, 122.27, 53.44, 35.17; Anal Calcd for C H BrN O, C,
24 18 5
1.03; H, 3.84; N, 14.83 found: C, 61.06; H, 3.78; N, 14.79.
N-((1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7j. White solid;
-1 1
isolated yield: 83%; mp 193-195 °C; IR (KBr, υ): 3288, 3051, 2917, 1622 cm ; H NMR (500
MHz, DMSO-d ) δ 9.55 (t, J = 5.6 Hz, 1H, NH-amide), 8.27 (s, 1H, H-triazole), 8.19 (d, J = 8.7
6
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Hz, 2H, Ar), 7.99 (d, J = 8.3 Hz, 2H, Ar), 7.90 – 7.85 (m, 2H, Ar), 7.63 – 7.59 (m, 3H, Ar), 7.58
7.55 (m, 1H, Ar), 7.37 (d, J = 5.0 Hz, 2H, Ar), 5.68 (s, 2H, CH2-Ph), 4.77 (d, J = 5.6 Hz, 2H,
–
1
3
CH -NH); C NMR (126 MHz, DMSO-d ) δ 166.54, 148.60, 145.17, 142.34, 139.30, 131.51,
2
6
1
31.45, 131.24, 131.06, 129.71, 127.56, 127.12, 126.06, 123.85, 122.34, 122.27, 52.44, 35.16;
Anal Calcd for C H BrN O, C, 61.03; H, 3.84; N, 14.83 found: C, 61.07; H, 3.86; N, 14.82.
2
4
18
5
N-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7k. White
-1 1
solid; isolated yield: 85%; mp 176-178 °C; IR (KBr, υ): 3292, 3057, 2920, 1639 cm ; H NMR
(
500 MHz, DMSO-d ) δ 9.55 (s, 1H, NH-amide), 8.27 – 8.10 (m, 3H, H-triazole& Ar), 7.98 (d, J
6
=
8.5 Hz, 2H, Ar), 7.89 (t, J = 7.3 Hz, 2H, Ar), 7.62 (d, J = 8.0 Hz, 4H, Ar), 7.34 (d, J = 7.9 Hz,
1
3
2
H, Ar), 5.66 (s, 2H, CH2-Ph), 4.76 (s, 2H, CH2-NH); C NMR (126 MHz, DMSO-d ) δ
6
1
66.52, 148.61, 145.14, 142.33, 136.07, 132.15, 131.02, 130.70, 129.71, 127.08, 126.05, 123.71,
22.27, 121.90, 52.54, 35.17; Anal Calcd for C H BrN O, C, 61.03; H, 3.84; N, 14.83 found:
1
2
4
18
5
C, 61.11; H, 3.82; N, 14.88.
N-((1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7l. White solid;
-1 1
isolated yield: 81%; mp 222-224 °C; IR (KBr, υ): 3293, 3056, 2911, 1639 cm ; H NMR (500
MHz, DMSO-d ) δ 9.56 (t, J = 5.5 Hz, 1H, NH-amide), 8.34 (s, 1H, H-triazole), 8.30 (s, 1H, Ar),
6
8
.25 – 8.16 (m, 3H, Ar), 8.00 (d, J = 8.6 Hz, 2H, Ar), 7.90 – 7.85 (m, 2H, Ar), 7.82 (d, J = 7.7
Hz, 1H, Ar), 7.71 (t, J = 7.9 Hz, 1H, Ar), 7.63 – 7.57 (m, 2H, Ar), 5.85 (s, 2H, CH2-Ph), 4.79 (d,
13
J = 5.6 Hz, 2H, CH -NH); C NMR (126 MHz, DMSO-d ) δ 166.57, 148.61, 148.36, 145.23,
2
6
1
42.34, 138.79, 135.19, 131.05, 130.89, 129.71, 127.10, 126.08, 124.02, 123.61, 123.30, 122.28,
2.23, 35.17; Anal Calcd for C H N O , C, 65.75; H, 4.14; N, 19.17 found: C, 65.72; H, 4.18;
5
2
4
18
6
3
N, 19.15.
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N-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)acridine-9-carboxamide 7m. White solid;
-1 1
isolated yield: 83%; mp 194-196 °C; IR (KBr, υ): 3298, 3050, 2926, 1631 cm ; H NMR (500
MHz, DMSO-d ) δ 9.61 – 9.47 (m, 1H, NH-amide), 8.31 (d, J = 1.6 Hz, 1H, H-triazole), 8.28 –
6
8
.23 (m, 2H, Ar), 8.19 (d, J = 9.6 Hz, 2H, Ar), 8.01 (d, J = 8.7 Hz, 2H, Ar), 7.91 – 7.85 (m, 2H,
1
3
Ar), 7.65 – 7.56 (m, 4H, Ar), 5.85 (s, 2H, CH2-Ph), 4.79 (d, J = 5.4 Hz, 2H, CH -NH); C NMR
2
(
126 MHz, DMSO-d ) δ 166.57, 148.61, 147.72, 145.21, 144.09, 142.33, 131.06, 129.72, 129.51,
6
+
1
27.13, 126.09, 124.38, 124.17, 122.28, 52.38, 35.19; MS (70 eV, m/z [M ]) = 438; Anal Calcd
for C H N O , C, 65.75; H, 4.14; N, 19.17 found: C, 65.68; H, 4.17; N, 19.09.
2
4
18
6
3
In vitro α-glucosidase inhibition assay and kinetic study. α-Glucosidase inhibition assay of
acridine-9-carboxamide-1,2,3-triazoles 7a-m and kinetic study of most potent compound 7j
were performed on yeast α-glucosidase (Saccharomyces cerevisiae, EC3.2.1.20, 20 U/mg)
according to previous works [18-21].
Docking study. For docking study, since Saccharomyces cerevisiae form of α-glucosidase has
not any crystallographic structure in protein data bank (PDB), homology model of it was
constructed by using SWISS-MODEL Repository according to our previous paper [19].
In vitro α-amylase inhibition assay. The α-amylase inhibitory activity of most potent
compounds was screened against porcine pancreatic α-amylase by a reported colorimetric
method and acarbose was used as reference drug [20].
In vitro cytotoxicity assay. In vitro cytotoxicity assay of most potent compounds was
determined by MTT assay according to literature [26]. For this study, two cell lines HDF and
MCF-7 as normal and cancer cells and etoposide as positive control were used.
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In silico ADME/T study. In silico ADME/Tox studies of most potent compounds and standard
drug acarbose were performed using the preADMET online server (http://preadmet.bmdrc.org/)
[
25].
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