Synthesis, in-vitro evaluation, molecular docking, and kinetic studies of pyridazine-triazole hybrid system as novel α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104670

In this study, we reported the discovery of pyridazine based 1,2,3-triazole derivatives as inhibitors of α-glucosidase. All target compounds exhibited significant inhibitory activities against yeast and rat α-glucosidase enzymes compared to positive control, acarbose. The most potent compound 6j, ethyl 3-(2-(1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)ethyl)-5,6-diphenylpyridazine-4-carboxylate exhibited IC₅₀ values of 58, and 73 μM. Docking studies indicated the responsibility of hydrophobic and hydrogen bonding interactions in the ligand-enzyme complex stability. The in-vitro safety against the normal cell line was observed by toxicity evaluation of the selected compounds.

Full text of DOI:10.1016/j.bioorg.2021.104670

Bioorganic Chemistry 109 (2021) 104670
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, in-vitro evaluation, molecular docking, and kinetic studies of
pyridazine-triazole hybrid system as novel -glucosidase inhibitors
α
a
b
c
a
Setareh Moghimi , Somayeh Salarinejad , Mahsa Toolabi , Loghman Firoozpour ,
Seyed Esmaeil Sadat Ebrahimi , Fatemeh Safari , Fatemeh Madani-Qamsari ,
Somayeh Mojtabavi , Mohammad Ali Faramarzi , Saeed Karima , Roya Pakrad ,
b
d
a
e
e
f
f
a,b,*
Alireza Foroumadi
a
Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran
b
c
d
e
f
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Department of Clinical Biochemistry, School of Medicine, Shahid Beheshti University of Medical Sciences (SBMU), Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, we reported the discovery of pyridazine based 1,2,3-triazole derivatives as inhibitors of -gluco-
α
Anti-diabetic drug
sidase. All target compounds exhibited significant inhibitory activities against yeast and rat
zymes compared to positive control, acarbose. The most potent compound 6j, ethyl 3-(2-(1-(4-nitrobenzyl)-1H-
,2,3-triazol-4-yl)ethyl)-5,6-diphenylpyridazine-4-carboxylate exhibited IC50 values of 58, and 73 µM. Docking
α
-glucosidase en-
α
-Glucosidase inhibitor
Click reaction
Triazole
1
studies indicated the responsibility of hydrophobic and hydrogen bonding interactions in the ligand-enzyme
complex stability. The in-vitro safety against the normal cell line was observed by toxicity evaluation of the
selected compounds.
Pyridazine
1
. Introduction
decades for the treatment of diabetes.
enzyme of intestinal brush border which catalyzes the hydrolysis of
-1,4-glucoside bond in oligosaccharides to form absorbable mono-
α
-Glucosidase is a catabolic
A progressive metabolic disorder, Diabetes (diabetes mellitus),
α
characterized by high glucose levels, affects millions of people’s life
worldwide. Assorted into type 1 and type 2, diabetes is resulted from the
unevenness in the action or in the secretion of insulin [1,2]. The type 2
diabetes mellitus (T2DM) and its total societal costs have the sensible
and tremendous impact on societies. Regarding the expectations about
the growing rate and high risks associated with diabetic patients, the
pharmacological treatment strategies along with the changes in lifestyle
should be considered to improve the well-being of patients. Most of
diabetes mellitus type-2 patients, a non-insulin dependent diabetes, are
suffering from hyperglycemia which led to the serious health disorders
including heart disease and stroke, renal failure, optic neuropathy, nerve
damage, foot problems, and skin complications. Due to the importance
of hyperglycemia and its serious long-term complications, many treat-
saccharides [3,4]. The inhibition of this enzyme could reduce or slow the
postprandial hyperglycemia by retarding the absorption of glucose
[5–9]. Biguanides [10], sulfonylureas [11], and thiazolidindiones
[12,13] were found effective in the treatment of diabetes. Moreover,
miglitol, nojirimycin, voglibose, and acarbose [14–17] are therapeuti-
cally glucosidase inhibitors in the market, effectively manage the
symptoms of this metabolic disorder by regulating the glucose level. The
reported side effects involving hepatic disorders, gastrointestinal, and
diarrhea [18,19] along with limited hyperglycemic activity have moti-
vated medicinal chemists to design selective and potent non-glycosidic
based compounds as potential therapeutic agents.
Click reaction is one of the most important chemical transformations,
employed in academic and pharmaceutical researches to form triazole
ring [20]. In particular, copper (I)-catalyzed azide-alkyne cycloaddition
(CuAAC) is known for its distinct advantages, such as exquisite
ment approaches have been devised.
α-Glucosidase inhibitors have been
considered as the subject of several research studies during the last
*
Corresponding author.
E-mail address: aforoumadi@yahoo.com (A. Foroumadi).
https://doi.org/10.1016/j.bioorg.2021.104670
Received 12 July 2020; Received in revised form 18 January 2021; Accepted 20 January 2021
Available online 1 February 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Moghimi et al.
Bioorganic Chemistry 109 (2021) 104670
selectivity, broad scope, and mild reaction condition [21–23]. Triazoles
are one of the most important toolkits in medicinal chemistry [24–29],
extensively used to synthesize lead analogues by providing the good
mimics of different functional groups involving amide bond, ester bond
and various heterocycles. This valuable heterocyclic core have also
found applications in functional materials [30–33]. Therefore, several
hybrid molecules containing 1,4-disubstituted-1,2,3-triazoles with
diverse range of bioactivities have been reported and found their way to
the market [34].
apparatus and are uncorrected. IR spectroscopy was performed using a
Nicolet FT-IR Magna 550 spectrograph (KBr disks).
2.1.1. Synthesis of ethyl-2,3-dihydro-3-oxo-5,6-diphenylpyridazine-4-
carboxylate (3)
A cooled flask was charged with sodium (0.05 mol) and ethanol (200
mL). Upon disappearance of sodium, compound 2 (0.05 mol) and
◦
diethyl malonate (0.075 mol) were added at 0 C. The mixture was
cooled to room temperature and was stirred at reflux temperature for 3
h. After this time, the mixture was evaporated under reduced pressure
and the residue was acidified with HCl (1 N). The slight yellow solid was
Pyridazine is the ubiquitous nitrogen containing heterocyclic core
found in a wide range of molecules with immense biological function-
alities [35–37]. Some investigations reported that applications of pyr-
idazinones as antihypertensive, antifungal, anti-inflammatory,
anticancer, and antimicrobial agents [38–43]. Accordingly, tremendous
effort has been devoted to the hybridization of this valuable core to
afford multi-target novel bioactive compounds. To date, different classes
of glucosidase inhibitors with different chemical scaffolds have been
developed. Given the importance of triazines in the inhibition of
glucosidase, we were keen to demonstrate the utility of pyridazine as its
bioisostere. In addition, many triazole-containing glucosidase inhibitors
have been found in the literature. Fig. 1, schematically presented the
previously reported compounds bearing these prominent cores [44–50].
Previously, we have reported on the synthesis of glucosidase in-
hibitors [51,52], so, in pursuit of our goal of discovering novel bioactive
agents [53–55], herein, we designed and synthesized a series of gluco-
sidase inhibitors based on the pyridazine core according to the bio-
isosterism principle. The sixteen final compounds showed better
activities compared to the activity of the positive control.
◦
simply filtered and washed with cold water. mp. lit. 218–219 C [56],
◦
mp. obtained; 217–219 C.
2.1.2. Synthesis of ethyl-3-mercapto-5,6-diphenylpyridazine-4-carboxylate
(4)
Compound 3 (20 mmol), toluene (150 mL), and Lawesson’s reagent
(10 mmol) were added to the flask and the mixture was refluxed for 18 h.
After this time, the mixture was concentrated and the residue was
◦
recrystallized from petroleum ether/ethyl acetate, mp. lit. 230 C [57],
◦
mp. obtained; 228–230 C.
2.1.3. Synthesis of ethyl-5,6-diphenyl-3-(prop-2-ynylthio)pyridazine-4-
carboxylate (5)
To the stirred solution of ethyl-3-mercapto-5,6-diphenylpyridazin-4-
2 3
carboxylate (5 mmol), K CO (5 mmol) in DMF (10 mL), propargyl
bromide (6 mmol) was added dropwise and the mixture was stirred at
◦
50 C. After completion, checked by TLC, the reaction was quenched
with ice/water solution and the resulted precipitate was filtered, washed
with water and used without further purification.
2
. Experimental
2
.1. General chemistry
2.1.3.1. Ethyl
5,6-diphenyl-3-(prop-2-yn-1-ylthio)pyridazine-4-carbox-
◦
ylate (5). Light-yellow solid; yield: 92%; mp: 79–81 C; IR (KBr,
ꢀ
1
–
–
1
All commercially available reagents and solvents, used without
cm ): 2173, 1734 (C
O), 1322, 1192; H NMR (500 MHz, DMSO‑d ) δ:
3
6
further purification, were purchased from Merck and Sigma-Aldrich
companies. Bruker FT-500 MHz spectrometer was used to confirm the
structure of the target compounds by 1H- and C NMR spectra in
1.34 (t, J = 6.9 Hz, 3H, CH ), 2.38 (t, J = 3.0 Hz, 1H), 4.00 (d, J = 2.9 Hz,
2H, S-CH ), 4.33 (q, J = 6.9 Hz, 2H, CH -CH ), 7.38–7.41 (m, 2H, Ar),
2
2
3
13
7.47–7.56 (m, 5H, Ar), 7.57–7.58 (m, 1H, Ar), 7.97 (d, J = 8.0 Hz, 2H,
Ar); 13C NMR (125 MHz, DMSO‑d ) δ: 14.22 (CH ), 18.66 (SCH ), 60.68
DMSO‑d
6
as the solvent. Chemical shifts reported in δ parts per million
6
3
2
–
–
(
ppm) downfield from tetramethylsilane (TMS) as the internal standard.
(OCH ), 72.36 (C
–
CH), 79.23 (C
–
CH), 127.06 (CH), 127.92 (CH),
2
–
–
All reactions were monitored through thin-layer chromatography (TLC)
on silica gel 250 mm, F254 plastic sheets. Elemental analysis was
recorded by a Perkin Elmer 2400 (automatic elemental analyzer).
Melting points were determined with a Kofler hot-plate microscope
128.45 (CH), 129.02 (CH), 129.85 (CH), 129.99 (CH), 130.37 (C),
134.87 (C), 135.95 (C), 138.35 (C), 155.72 (C N), 159.20 (C N),
166.88 (C O); Anal. Calcd. For C22H18N O S: C, 70.57; H, 4.85; N,
–
–
–
2
2
+
7.48. Found: C, 70.16; H, 4.83; N, 7.52; ESI-MS m/z: 375.11 [M + H] .
R1
OH
R
O
Ar
Ar
N
N
S
N
N N
R2
Ar
Ar
N
N
H
N
S
N
N
N
Ph
Ph
N
X
OH
N
Ar
Ar
N
S
O
S
O
O
N
N
R1
N
N
N
R
O
N
R1
This work
N
Ph
Ph
N
N
N
N
S
O
N
R2
X
R
HN
Fig. 1. Previously described triazole and triazine-containing analogues with alpha-glucosidase inhibitory activity.
2

S. Moghimi et al.
Bioorganic Chemistry 109 (2021) 104670
2
.1.4. General procedure for the preparation of compounds (6a-6 m)
59.28; H, 4.15; N, 12.02. ESI-MS m/z: 586.09 [M + H]+.
A solution of benzyl bromide/chloride derivatives (1 mmol), sodium
azide (1 mmol) were stirred in a H
2
O/tert-buthanol (1:1, 10 mL) for 1 h.
2.1.4.5. Ethyl 3-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
After this time, compound 5 (1 mmol), triethyl amine (1 mmol), sodium
ascorbate (10 mol%) and copper (II) sulfate (10 mol%) were added to
the reaction mixture and the reaction was stirred for 6–10 h. After this
time, the ice/water solution was added to the mixture and the resultant
solid was recrystallized from ethanol.
5,6-diphenylpyridazine-4-carboxylate (6e). White solid; yield: 85%; mp:
◦
ꢀ 1
–
1
124–126 C; IR (KBr, cm ): 1721 (C
–
O), 1639, 1456, 1309, 1214; H
NMR (500 MHz, DMSO‑d ) δ: 0.84 (t, J = 7.1 Hz, 3H, CH ), 4.02 (q, J =
6
3
7.1 Hz, 2H, CH -CH ), 4.73 (s, 2H, S-CH ), 5.57 (s, 2H, N-CH ), 7.12 (dt,
2
3
2
2
J = 6.8, 1.6 Hz, 2H, Ar), 7.16–7.20 (m, 2H, Ar), 7.29–7.39 (m, 10H, Ar),
1
3
8
.15 (d, J = 2.4 Hz, 1H, N-CH); C NMR (125 MHz, DMSO‑d
6
) δ: 13.79
2
2
.1.4.1. Ethyl 3-(((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-diphe-
(CH ), 25.26 (SCH ), 50.16 (NCH ), 52.40 (OCH ), 115.24 (d,
3
2
2
2
J
C-F
=
nylpyridazine-4-carboxylate (6a). White solid; yield: 83%; mp:
20.0 Hz), 122.02 (2C), 127.17, 128.09 (2C), 128.11, 129.07 (2C),
◦
ꢀ 1
–
1
3
1
18–120 C; IR (KBr, cm ): 1723 (C
–
O), 1639, 1458, 1319, 1194; H
) δ: 0.84 (t, J = 7.2 Hz, 3H, CH ), 4.02 (q, J =
), 5.58 (s, 2H, N-CH ), 7.12 (d,
J = 7.1 Hz, 2H, Ar), 7.27–7.35 (m, 13H, Ar), 8.16 (s, 1H, N-CH);
NMR (125 MHz, DMSO‑d ), 25.23 (SCH ), 53.22 (NCH
129.88 (2C), 129.99 (2C), 130.01, 130.30 (d, JC-F = 7.9 Hz, 2C), 133.09
4
NMR (500 MHz, DMSO‑d
6
3
(d,
J
C-F = 3.1 Hz), 134.97, 136.19, 138.45, 144.03 (S-CH2-C), 155.67
–
–
–
–
1
–
–
O);
7
.2 Hz, 2H, CH
2
-CH
3
), 4.74 (s, 2H, S-CH
2
2
(C
N), 158.66 (S-C
N), 163.13 (d, JC-F = 252.1 Hz), 164.49 (C
1
3
C
),
5 2
Anal. Calcd. For C29H24FN O S. C, 66.27; H, 4.60; N, 13.32. Found: C,
+
6
) δ: 13.78 (CH
3
2
2
65.93; H, 4.84; N, 13.06; ESI-MS m/z: 526.17 [M + H] .
6
1
1
2.56 (OCH
2
), 124.43, 124.47, 128.37, 128.57, 128.79, 129.10, 129.18,
29.31, 129.45, 129.47, 130.13, 130.90, 134.08, 135.64, 136.42,
2.1.4.6. Ethyl 3-(((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
–
–
–
N), 164.51
S. C, 68.62; H, 4.96; N, 13.80.
36.49, 143.36 (S-CH2-C), 155.88 (C
–
N), 157.81 (S-C
5,6-diphenylpyridazine-4-carboxylate (6f). White solid; yield: 81%; mp:
–
–
◦
ꢀ 1
–
1
(
C
O); Anal. Calcd. For C29
H
25
N
5
O
2
161–162 C; IR (KBr, cm ): 1724 (C
–
O), 1632, 1454, 1324, 1166; H
+
Found: C, 68.47; H, 4.67; N, 13.96; ESI-MS m/z: 508.18 [M + H] .
NMR (500 MHz, DMSO‑d ) δ: 0.88 (t, J = 7.1 Hz, 3H, CH ), 4.04 (q, J =
6
3
7
.1 Hz, 2H, CH
2
-CH
3
), 4.78 (s, 2H, S-CH
2
2
), 5.46 (s, 2H, N-CH ), 7.28 (d,
2
5
1
.1.4.2. Ethyl 3-(((1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
J = 8.5 Hz, 2H, Ar), 7.38–7.46 (m, 6H, Ar), 7.53–7.58 (m, 4H, Ar), 7.97
13
,6-diphenylpyridazine-4-carboxylate (6b). Gray solid; yield: 63%; mp:
(d, J = 8.5 Hz, 2H, Ar), 8.11 (s, 1H, N-CH); C NMR (125 MHz,
DMSO‑d ), 31.64 (SCH ), 52.85 (NCH ), 59.62 (OCH ),
◦
ꢀ 1
–
1
16–118 C; IR (KBr, cm ): 1728 (C
) δ: 0.87 (t, J = 7.1 Hz, 3H, CH
), 4.76 (s, 2H, S-CH ), 5.66 (s, 2H, N-CH
2
–
O), 1640, 1456, 1322, 1184; H
), 4.04 (q, J =
), 7.14 (d,
6
) δ: 13.82 (CH
3
2
2
2
NMR (500 MHz, DMSO‑d
.1 Hz, 2H, CH -CH
6
3
122.05, 127.23, 128.09 (2C), 128.12, 128.66 (2C), 129.07 (2C), 129.88
(2C), 129.91 (2C), 129.99 (2C), 130.13, 132.37, 134.97 (C-Cl), 135.05,
7
2
3
2
–
N), 156.63 (S-C–
J = 6.7 Hz, 2H, Ar), 7.19–7.35 (m, 11H, Ar), 7.40–7.42 (m, 1H, Ar), 8.14
136.19, 138.45, 144.03 (S-CH2-C), 154.62 (C
–
–
N),
(
(
s, 1H, N-CH); 13C NMR (125 MHz, DMSO‑d
SCH ), 47.36 (NCH ), 62.57 (OCH
), 116.06 (d, 2
23.30 (NCH), 124.56, 125.28, 128.36 (2C), 128.79, 129.11 (2C),
29.32 (2C), 129.47 (2C), 130.14, 130.96, 131.15, 131.21, 134.10,
6
) δ: 13.79 (CH
C-F = 20.0 Hz),
3
), 25.24
164.90 (C O); Anal. Calcd. For C29
–
H24ClN O S: C, 64.26; H, 4.46; N,
5 2
2
2
2
J
12.92. Found: C, 64.41; H, 4.14; N, 12.67; ESI-MS m/z: 542.14 [M +
+
1
1
1
1
H] .
–
–
35.65, 136.45, 143.32 (S-CH2-C), 155.90 (C
–
N), 157.84 (S-C
–
N),
–
–
O); Anal. Calcd. For
2.1.4.7. Ethyl 3-(((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
1
60.54 (d,
24FN
J
C-F = 246.7 Hz), 164.51 (C
5,6-diphenylpyridazine-4-carboxylate (6g). White solid; yield: 84%; mp:
◦
ꢀ 1
–
–
1
C
29
H
5
O
2
S. C, 66.27; H, 4.60; N, 13.32; Found: C, 66.01; H, 4.82; N,
129–131 C; IR (KBr, cm ): 1723 (C
O), 1636, 1457, 1323, 1156; H
+
1
3.06; ESI-MS m/z: 526.17 [M + H] .
NMR (500 MHz, DMSO‑d ) δ: 0.84 (t, J = 7.1 Hz, 3H, CH ), 4.01 (q, J =
6
3
7
.1 Hz, 2H, CH
2
-CH
3
), 4.72 (s, 2H, S-CH
2
2
), 5.55 (s, 2H, N-CH ), 7.11 (d,
2
5
1
.1.4.3. Ethyl 3-(((1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
J = 7.3 Hz, 2H, Ar), 7.22–7.24 (m, 2H, Ar), 7.26–7.33 (m, 8H, Ar), 7.53
13
,6-diphenylpyridazine-4-carboxylate (6c). Gray solid; yield: 79%; mp:
(d, J = 8.2 Hz, 2H, Ar), 8.14 (s, 1H, N-CH); C NMR (125 MHz,
DMSO‑d ), 32.56 (SCH ), 51.41 (NCH ), 59.46 (OCH ),
◦
ꢀ 1
–
1
25–127 C; IR (KBr, cm ): 1727 (C
) δ: 0.87 (t, J = 7.1 Hz, 3H, CH
), 4.77 (s, 2H, S-CH ), 5.63 (s, 2H, N-CH
.21–7.23 (m, 5H, Ar), 7.29–7.35 (m, 8H, Ar), 7.40–7.44 (m, 1H, Ar),
.21 (s, 1H, N-CH); 13C NMR (125 MHz, DMSO‑d
) δ: 13.79 (CH ), 25.25
), 52.56 (NCH ), 62.57 (OCH ), 115.14, 115.34, 115.53, 124.46
–
O), 1639, 1458, 1321, 1183; H
), 4.05 (q, J =
),
6
) δ: 13.74 (CH
3
2
2
2
NMR (500 MHz, DMSO‑d
6
3
112.86, 121.06 (C-Br), 122.10, 127.55 (2C), 127.73 (2C), 128.06,
128.30 (2C), 128.48 (2C), 128.79, 131.14 (2C), 131.90 (2C), 132.39,
7
7
8
.1 Hz, 2H, CH
2
-CH
3
2
2
–
132.45, 136.10, 137.96, 143.40 (S-CH2-C), 146.76 (C
–
N), 153.87 (S-
–
–
–
6
3
C
–
N), 164.73 (C
O); Anal. Calcd. For C29
H24BrN O S. C, 59.39; H,
5 2
(
(
SCH
2
2
2
4.12; N, 11.94. Found: C, 59.15; H, 4.39; N, 12.10; ESI-MS m/z: 586.09
[M + H]+.
4
2
d, JC-F = 3.75 Hz), 124.61, 128.35 (2C), 128.79 (d, JC-F = 25.8 Hz),
2
1
1
29.21 (d,
J
C-F = 25.8 Hz), 129.47 (2C), 130.14 (2C), 130.95 (2C),
31.28 (d, 3
J
C-F = 8.5 Hz), 134.00, 135.65, 136.45, 139.18 (d,
3
J
=
C-F
2.1.4.8. Ethyl 3-(((1-(2,6-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
–
–
–
7
.6 Hz), 143.49 (S-CH2-C), 155.87 (C
–
N), 157.84 (S-C
N), 162.58 (d,
24FN S. C,
thio)-5,6-diphenylpyridazine-4-carboxylate (6h). Off-white solid; yield:
1
–
–
ꢀ 1
◦
–
J
C-F = 244.3 Hz), 164.51 (C
O); Anal. Calcd. For C29
H
5
O
2
66%; mp: 148–150 C; IR (KBr, cm ): 1723 (C
–
O), 1636, 1457, 1323,
1156; 1H NMR (500 MHz, DMSO‑d ) δ: 0.87 (t, J = 7.0 Hz, 3H, CH ),
6
6.27; H, 4.60; N, 13.32; Found: C, 66.52; H, 4.43; N, 13.51. ESI-MS m/
6
3
+
z: 526.17 [M + H] .
4.04 (q, J = 7.1 Hz, 2H, CH -CH ), 4.76 (s, 2H, S-CH ), 5.67 (s, 2H, N-
2
3
2
CH
2
), 7.14–7.19 (m, 4H, Ar), 7.28–7.36 (m, 8H, Ar), 7.48–7.56 (m, 1H,
13
2
5
.1.4.4. Ethyl 3-(((1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
Ar), 8.14 (s, 1H, N-CH); C NMR (125 MHz, DMSO‑d
6
) δ: 13.78 (CH
3
),
,6-diphenylpyridazine-4-carboxylate (6d). Off-white solid; yield: 76%;
25.18 (SCH
2
), 41.32 (NCH
2
), 62.56 (OCH
2
), 112.30 (d, 2JC-F = 4.9 Hz),
◦
ꢀ 1
–
mp: 136–137 C; IR (KBr, cm ): 1721 (C
–
O), 1631, 1453, 1319, 1149;
) δ: 0.88 (t, J = 7.1 Hz, 3H, CH ), 4.05 (q, J
), 4.77 (s, 2H, S-CH ), 5.61 (s, 2H, N-CH ),
.14–7.15 (m, 2H, Ar), 7.30–7.36 (m, 10H, Ar), 7.54–7.55 (m, 2H, Ar),
.21 (s, 1H, N-CH); 13C NMR (125 MHz, DMSO‑d
) δ: 13.27 (CH ), 33.94
), 52.03 (NCH ), 58.64 (OCH ), 112.44, 122.79 (C-Br), 122.93,
112.46, 124.56, 128.37 (2C), 128.79 (2C), 129.11, 129.31, 129.47 (2C),
1
3
H NMR (500 MHz, DMSO‑d
7.1 Hz, 2H, CH -CH
6
3
130.14 (2C), 130.97, 132.18 (d, JC-F = 10.4 Hz), 134.11, 135.66 (2C),
–
–
–
–
=
2
3
2
2
136.46, 143.22 (S-CH2-C), 155.89 (C
N), 157.85 (S-C
N), 161.28 (d,
23 2 5 2
H F N O S: C,
1
–
–
7
8
J
C-F = 249.1 Hz), 164.51 (C
O); Anal. Calcd. For C29
6
3
64.08; H, 4.26; N, 12.88; Found: C, 63.88; H, 4.48; N, 12.92; ESI-MS m/
z: 544.16 [M + H]+.
(
SCH
2
2
2
1
1
1
27.58 (2C), 127.96 (2C), 128.03, 128.27 (2C), 128.40, 128.44 (2C),
28.68, 129.89, 131.00, 132.25, 132.39, 132.45, 136.54, 137.96,
2.1.4.9. Ethyl 3-(((1-(2,5-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
–
–
–
43.40 (S-CH2-C), 146.76 (C
24BrN
–
N), 153.87 (S-C
–
N), 164.32 (C
–
O);
thio)-5,6-diphenylpyridazine-4-carboxylate (6i). Off-white solid; yield:
◦
ꢀ 1
–
Anal. Calcd. For C29
H
5
O
2
S. C, 59.39; H, 4.12; N, 11.94. Found: C,
64%; mp: 110–112 C; IR (KBr, cm ): 1721 (C O), 1632, 1449, 1300,
–
3

S. Moghimi et al.
Bioorganic Chemistry 109 (2021) 104670
1
4
187; 1H NMR (500 MHz, DMSO‑d
.04 (q, J = 7.1 Hz, 2H, CH -CH ), 4.78 (s, 2H, S-CH
), 7.13–7.14 (m, 2H, Ar), 7.27–7.37 (m, 9H, Ar), 7.48 (dd, J = 8.6,
6
) δ: 0.87 (t, J = 7.1 Hz, 3H, CH
3
),
128.03, 128.10 (2C), 128.44 (2C), 128.50, 128.65, 125.68, 132.39,
–
2
3
2
), 5.70 (s, 2H, N-
135.55, 137.03, 138.27, 146.76 (S-CH2-C), 147.59 (C
–
N), 154.94 (S-
S:
–
–
–
–
CH
.6 Hz, 2H), 8.18 (s, 1H, N-CH); C NMR (125 MHz, DMSO‑d
CH ), 25.19 (SCH ), 50.68 (NCH ), 62.57 (OCH ), 125.02, 128.36 (2C),
2
C
N), 159.16 (C-OMe), 164.27 (C
O); Anal. Calcd. For C30
H
27
N
5
O
3
1
3
2
6
) δ: 13.79
C, 67.02; H, 5.06; N, 13.03. Found: C, 67.22; H, 5.28; N, 13.21; ESI-MS
(
3
2
2
2
m/z: 538.19 [M + H]+.
1
1
1
28.80 (2C), 129.12, 129.32, 129.46 (2C), 130.13 (2C), 130.51, 130.68,
30.98, 131.81, 131.97 (C-Cl), 132.53 (C-Cl), 134.10, 135.63, 135.79,
2.1.5. General procedure for the preparation of compounds (7a-7c)
The appropriate compounds (6a, 6e, 6 g, 1 mmol) were dissolved in
ethanol (5 mL) and the NaOH solution (1 M, 5 mL) was added and the
mixture was refluxed. Upon completion (1–2 h), checked by TLC, the
mixture was acidified to pH = 1 and the resultant solid was filtered and
washed with water.
–
–
36.44, 143.38 (S-CH2-C), 155.80 (C
–
N), 157.82 (S-C
–
N), 164.50
–
–
(
C
O); Anal. Calcd. For C29
H
23Cl
N
2 5
O
2
S: C, 60.42; H, 4.02; N, 12.15;
+
Found: C, 60.09; H, 3.90; N, 12.28; ESI-MS m/z: 579.09 [M + H] .
2
5
1
.1.4.10. Ethyl 3-(((1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
,6-diphenylpyridazine-4-carboxylate (6j). Gray solid; yield: 82%; mp:
◦
ꢀ 1
–
1
37–139 C; IR (KBr, cm ): 1728 (C
) δ: 0.87 (t, J = 7.1 Hz, 3H, CH
), 4.77 (s, 2H, S-CH ), 5.78 (s, 2H, N-CH
–
O), 1641, 1459, 1325, 1188; H
), 4.05 (q, J =
),
2.1.5.1. 3-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-diphe-
NMR (500 MHz, DMSO‑d
6
3
nylpyridazine-4-carboxylic acid (7a). White solid; yield: 76%; mp:
◦
ꢀ 1
–
7
.1 Hz, 2H, CH
2
-CH
3
2
2
192–194 C; IR (KBr, cm ): 3056 (COOH), 1719 (C
–
O), 1642, 1463,
) δ: 4.74 (s, 2H, S-CH ), 5.59
), 7.15–7.17 (m, 2H, Ar), 7.28–7.38 (m, 13H, Ar), 8.16 (s,
1
7
.14–7.15 (m, 2H, Ar), 7.29–7.37 (m, 8H, Ar, N-CH), 7.67–7.70 (m, 1H,
1326, 1200; H NMR (500 MHz, DMSO‑d
(s, 2H, N-CH
1H, N-CH), 14.12 (s, 1H, OH); C NMR (125 MHz, DMSO‑d
(SCH ), 52.10 (NCH ), 121.99, 128.03, 128.04 (2C), 128.07 (2C),
128.09, 128.42 , 129.08, 129.67, 129.85, 129.98, 130.02, 134.78,
6
2
1
3
Ar), 7.77 (d, J = 7.7 Hz, 1H, Ar), 8.20–8.22 (m, 2H), 8.27 (s, 1H, Ar);
C
),
2
1
3
NMR (125 MHz, DMSO‑d
6
) δ: 13.79 (CH
3
), 25.19 (SCH
2
), 52.20 (NCH
2
6
) δ: 21.48
6
1
1
2.57 (OCH
29.12 (2C), 129.63, 129.88 (2C), 129.98 (2C), 130.38, 133.73, 134.97,
36.09, 137.49, 138.45, 144.03 (S-CH2-C), 148.14 (C-NO ), 155.83
N), 157.85 (S-C–
2
), 122.02, 122.19, 123.45, 127.17, 128.12, 128.19 (2C),
2
2
–
2
136.21, 136.26, 138.36, 144.03 (S-CH2-C), 157.65 (C
–
N), 158.37 (S-
–
–
–
–
–
–
–
(
C
–
N), 164.50 (C
O); Anal. Calcd. For C29
H
24
N
6
O
4
S:
C
N), 166.88 (C O); Anal. Calcd. For C27
–
H N O S: C, 67.62; H, 4.41;
21 5 2
C, 63.03; H, 4.38; N, 15.21. Found: C, 62.90; H, 4.60; N, 15.40; ESI-MS
m/z: 553.16 [M + H]+.
N, 14.60. Found: C, 67.43; H, 4.29; N, 14.75; ESI-MS m/z: 480.14 [M +
+
H] .
2
.1.4.11. Ethyl 3-(2-(1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)ethyl)-5,6-
2.1.5.2. 3-(((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-
diphenylpyridazine-4-carboxylate (6k). Gray solid; yield: 89%; mp:
diphenylpyridazine-4 carboxylic acid (7b). White solid; yield: 93%; mp:
◦
ꢀ 1
–
1
◦
ꢀ 1
–
1
49–151 C; IR (KBr, cm ): 1730 (C
–
O), 1642, 1459, 1325, 1187; H
) δ: 0.85 (t, J = 7.0 Hz, 3H, CH ), 4.01 (q, J =
), 5.76 (s, 2H, N-CH ), 7.11 (d,
J = 7.0 Hz, 2H, Ar), 7.26–7.34 (m, 8H, Ar), 7.50 (d, J = 8.3 Hz, 2H, Ar),
212–213 C; IR (KBr, cm ): 3051 (COOH), 1705 (C
–
O), 1636, 1468,
) δ: 4.74 (s, 2H, S-CH ), 5.58
), 7.16–7.21 (m, 4H, Ar), 7.28–7.33 (m, 8H, Ar), 7.37–7.40
1
NMR (500 MHz, DMSO‑d
6
3
1301, 1166; H NMR (500 MHz, DMSO‑d
(s, 2H, N-CH
(m, 2H, Ar), 8.17 (d, J = 2.5 Hz, 1H, N-CH), 13.02 (s, 1H, OH); C NMR
6
2
7
.1 Hz, 2H, CH
2
-CH
3
), 4.75 (s, 2H, S-CH
2
2
2
1
3
1
3
2
8
.20 (d, J = 8.7 Hz , 2H, Ar), 8.23 (s, 1H, N-CH); C NMR (125 MHz,
DMSO‑d ), 22.20 (SCH ), 52.42 (NCH ), 60.61 (OCH ),
) δ: 14.24 (CH
22.02, 123.76 (2C), 127.17, 128.12, 128.19 (2C), 129.12 (2C), 129.17
2C), 129.88 (2C), 129.98 (2C), 130.38, 134.97, 136.09, 138.45,
(125 MHz, DMSO‑d
20 Hz, 2C), 122.02, 128.07, 128.09, 129.03, 129.67, 129.85, 130.03
(2C), 130.06, 130.30 (d, 3
C-F = 7.5 Hz, 133.09 (2C), 134.78, 136.21,
138.36, 144.03 (S-CH2-C), 157.52 (C
6
) δ: 22.20 (SCH
2
), 52.17 (NCH
2
), 115.24 (d, JC-F
=
6
3
2
2
2
1
J
–
–
–
–
(
N), 158.23 (S-C
–
–
O); Anal. Calcd. For C27
N), 162.27 (d,
20FN S: C,
–
1
1
40.28, 144.03 (S-CH2-C), 147.03 (C-NO
2
), 155.68 (C
–
N), 158.66 (S-
J
C-F = 252.1 Hz), 167.42 (C
H
5 2
O
–
–
–
–
C
N), 164.49 (C
O); Anal. Calcd. For C29
H
24
N
6
O
4
S: C, 63.03; H, 4.38;
65.18; H, 4.05; N, 14.08. Found: C, 65.36; H, 3.87; N, 14.22; ESI-MS m/
z: 498.14 [M + H]+.
N, 15.21. Found: C, 63.22; H, 4.51; N, 15.35; ESI-MS m/z: 553.16 [M +
+
H] .
2
.1.5.3. 3-(((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-
2
.1.4.12. Ethyl 3-(((1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
diphenylpyridazine-4-carboxylic acid (7c). White solid; yield: 81%; mp:
◦
ꢀ 1
–
211–213 C; IR (KBr, cm ): 3105 (COOH), 1695 (C
–
thio)-5,6-diphenylpyridazine-4-carboxylate (6l). Gray solid; yield: 71%;
O), 1649, 1451,
) δ: 4.74 (s, 2H, S-CH ), 5.58
), 7.14 (dd, J = 7.7, 1.9 Hz, 2H, Ar), 7.25 (d, J = 8.4 Hz,
2H, Ar), 7.28–7.34 (m, 8H, Ar), 7.56 (d, J = 8.4 Hz, 2H, Ar), 8.17 (s, 1H,
N-CH), 12.99 (s, 1H, OH); 13C NMR (125 MHz, DMSO‑d
) δ: 23.29
(SCH ), 53.79 (NCH ), 121.87 (C-Br), 122.16, 128.09, 128.10, 129.09,
129.68, 129.88, 130.02, 130.03, 130.28, 131.57, 134.79, 135.31,
N), 158.23 (S-C–
◦
ꢀ 1
–
–
1
mp: 154–156 C; IR (KBr, cm ): 1730 (C
O), 1641, 1462, 1325, 1188;
) δ: 0.86 (t, J = 7.1 Hz, 3H, CH ), 3.71 (s,
), 4.03 (q, J = 7.0 Hz, 2H, CH -CH ), 4.74 (s, 2H, S-CH ), 5.54
s, 2H, N-CH
), 6.84–6.90 (m, 3H, Ar), 7.12–7.15 (m, 3H, Ar), 7.25–7.36
m, 8H, Ar), 8.15 (s, 1H, N-CH); C NMR (125 MHz, DMSO‑d
CH ), 22.20 (SCH ), 52.84 (NCH ), 55.15 (OCH ), 60.61 (OCH
12.78, 113.00, 121.87, 122.02, 127.17, 128.12, 128.15 (2C), 129.12
2C), 129.50, 129.88 (2C), 129.99 (2C), 130.13, 134.97, 136.19,
1309, 1217; H NMR (500 MHz, DMSO‑d
(s, 2H, N-CH
6
2
1
H NMR (500 MHz, DMSO‑d
H, OCH
6
3
2
3
3
2
3
2
(
(
(
2
6
1
3
6
) δ: 14.24
),
2
2
3
2
2
3
2
–
1
136.11, 138.36, 144.03 (S-CH2-C), 157.49 (C
–
–
N),
–
5 2
167.51 (C O); Anal. Calcd. For C27H20BrN O S: C, 58.07; H, 3.61; N,
(
–
–
–
3
1
1
6
36.58, 138.45, 144.03 (S-CH2-C), 155.67 (C
–
N), 158.66 (S-C
–
N),
S: C,
12.54. Found: C, 57.81; H, 3.44; N, 12.71; ESI-MS m/z: 558.06 [M +
–
+
59.53 (C-OMe), 167.00 (C
–
O); Anal. Calcd. For C30
H
27
N
5
O
H] .
7.02; H, 5.06; N, 13.03. Found: C, 66.92; H, 5.31; N, 13.22; ESI-MS m/
+
z: 538.19 [M + H] .
2
2
.2. Biological results
2
.1.4.13. Ethyl 3-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
.2.1. In-vitro
α
-glucosidase inhibition assay
thio)-5,6-diphenylpyridazine-4-carboxylate (6m). Off-white solid; yield:
◦
ꢀ 1
–
α-Glucosidase (Saccharomyces cerevisiae, EC3.2.1.20, 20 U/mg) and
7
1
3
4%; mp: 123–125 C; IR (KBr, cm ): 1722 (C
–
O), 1639, 1458, 1300,
) δ: 0.87 (t, J = 7.1 Hz, 3H, CH ),
), 4.04 (q, J = 7.2 Hz, 2H, CH -CH ), 4.74 (s, 2H, S-
), 5.50 (s, 2H, N-CH
), 6.92 (d, J = 8.7 Hz, 2H, Ar), 7.13 (d, J = 7.1
Hz, 2H, Ar), 7.28–7.37 (m, 10H, Ar), 8.10 (s, 1H, N-CH); C NMR (125
MHz, DMSO‑d ), 23.60 (SCH ), 52.03 (NCH ), 55.23
) δ: 14.25 (CH
OCH ), 60.67 (OCH ), 112.44, 113.80 (2C), 122.93, 127.72 (2C),
1
substrate (p-nitrophenyl glucopyranoside) were purchased from Sigma-
Aldrich. Desired concentrations of enzyme were prepared by potassium
phosphate buffer (pH 6.8, 50 mM), and the target compounds were
dissolved in DMSO (10% final concentration). The enzyme solution (20
161; H NMR (500 MHz, DMSO‑d
.73 (s, 3H, OCH
6
3
3
2
3
CH
2
2
1
3
μ
L), different concentrations of compounds (20
μ
L), and potassium
6
3
2
2
phosphate buffer (135
μL) were added to the 96-well plate and incu-
(
3
2
◦
bated at 37 C for 10 min. Then, p-nitrophenyl glucopyranoside as
4

S. Moghimi et al.
Bioorganic Chemistry 109 (2021) 104670
substrate (25
μ
L, 4 mM) was added to each well and allowed to be
reciprocal of the substrate concentration (1/[S]) and reciprocal of
enzyme rate (1/V) over various inhibitor concentrations. Experimental
◦
incubated at 37 C for 20 min. Finally, the change in the absorbance was
measured at 405 nm by using spectrophotometer (Gen5, Power wave
xs2, BioTek, America). DMSO and acarbose were used as the control and
standard inhibitor, respectively. The percentage of inhibition for target
compounds, control, and the standard inhibitor was calculated by using
the following formula:
(K
i
) value was constructed by secondary plots of the inhibitor concen-
tration [I] versus K
m
.
2.2.4. Cytotoxic studies
The cytotoxic studies of selected compounds were performed ac-
cording to the previously reported literature [52].
%Inhibition = [(Abs control Abs sam ple)/Abs control] × 100
IC50 values of tested compounds were obtained from the nonlinear
2.2.5. Docking studies
regression curve using the Logit method.
Docking study of compound 6j was done using Autodock 4.2.1
software. The structure of S. cerevisiae isomaltase (PDB: 3A4A with 1.6 Å
resolution) was taken from RCSB data bank and the docking procedure
was carried out as previously reported [52]. The docking results were
analyzed by Discovery Studio visualizer 4.5.
2
.2.2. Rat
α
- glucosidase assay
-glucosidase (EC 3.2.1.20) was prepared ac-
Rat small intestine
α
cording to the method published by Lossow et al. (1964). Enzyme in
vitro activity was determined by recording the release of 4-nitrophenol
from Pnitrophenyl
et al. [59,60]. Final volume of 200
6-well plate as follow: the enzyme solution (190
different concentrations of compounds 1, 10, 20, 50, 100, 500 and 1000
M (5 L), and potassium phosphate buffer. Test compounds were dis-
α
-D glucopyranoside as previously described by Kim
L of assay solution was prepared in a
L, 0.15 units/ml),
3. Results and discussion
μ
9
μ
3.1. Chemistry
μ
μ
The synthesis of final compounds (6a-m) was achieved through the
five step reaction. Scheme 1 showed the synthetic route used to prepare
final compounds, starting from benzil. Benzil constituted a useful
starting material for the synthesis of heterocyclic compounds. According
to the related reports on the construction of pyridazine core, the reaction
of benzil and hydrazine gave 2-hydrazonoe-1,2-diphenyl ethanone 2
which upon treatment with diethyl malonate by using sodium ethano-
late afforded cyclized product 3. Ethyl-2,3-dihydro-3-oxo-5,6-
diphenylpyridazine-4-carboxylate was converted to compound 4 by
the action of Lawesson’s reagent. The propargylic unit at the side chain
was installed by the treatment of compound 4 in N,N-dimethyl form-
amide (DMF) with propargyl bromide in the presence of potassium
carbonate. The click reaction proceeded smoothly by employing 10 mol
percent sodium ascorbate/copper sulfate and 1 equiv. of triethyl amine
in water/tert-buthanol, affording 1,4-disubstituted triazole ring in good
yields with complete regioselectivity. The corresponding acids could be
easily obtained from the saponification reaction with ethanolic sodium
hydroxide solution. The synthetic protocol was examined on a series of
substituted benzyl bromides/chlorides and the data are collated in
Table 1.
solved in DMSO (not exceed than 5% of final volume). After 10 min. of
◦
pre-incubation at 37 C, p-nitrophenyl glucopyranoside as substrate (5
μ
L, 3 mM), was added to the enzyme solution and let to be incubated for
◦
one hour at 37 C. Finally, the change in the absorbance was followed at
4
05 nm using Cytation 3 hybrid microplate reader (BioTek, USA). DMSO
and acarbose were used as the control and standard inhibitor, respec-
tively. The extent of enzyme inhibition was calculated in the presence of
different concentrations of compounds by using the following formula
and data presented as percentage of inhibition.
%Inhibition = [(Abs control Abs sam ple)/Abs control] × 100
IC50 values of tested compounds were obtained from the nonlinear
regression curve using GraphPadprism 6.0 (San Diego, California, USA).
2
.2.3. Kinetic studies
The mode of inhibition of the most active compound 6j, identified
with the lowest IC50, was investigated against
α
-glucosidase activity
with different concentrations of p-nitrophenyl
α-D-glucopyranoside
(
2–10 mM) as substrate in the absence and presence of sample 6j at
different concentrations (0, 45, 65, and 85 µM). A Lineweaver–Burk plot
was generated to identify the type of inhibition and the Michae-
m
lis–Menten constant (K ) value was determined from plot between
N N
O
Ph
O
N N
H NN
Ph
O
N NH
c
2
d
a
b
Ph
S
Ph
SH
Ph
O
Ph
Ph
Ph
COOEt
Ph
COOEt
Ph
COOEt
1
2
3
4
5
N
R
N N
N
N
R
N
N N
Ph
S
N
f
e
Ph
S
N
Ph
COOEt
a-m
Ph
COOH
7
a-c
6
R = H, 4-F, 4-Br
R = H, 2-F, 3-F, 4-F, 3-Br, 4-F, 4-Cl, 4-Br, 2,6-dif,
,5-diCl, 3-NO , 4-NO , 3-OMe, 4-OMe
2
2
2
◦
Scheme 1. Synthesis of target compounds. Reagents and conditions: a) hydrazine hydrate, methanol, reflux, 15 min., 79%, m.p. lit. 149–151 C [58], m.p. obtained;
◦
1
48–150 ◦C; b) Na, EtOH, diethyl malonate, reflux, 3 h, 47%; c) Lawesson’s reagent, toluene, reflux, 18 h, 94%; d) propargyl bromide, DMF, K
2
CO
3
, 50 C, 2 h, 92%;
e) (i) benzyl bromide/chloride derivatives, NaN
3
, H
2
O/tert-buthanol, Et
3
N, 1 h; (ii) sodium ascorbate, copper (sulfate, 5, 6–10 h; f) NaOH (1 M), EtOH, reflux, 1–2 h.
5

S. Moghimi et al.
Bioorganic Chemistry 109 (2021) 104670
Table 1
rat enzyme, all compounds except 6l and 6 m were more active
α
-Glucosidase inhibitory activity of the target compounds, presented as IC50
compared to acarbose. The IC50 inhibition of acarbose against rat a-
glucosidase was found to be 318.2 ± 23.9
μ
M. Comparing the presence
a
(
μ
M).
of halogens at para position, the less electronegative atom, meaning
bromine (6 g) exhibited the less inhibitory activity. The reverse trend
was observed by replacing fluorine with bromine at meta position. The
introduction of second fluorine enhanced the inhibitory activity (6b vs
6h). The most active compounds was 6j. The interesting point is moving
this group to para position decreased the activity. The less active com-
pounds contained methoxy group at meta and para positions (Table 1).
b
c
Compound
X
R
IC50
(
μM)
IC50
(μM)
6
6
6
6
6
6
6
6
6
6
6
6
6
7
7
7
a
b
c
d
e
f
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOH
COOH
COOH
Acarbose
H
307.4 ± 4.3
198.0 ± 2.0
321.5 ± 4.5
238.4 ± 3.7
449.5 ± 6.6
213.3 ± 3.3
190.7 ± 1.9
202.5 ± 3.1
107.7 ± 1.2
85.6 ± 0.5
220.7 ± 52.6
119.7 ± 12.9
148.3 ± 16.3
278.5 ± 57.2
166.2 ± 31.3
284.5 ± 59.1
304.7 ± 46.9
89.1 ± 15.7
193.1 ± 42.6
73.7 ± 11.4
118.4 ± 21.2
466.8 ± 39.1
412.5 ± 45.4
294.6 ± 43.7
179.1 ± 33.2
215.4 ± 20.8
318.2 ± 23.9
2-F
3-F
3-Br
4-F
4-Cl
4-Br
2,6-diF
2,5-diCl
3.3. Kinetic studies
g
h
i
The mode of inhibition of the target derivatives was investigated by
kinetic studies on compound 6j (Fig. 2). The competitive mode of in-
hibition was determined for the most active compound. The unchanged
j
3-NO
4-NO
2
2
k
l
414.2 ± 6.3
220.6 ± 3.5
634.0 ± 9.2
489.2 ± 7.0
453.0 ± 6.8
249.4 ± 3.9
750.0 ± 10.0
3-OMe
4-OMe
H
V
max value and increased
Lineweaver-Burk plot, indicated this fact. Moreover, by drawing the plot
of the K versus different concentration of inhibitor, K
m
k value which were determined by
m
a
b
c
m
i
of 82 µM, was
4-F
determined for compound 6j.
4-Br
–
a
b
c
3.4. Cytotoxic studies
Values are the means of three replicates ± standard deviation (SD).
The activity against Saccharomyces cerevisiae.
In order to investigate the cytotoxicity of these compounds, the
active ones, 6g, 6j, and 7c, from both series were tested against the
normal cell line. In case of this cell line, HDF, no toxicity was observed
with either compound.
The activity against rat small intestine
α
-glucosidase.
3
.2.
α
-Glucosidase inhibitory activity
All target compounds were evaluated for their abilities to inhibit
3
.5. Docking study
α
-glucosidase activity of the extracted enzyme from Saccharomyces cer-
evisiae and rat small intestine
α-glucosidase. The activity against both
According to the biological results, the most active compound 6j was
enzymes showed same trend and the most active compound was 6j.
From the reported data, the preliminary structure–activity relationship
against Saccharomyces cerevisiae can be drawn. All synthesized com-
pounds had better inhibitory activities than acarbose, exhibiting IC50
values ranging from 85.6 to 634.0 µM. The most potent compound was
selected and docked into the active site of
α
-glucosidase enzyme to
identify the binding modes and interactions. Molecular docking study
was accomplished by Autodock 4.2.1 software package and the 3D and
2
D structure of docking interactions were depicted by Discovery Studio
visualizer 4.5. Crystal structure of S. cerevisiae -glucosidase is not pre-
pared yet, so the crystal structure of S. cerevisiae isomaltase (PDB:
A4A) was used due to its high similarity to S. cerevisiae -glucosidase.
The best score docking conformation of 6j was shown in Fig. 3.
α
3
-nitro containing derivative, 6j. The influence of halogens involving
fluorine, chlorine, and bromine at different positions was examined.
Moving fluorine from ortho to meta led to the decreased potency. Sub-
stitution of the most electronegative atom, fluorine as the lipophilic
group, at para position was not tolerated and led to the less potent
compound 6e with IC50 = 449.5 µM among halogen containing
derivatives.
3
α
Compound 6j established two conventional hydrogen bonds and several
hydrophobic interactions with the active pocket of enzyme which
enhanced the stability of the ligand-enzyme complex. The triazole ring
of 6j formed a hydrogen bond with His 280 of distance 2.46 Å and the
nitro group made the second hydrogen bond with Asn 415 of distance
Replacing fluorine at meta position (6c, IC50 = 321.5 µM) with
bromine resulted in increased potency, (6d, IC50 = 238.4 µM). The
replacement of the fluorine at para position with chlorine and bromine
yielded more potent compounds with IC50 values of 213.3, 190.7 µM,
respectively. The introduction of second fluorine atom at ortho position,
2
.87 Å. This ligand exhibited
42 and Asp 352, respectively. Moreover, the sulfide group of this
-sulfur interaction with Phe 303. Other interactions
stacked, T-shaped, and van der Waals were observed
between two phenyl moieties of this ligand and a number of residues.
π-cation and
π
-anion interactions with Arg
4
compound made
including
π
π-
π
π-π
6
h, did not lead to the significant enhancement in the activity compared
to mono substituted derivative 6b. 2,5-Dichloro-substituted compound
i vs 6h exhibited 2-fold improvements in activity indicating that
6
4
. Conclusion
dichloro substitution was more favorable than difluoro substituted one.
Interestingly, the introduction of nitro group at meta position produced
the most potent compound 6j, IC50 = 85.6 µM, with almost 10-fold more
potency compared to acarbose. IC50 = 750.0 µM. While, moving this
functional group to the para position led to the compound with 5-times
less activity compared to 6j. The presence of electron-donating group at
para position, 6m, dramatically decreased the enzyme inhibition
compared to unsubstituted and meta-substituted analogue, resulted in
the weakest compound with IC50 value of 634.0 µM.
We expanded the scope of our investigation by the replacement of
ester with acidic group. Compared with ester containing compounds, no
improvement in the inhibitory profile was observed. For compounds
bearing hydrogen, chlorine and fluorine at para position, the presence of
acidic group led to the decreased inhibitory effects, (7a, 7b, and 7c; IC50s
In conclusion, for the first time, we have demonstrated the -gluco-
α
sidase inhibitory activity of pyridazine-containing compounds. By this
method, sixteen compounds have been synthesized through the five-step
approach and their in-vitro inhibitory activity against yeast and rat small
intestine
α-glucosidase enzymes were investigated. Structure-activity
relationship investigation indicated compound 6j with nitro group at
meta position as the most active compound. The enhanced activity of
final compounds suggested this series as the good candidates for further
development. Our group is also engaged in expanding the library of
pyridazine-containing compounds as glucosidase inhibitors.
Declaration of Competing Interest
=
489.2, 453.0, 249.4 µM, respectively). Based on the evaluation against
The authors declare that they have no known competing financial
6

S. Moghimi et al.
Bioorganic Chemistry 109 (2021) 104670
a)
b)
3
0
8
6
4
2
0
Concentration of Inhibition
0
4
6
8
μM
20
5 μM
5 μM
5 μM
10
0
-
1.5
-1.0
-0.5
0.0
0.5
1.0
-1
1
/[S](PNPG, mM )
-
10
-
100
-80
-60
-40
-20
0
20
40
60
80
100
Compound (6)
Fig. 2. Kinetics of
α
-gluosidase inhibition by 6j. (a) The Lineweaver-Burk plot in the absence and presence of different concentrations of 6j (
μM); (b) the secondary
plot between K and various concentrations of 6j.
m
Fig. 3. The 3D binding conformation and 2D binding conformation of compound 6j.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported and funded by a grant from National
Institute for Medical Research Development (NIMAD); grant no.
9
95866.
7

S. Moghimi et al.
Bioorganic Chemistry 109 (2021) 104670
Appendix A. Supplementary data
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