Design and synthesis of novel pyridazine N-aryl acetamides: In-vitro evaluation of α-glucosidase inhibition, docking, and kinetic studies

Article abstract of DOI:10.1016/j.bioorg.2020.104071

We herein applied the four step-synthetic route to prepare the pyridazine core attached to the various N-aryl acetamides. By this approach, a new series of pyridazine-based compounds were synthesized, characterized and evaluated for their activities against α-glucosidase enzyme. In-vitro α-glucosidase assay established that twelve compounds are more potent than acarbose. Compound 7a inhibited α-glucosidase with the IC₅₀ value of 70.1 μM. The most potent compounds showed no cytotoxicity against HDF cell line. Molecular docking and kinetic studies were performed to determine the modes of interaction and inhibition, respectively.

Full text of DOI:10.1016/j.bioorg.2020.104071

Journal Pre-proofs
Design and synthesis of novel pyridazine N-aryl acetamides: In-vitro evalua-
tion of α-glucosidase inhibition, docking, and kinetic studies
Setareh Moghimi, Mahsa Toolabi, Somayeh Salarinejad, Loghman
Firoozpour, Seyed Esmaeil Sadat Ebrahimi, Fatemeh Safari, Somayeh
Mojtabavi, Mohammad Ali Faramarzi, Alireza Foroumadi
PII:
S0045-2068(20)31368-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.104071
YBIOO 104071
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
21 April 2020
21 June 2020
2 July 2020
Please cite this article as: S. Moghimi, M. Toolabi, S. Salarinejad, L. Firoozpour, S. Esmaeil Sadat Ebrahimi, F.
Safari, S. Mojtabavi, M. Ali Faramarzi, A. Foroumadi, Design and synthesis of novel pyridazine N-aryl
acetamides: In-vitro evaluation of α-glucosidase inhibition, docking, and kinetic studies, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.104071
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Design and synthesis of novel pyridazine N-aryl acetamides: In-vitro evaluation of α-
glucosidase inhibition, docking, and kinetic studies
a
b
c
a
Setareh Moghimi , Mahsa Toolabi , Somayeh Salarinejad , Loghman Firoozpour , Seyed
Esmaeil Sadat Ebrahimi c, Fatemeh Safari d, Somayeh Mojtabavi,e Mohammad Ali Faramarzi
e, Alireza Foroumadi a, c, **
a Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS),
Tehran University of Medical Sciences, Tehran, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran
c
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
d Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran
e Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
Acknowledgement
This work was supported and funded by a grant from Iran National Science Foundation (INSF);
grant no. 96011863.
Conflict of Interest
The authors declare no conflict of interest.
Corresponding author:
** E-mail: aforoumadi@yahoo.com (A. Foroumadi); Tel.: 98 21 66406757; Fax: 98 21 66461178.

Abstract
We herein applied the four step-synthetic route to prepare the pyridazine core attached
to the various N-aryl acetamides. By this approach, a new series of pyridazine-based
compounds were synthesized, characterized and evaluated for their activities against α-
glucosidase enzyme. In-vitro α-glucosidase assay established that twelve compounds are more
potent than acarbose. Compound 7a inhibited α-glucosidase with the IC50 value of 70.1 µM.
The most potent compounds showed no cytotoxicity against HDF cell line. Molecular docking
and kinetic studies were performed to determine the modes of interaction and inhibition,
respectively.
.
Keywords: Pyridazine; Glucosidase inhibitor; Lawesson’s reagent; Antidiabetic drug
1. Introduction
Diabetes (diabetes mellitus) is a chronic metabolic disease, characterized by the
increased blood sugar, resulted from defects in the action or secretion of insulin. The
cardiovascular, retinopathy, kidney failure, amputation, eyesight difficulties, and diabetic
neuropathy problems are known as the complicated symptoms resulted from diabetes. This
health problem is divided to type 1 and type 2 and nearly 90% of all cases of diabetes are type
2. This disease is affecting millions of people globally and the increasing number of patients
up to 642 million is expected by 2040 [1,2]. In addition, this disease will become the seventh
cause of death by 2030. The public health crisis stemming from long term complications of
diabetes, the increasing rate of prevalence among people specifically type 2 (T2DM) and its
long term complications urged the pressing need for novel chemotherapies with better safety
profiles to combat diabetes [3]. The efforts in the discovery of novel agents should be
accompanied by exercise and dietary modifications. The antidiabetic agents currently used to
control blood glucose level are sulfonyl ureas, thiazolidinediones [4], biguanides [5], dipeptidyl
peptidase-4 (DPP-4) inhibitors [6], GLP-1 agonists [7], and SGLT2 inhibitors [8-11]. The
glycemic control in type 2 diabetes is also achieved by α-glucosidase inhibitors [12,13]
involving acarbose, miglitol, and vaglibose, used as monotherapy or in combination with
insulin or other oral drugs. The function of this class of inhibitors led to the reduced
postprandial hyperglycemia by retarding the digestion of carbohydrates and release of glucose.
Despite the therapeutic benefits associated with currently available drugs, the side effects
including decreased carbohydrate digestion, increased hyperglycemia risk, obesity and
possible cancer risk limited their uses and highlighted the ongoing needs to survey for new
drug candidates.

Pyridazines and their oxo-derivatives are known as the privileged heteroaromatic
skeleton due to the important bioactivities associated with these compounds involving
antihypertensive, antifungal, anti-inflammatory, anticancer, and antimicrobial activities [14-
19]. Regarding this fact, there has been extensive attention toward the synthesis of novel
compounds containing pyridazine moiety and evaluation their bioactivities. Moreover, amide
bond is ubiquitously present in drug molecules and biologically active compounds because of
its unique features including the polarity, amide proton exchange rate, and protein binding. For
instance, this functional group is found in lidocaine, mepivacaine, prilocaine, ceramide drugs
used as anesthetics [20].
Bioisosterism is a fundamental concept for medicinal chemists to rationally modify and
optimize the compounds to design more potent drug candidates [21,22]. Regarding the previous
reports on the α-glucosidase inhibitory activity of triazine-containing compounds [23-27], we
have chosen pyridazine ring as triazine bioisoster to study their glucosidase inhibitory activity.
The purpose of the current work is to synthesize a series of pyridazine-N-aryl acetamide hybrid
system and evaluate them as α-glucosidase inhibitors, so, in continuation of our work focused
on the synthesis and biological evaluation of novel heterocyclic compounds [28-31], herein,
our results are presented.
Some compounds with α-glucosidase inhibitory activity are depicted in Figure 1.
R1
R
O
N
N
N
N
OH
H
Ar
Ar
N
N
S
Ar
Ar
N
N
S
S
N
O
R2
N
N
N
A
B
O
R
OH
S
Ph
Ph
N
N
N
Ar
Ar
N
N
H
N
O
N
O
S
C
D
Ph
Ph
N
O
X
R
HN
This work
Figure 1. Some reported α-glucosidase inhibitors containing the triazine skeleton.
2. Experimental
2.1 General chemistry
All commercially available reagents and solvents, used without further purification,
were purchased from Merck and Sigma-Aldrich companies. Bruker FT-500 MHz spectrometer
1
13
was used to confirm the structure of the target compounds by H- and C-NMR spectra in

DMSO-d6 as the solvent. Chemical shifts reported in δ parts per million (ppm) downfield from
tetramethylsilane (TMS) as the internal standard. 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 apparatus and are uncorrected. IR spectroscopy
was performed using a Nicolet FT-IR Magna 550 spectrograph (KBr disks).
2.1.1. General procedure for the synthesis of 2-hydrazono-1,2-diphenylethanone (2)
To the warm solution of benzil (10 mmol) in methanol (50 ml), hydrazine hydrate (12
mmol) was added dropwise. The reaction was stirred at reflux temperature for 10 minutes.
Upon cooling, the precipitate was filtered and washed with cold methanol [32].
2.1.2 General procedure for the synthesis of ethyl-2,3-dihydro-3-oxo-5,6-
diphenylpyridazine-4-carboxylate (3)
To the chilled solution of absolute ethanol (200 mL), sodium (0.05 mol) was carefully
added. After obtaining sodium ethoxide solution, 0.05 mol of 2-hydrazono-1,2-diphenyl
ethanone was added followed by the addition of diethyl malonate (0.075 mol). Then, the
reaction was refluxed for 3 h. After this time, the mixture was concentrated under vaccum and
acidifed by HCl (1M). The desired product was collected and washed with water [33].
2.1.3. General procedure for the synthesis ethyl-3-mercapto-5,6-diphenylpyridazie-4-
carboxylate (4)
To the solution of compound 3 (20 mmol) in toluene (150 mL) was added Lawesson’s
reagent (10 mmol) and the mixture was allowed to reflux for 18 h. The reaction was then
concentrated and the residue was purified by crystallization from petroleum ether/ethyl acetate
[34].
2.1.4. General procedure for the synthesis of N-aryl acetamide (6)
To the chilled solution of the appropriate amine (10 mmol) and triethyl amine (10
mmol), chloroacetyl chloride (10 mmol) was added dropwise. The reaction was continued at
room temperature upon completion, checked by TLC. Then, petroleum ether was added to the
mixture and the resultant solid was filtered, washed and used without further purification.
2.1.5. General procedure for the synthesis of compounds (7a-i)
The mixture of compound 4 (1 mmol) and K2CO3 (1 mmol) in DMF (10 mL) was stirred
for 10 min. at room temperature. Then, N-aryl acetamides (1 mmol) and catalytic amounts of
o
KI were added to the mixture and the reaction was continued at 50 C. Upon completion,
checked by TLC, the ice/water solution was added to the mixture. The precipitate was filtered
and recrystallized from petroleum ether/ethyl acetate. If no solid obtained, the mixture would
be extracted with ethyl acetate and the residue was purified by column chromathography
(different ratios of petroleum ether/ethyl acetate).

2.1.5.1.
Ethyl
3-((2-oxo-2-(phenylamino)ethyl)thio)-5,6-diphenylpyridazine-4-
carboxylate (7a)
White solid; yield: 80%; mp: 109-111 °C; IR (KBr, cm-1): 3354 (N-H), 1724 (C=O), 1690
(C=O), 1319, 1194; 1H NMR (500 MHz, DMSO-d6) δ: 0.90 (t, J = 7.1 Hz, 3H), 4.09 (q, J = 7.2
Hz, 2H), 4.42 (s, 2H), 7.06 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 6.8 Hz, 2H), 7.25-7.35 (m, 10H),
7.61 (d, J = 8.1 Hz, 2H), 10.41 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 13.2, 34.7, 59.2,
111.2, 123.3 (2C), 124.2, 126.3 (2C), 126.4 (2C), 126.7, 127.1 (2C), 127.3 (2C), 127.4, 127.8
(2C), 131.2, 137.3, 137.4, 146.5, 149.0, 164.5, 168.3; Anal. Calcd. For C27H23N3O3S: C, 69.06;
H, 4.94; N, 8.95. Found: C, 69.28; H, 4.61; N, 8.71; ESI-MS m/z: calculated: 469.1 [M]+;
obtained: 470.2 [M+H]+.
2.1.5.2.
Ethyl
3-((2-oxo-2-(p-tolylamino)ethyl)thio)-5,6-diphenylpyridazine-4-
carboxylate (7b)
Off-white solid; yield: 80%; mp: 160-162 °C; IR (KBr, cm-1): 3317 (N-H), 1728 (C=O), 1674
(C=O), 1298, 1201; 1H NMR (500 MHz, DMSO-d6) δ: 0.90 (t, J = 7.1 Hz, 3H), 2.25 (s, 3H),
4.08 (q, J = 7.2 Hz, 2H), 4.40 (s, 2H), 7.11-7.15 (m, 4H), 7.26-7.36 (m, 8H), 7.49 (d, J = 7.9
Hz, 2H), 10.33 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 12.8, 19.8, 34.5, 61.5, 118.5 (2C),
127.3 (2C), 127.7 (2C), 128.0, 128.2, 128.4 (2C), 128.6 (2C), 129.0 (2C), 129.6, 131.7, 133.1,
134.6, 135.4, 135.9, 155.1, 156.8, 163.5, 164.8; Anal. Calcd. For C28H25N3O3S: C, 69.54; H,
5.21; N, 8.69. Found: C, 69.75; H, 5.49; N, 8.81; ESI-MS m/z: calculated: 483.1 [M]+; obtained:
484 .2 [M+H]+.
2.1.5.3. Ethyl 3-((2-((4-isopropylphenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-
4-carboxylate (7c)
Off-white solid; yield: 75%; mp: 164-166 °C; IR (KBr, cm-1): 3347 (N-H), 1730 (C=O), 1669
(C=O), 1325, 1188; 1H NMR (500 MHz, DMSO-d6) δ: 0.90 (t, J = 7.1 Hz, 3H), 1.17 (d, J =
6.8 Hz, 6H), 2.80-2.86 (m, 1H), 4.09 (q, J = 7.1 Hz, 2H), 4.41 (s, 2H), 7.15 (d, J = 7.1 Hz, 2H),
7.19 (d, J = 8.0 Hz, 2H), 7.24-7.45 (m, 8H), 7.53 (d, J = 8.0 Hz, 2H), 10.36 (s, 1H); 13C NMR
(125 MHz, DMSO-d6) δ: 12.8, 23.3, 32.3, 34.5, 38.9, 61.5, 118.6 (2C), 125.9 (2C), 127.3 (2C),
127.7 (2C), 128.0, 128.2, 128.4 (2C), 129.0 (2C), 129.6, 133.1, 134.6, 135.4, 136.2, 142.9,
155.1, 156.8, 163.5, 164.8; Anal. Calcd. For C30H29N3O3S: C, 70.43; H, 5.71; N, 8.21. Found:
C, 70.12; H, 5.47; N, 8.54; ESI-MS m/z: calculated: 511.2 [M]+; obtained: 512.3 [M+H]+.
2.1.5.4. Ethyl 3-((2-((3-ethynylphenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylate (7d)
Off-white solid; yield: 70%; mp: 83-85 °C; IR (KBr, cm-1): 3288 (N-H), 1727 (C=O), 1688
(C=O), 1326, 1195; 1H NMR (500 MHz, DMSO-d6) δ: 0.89 (t, J = 7.1 Hz, 3H), 4.09 (q, J =
7.1 Hz, 2H), 4.19 (s, 1H), 4.43 (s, 2H), 7.14-7.18 (m, 3H), 7.22-7.36 (m, 9H), 7.59 (d, J = 8.3
Hz, 1H), 7.83 (s, 1H), 10.56 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 12.8, 34.5, 61.6, 80.0,
82.7, 119.1, 121.3, 121.5, 126.0, 127.3 (2C), 127.7 (2C), 128.0, 128.2, 128.4 (2C), 128.7, 129.0
(2C), 129.6, 133.1, 134.6, 135.4, 138.6, 155.0, 156.8, 163.5, 165.4; Anal. Calcd. For

C29H23N3O3S: C, 70.57; H, 4.70; N, 8.51. Found: C, 70.21; H, 4.59; N, 8.84; ESI-MS m/z:
calculated: 493.1 [M]+; obtained: 494.1 [M+H]+.
2.1.5.5. Ethyl 3-((2-((4-methoxyphenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-
4-carboxylate (7e)
Off-white solid; yield: 82%; mp: 142-145 °C; IR (KBr, cm-1): 3312 (N-H), 1722 (C=O), 1672
(C=O), 1300, 1161; 1H NMR (500 MHz, DMSO-d6) δ: 0.89 (t, J = 6.6 Hz, 3H), 3.71 (s, 3H),
4.09 (q, J = 7.2 Hz, 2H), 4.39 (s, 2H), 6.89 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 5.9 Hz, 2H), 7.23-
7.33 (m, 8H), 7.52 (d, J = 7.9 Hz, 2H), 10.33 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 13.3,
34.9, 55.1, 62.1, 113.9 (2C), 120.6 (2C), 127.8 (2C), 128.3 (2C), 128.5, 128.8 (2C), 128.9,
129.6 (2C), 130.2, 132.1, 133.6, 135.1, 135.9, 155.2, 155.7, 157.3, 164.1, 165.0; Anal. Calcd.
For C28H25N3O4S: C, 67.32; H, 5.04; N, 8.41; Found: C, 67.59; H, 5.21; N, 8.12; ESI-MS m/z:
calculated: 499.2 [M]+; obtained: 500.1 [M+H]+.
2.1.5.6. Ethyl 3-((2-((4-fluorophenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylate (7f)
Yellow solid; yield: 77%; mp: 159-161 °C; IR (KBr, cm-1): 3342 (N-H), 1721 (C=O), 1679
(C=O), 1309, 1214; 1H NMR (500 MHz, DMSO-d6) δ: 0.90 (t, J = 7.2 Hz, 3H). 4.09 (q, J =
7.2 Hz, 2H), 4.41 (s, 2H), 7.14-7.17 (m, 4H), 7.24-7.36 (m, 8H), 7.64 (dd, J = 9.4, 5.1 Hz, 2H),
10.46 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 12.8, 34.4, 61.5, 114.8 (d, J = 22.0 Hz, 2C),
120.3 (d, J = 7.6 Hz, 2C), 127.3 (2C), 127.7 (2C), 128.0, 128.2, 128.4 (2C), 129.0 (2C), 129.6,
133.1, 134.6, 134.8, 135.4, 155.0, 156.8, 157.5 (d, J = 238.5 Hz), 163.5, 165.0; Anal. Calcd.
For C27H22FN3O3S: C, 66.52; H, 4.55; N, 8.62; Found: C, 66.75; H, 4.29; N, 8.81; ESI-MS
m/z: calculated: 487.1 [M]+; obtained: 488.2 [M+H]+.
2.1.5.7. Ethyl 3-((2-((4-chlorophenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylate (7g)
Yellow solid; yield: 75%; mp: 149-151 °C; IR (KBr, cm-1): 3331 (N-H), 1724 (C=O), 1683
(C=O), 1324, 1166; 1H NMR (500 MHz, DMSO-d6) δ: 0.90 (t, J = 7.2 Hz, 3H), 4.09 (q, J =
7.1 Hz, 2H), 4.42 (s, 2H), 7.15 (d, J = 7.1 Hz, 2H), 7.23-7.27 (m, 5H), 7.31-7.41 (m, 5H), 7.65
(d, J = 8.5 Hz, 2H), 10.56 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 12.8, 34.5, 61.6, 120.0
(2C), 126.4, 127.3 (2C), 127.7 (2C), 128.0, 128.1 (2C), 128.2, 128.4 (2C), 129.0 (2C), 129.6,
133.1, 134.6, 135.4, 137.4, 155.0, 156.8, 163.5, 165.3; Anal. Calcd. For C27H22ClN3O3S: C,
64.34; H, 4.40; N, 8.34. Found: C, C, 64.15; H, 4.61; N, 8.54; ESI-MS m/z: calculated: 503.1
[M]+; obtained: 504.1 [M+H]+.
2.1.5.8.
Ethyl
3-((2-((3,5-dichlorophenyl)amino)-2-oxoethyl)thio)-5,6-
diphenylpyridazine-4-carboxylate (7h)
Off-white solid; yield: 74%; mp: 133-135°C; IR (KBr, cm-1): 3412 (N-H), 1721 (C=O), 1664
(C=O), 1300, 1187; 1H NMR (500 MHz, DMSO-d6) δ: 0.89 (t, J = 7.7 Hz, 3H), 4.09 (q, J =
7.6 Hz, 2H), 4.45 (s, 2H), 7.14 (d, J = 7.1 Hz, 2H), 7.24-7.34 (m, 9H), 7.70 (s, 2H), 10.80 (s,
1H); 13C NMR (125 MHz, DMSO-d6) δ: 12.7, 34.6, 61.6, 116.6 (2C), 122.0, 127.3 (2C), 127.7
(2C), 128.0, 128.2, 128.4 (2C), 129.0 (2C), 129.6, 133.1, 133.6 (2C), 134.7, 135.3, 140.7,

154.9, 156.8, 163.5, 165.9; Anal. Calcd. For C27H21Cl2N3O3S: C, 60.23; H, 3.93; N, 7.80.
Found: C, 60.51; H, 3.67; N, 7.71; ESI-MS m/z: calculated: 537.1 [M]+; obtained: 538.3
[M+H]+.
2.1.5.9. Ethyl 3-((2-((4-cyanophenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylate (7i)
Yellow solid; yield: 71%; mp: 194-196 °C; IR (KBr, cm-1): 3291 (N-H), 2216 (C N), 1724
(C=O), 1671 (C=O), 1324, 1199; 1H NMR (500 MHz, DMSO-d6) δ: 0.91 (t, J = 7.0 Hz, 3H),
4.06-4.13 (q, J = 7.1 Hz, 2H), 4.46 (s, 2H), 7.15 (d, J = 7.0 Hz, 2H), 7.25-7.33 (m, 8H), 7.77-
7.82 (m, 4H), 10.87 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 12.7, 34.6, 61.6, 104.6, 118.4,
118.5 (2C), 127.3 (2C), 127.7 (2C), 128.0, 128.2, 128.4 (2C), 129.0 (2C), 129.5, 132.8 (2C),
133.1, 134.7, 135.3, 142.6, 154.9, 156.9, 163.5, 166.0; Anal. Calcd. For C28H22N4O3S: C,
68.00; H, 4.48; N, 11.33. Found: C, 68.24; H, 4.19; N, 11.58; ESI-MS m/z: calculated: 494.1
[M]+; obtained: 495.4 [M+H]+.
2.1.6. General procedure for the preparation of compounds 8a-8e
The appropriate compounds (1 mmol) were dissolved in ethanol (5 mL) and after that
NaOH solution (1M, 5 mL) was added and the mixture was refluxed. Upon completion,
checked by TLC, the mixture was acidified to pH = 1 and the resultant solid was filtered and
washed with water.
2.1.6.1. 3-((2-Oxo-2-(phenylamino)ethyl)thio)-5,6-diphenylpyridazine-4-carboxylic acid
(8a)
Off-white solid; yield: 82%; mp: 122-124 °C; IR (KBr, cm-1): 3376 (N-H), 3056 (COOH),
1719 (C=O), 1660 (C=O), 1326, 1200; 1H NMR (500 MHz, DMSO-d6) δ: 4.45 (s, 2H), 7.16-
7.20 (m, 2H), 7.23-7.36 (m, 9H), 7.79-7.83 (m, 4H), 10.88 (s, 1H), 12.25 (brs, 1H). 13C NMR
(125 MHz, DMSO-d6) δ: 35.8, 115.3, 121.2 (2C), 125.3, 127.4 (2C), 127.9, 128.1 (2C), 128.1
(2C), 128.3 (2C), 128.5, 129.0 (2C), 132.4, 133.3, 136.2, 138.6, 147.6, 148.9, 166.9, 169.5;
Anal. Calcd. For C25H19N3O3S: C, 68.01; H, 4.34; N, 9.52. Found: C, 68.32; H, 4.15; N, 9.84.
ESI-MS m/z: calculated: 441.1 [M]+; obtained: 442.4 [M+H]+.
2.1.6.2.
3-((2-((3-Ethynylphenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylic acid (8b)
Off-white solid; yield: 78%; mp: 201-203 °C; IR (KBr, cm-1): 3308 (N-H), 3105 (COOH),
1695 (C=O), 1681 (C=O), 1309, 1217; 1H NMR (500 MHz, DMSO-d6) δ: 4.19 (s, 1H), 4.41
(s, 2H), 7.17-7.19 (m, 3H), 7.25-7.37 (m, 9H), 7.60 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 10.54 (s,
1H), 12.14 (brs, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 35.1, 77.5, 79.3, 122.4, 122.6, 123.9,
126.5 (2C), 128.5, 128.6, 129.0, 129.3, 129.6 (2C), 130.3, 130.4 (2C), 130.5 (2C), 135.2, 136.6,
138.6, 138.8, 158.0, 159.2, 167.8, 168.1; Anal. Calcd. For C27H19N3O3S: C, 69.66; H, 4.11; N,
9.03. Found: C, 69.45; H, 4.31; N, 9.37; ESI-MS m/z: calculated: 465.1 [M]+; obtained: 466.2
[M+H]+.

2.1.6.3.
3-((2-((4-Chlorophenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylic acid (8c)
Off-white solid; yield: 80%; mp: 169-171 °C; IR (KBr, cm-1): 3425 (N-H), 3081 (COOH),
1691 (C=O), 1683 (C=O), 1321, 1200; 1H NMR (500 MHz, DMSO-d6) δ: 4.41 (s, 2H), 7.18
(d, J = 8.1 Hz, 2H), 7.26-7.34 (m, 8H), 7.39 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.6 Hz, 2H), 10.57
(s, 1H), 12.17 (brs, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 34.7, 124.4 (2C), 128.6, 128.8
(2C), 129.9 (2C), 130.1 (2C), 130.4, 130.6 (2C), 131.1 (2C), 132.4, 134.4, 135.0, 136.3, 140.6,
141.7, 154.9, 161.9, 168.8, 169.6; Anal. Calcd. For C25H18ClN3O3S: C, 63.09; H, 3.81; N, 8.83.
Found: C, 63.27; H, 3.52; N, 8.99; ESI-MS m/z: calculated: 475.1 [M]+; obtained: 476.1
[M+H]+.
2.1.6.4.
3-((2-((3,5-Dichlorophenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylic acid (8d)
Off-white solid; yield: 79%; mp: 161-163 °C; IR (KBr, cm-1): 3485 (N-H), 3244 (COOH),
1687 (C=O), 1677 (C=O), 1326, 1203; 1H NMR (500 MHz, DMSO-d6) δ: 4.42 (s, 2H), 7.16-
7.20 (m, 2H), 7.25-7.34 (m, 9H), 7.70 (d, J = 1.9 Hz, 2H), 10.79 (s, 1H), 12.24 (brs, 1H); 13C
NMR (125 MHz, DMSO-d6) δ: 33.2, 118.0 (2C), 122.4, 128.4 (2C), 128.6 (2C), 128.9, 129.1
(2C), 129.6 (2C), 130.3, 132.9, 133.5, 134.8, 134.9 (2C), 140.0, 140.2, 153.4, 160.4, 167.3,
168.1; Anal. Calcd. For C25H17Cl2N3O3S: C, 58.83; H, 3.36; N, 8.23. Found: C, 58.98; H, 3.51;
N, 8.01. ESI-MS m/z: calculated: 475.1 [M]+; obtained:510.4 [M+H]+.
2.1.6.5.
3-((2-((4-cyanophenyl)amino)-2-oxoethyl)thio)-5,6-diphenylpyridazine-4-
carboxylic acid (8e)
Off-white solid; yield: 75%; mp: 182-184 °C; IR (KBr, cm-1): 3395 (N-H), 3051 (COOH),
2221 (C N), 1705 (C=O), 1671 (C=O), 1301, 1166; 1H NMR (500 MHz, DMSO-d6) δ: 4.43
(s, 2H), 7.07 (t, J = 7.4 Hz, 1H), 7.17-7.21 (m, 2H), 7.25-7.34 (m, 9H), 7.64 (d, J = 8.0 Hz,
2H), 10.44 (s, 1H), 12.33 (s, 1H); 13C NMR (125 MHz; DMSO-d6) δ: 33.2, 108.7, 118.5, 120.1
(2C), 128.6 (2C), 128.9, 129.1 (2C), 129.6 (2C), 130.9, 132.0 (2C), 132.9 (2C), 133.5, 134.8,
140.2, 141.2, 153.4, 160.4, 167.3, 168.1; Anal. Calcd. For C26H18N4O3S: C, 66.94; H, 3.89; N,
12.01. Found: C, 66.71; H, 3.62; N, 12.37; ESI-MS m/z: calculated: 466.1 [M]+; obtained:
467.4 [M+H]+.
2.2. Biological Results
2.2.1. In-vitro α-glucosidase inhibition assay
α-Glucosidase (Saccharomyces cerevisiae, EC3.2.1.20, 20 U/mg) and 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 μL),
different concentrations of compounds (20 μL), and potassium phosphate buffer (135 μL) were
added to the 96-well plate and incubated at 37 °C for 10 min. Then, p-nitrophenyl
glucopyranoside as substrate (25 μL, 4 mM) was added to each well and allowed to be
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:
%Inhibition = [(Abs control Abs sample)/Abs control] × 100
IC50 values of tested compounds were obtained from the nonlinear regression curve using the
Logit method.
2.2.2. Kinetic studies
The mode of inhibition of the most active compound 7a, 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 7a at
different concentrations (0, 30, 50, and 70 µM). A Lineweaver–Burk plot was generated to
identify the type of inhibition and the Michaelis–Menten constant (Km) value was determined
from plot between reciprocal of the substrate concentration (1/[S]) and reciprocal of enzyme
rate (1/V) over various inhibitor concentrations. Experi (Ki) value was constructed by
secondary plots of the inhibitor concentration [I] versus Km.
2.2.3. Cytotoxic studies
The cytotoxic studies of selected compounds were performed according to the
previously reported literature [28].
2.2.4. Docking studies
Molecular docking study of compound 7a and 8a were conducted using Autodock
4.2.1. The crystal structure of S. cerevisiae isomaltase (PDB: 3A4A) with 1.6 Å resolution was
downloaded from the RCSB data bank. The docking procedure was performed as previously
reported. The detailed docking analysis including the 3D and 2D diagram interactions were
depicted by Discovery Studio visualizer 4.5 [35].
3. Results and discussion
3.1 Chemistry
The overall synthetic approach is shown in scheme 1. At first, benzil, dissolved in
methanol, was treated with hydrazine hydrate to generate compound 2. Then, the isolated white
solid was added to the sodium ethanolate solution, prepared in situ, followed by the addition

of diethyl malonate. After stirring at reflux temperature for 3 hours and acidification of the
residue, the desired pyridazine 3 was obtained by a tandem one-pot cyclization. Finally, ethyl-
3-mercapto-5,6-diphenylpyridazie-4-carboxylate was obtained by the reaction of ethyl-2,3-
dihydro-3-oxo-5,6-diphenylpyridazine-4-carboxylate with Lawesson’s reagent in toluene at
reflux temperature. The procedure for the synthesis of N-aryl acetamides was carried out by
using the appropriate amines and chloroacetyl chloride using triethyl amine. Compound 5 with
different substituents e.g. electron-donating and electron withdrawing at different positions on
the phenyl ring could react and provide the corresponding N-chloro acetamide derivatives in
satisfactory yields. This procedure, called route B and presented in Scheme 1. As schematically
presented in route C, the nucleophilic substitution reaction between compounds 4 and 6 in
dimethyl formamide (DMF), K2CO3, and catalytic amounts of KI afforded final compounds
7a-i in good yields. The acid analogues of final compounds 8a-e were obtained by the basic
hydrolysis of compounds 7a-i in ethanol. Our tries to perform the reaction between acid
analogue of compound 4 and N-phenyl acetamide led to the product in much lower yields.
A series of substituents including methyl, methoxy, ethynyl, isopropyl, fluoro, chloro,
and cyano were examined to explore the substrate scope of the reaction and the results are
presented in table 1.
A
:
O
Ph
O
N N
H2NN
Ph
Ph
O
N NH
c
a
b
Ph
SH
Ph
O
Ph
Ph
COOEt
Ph
COOEt
1
2
3
4
H
N
NH2
B
C
:
d
Cl
R
R
O
5
6
R
R
HN
HN
O
N N
:
N N
f
e
Ph
S
O
Ph
S
4
+
6
Ph
COOEt
7a-i
R = H, 4-Me, 4-i-Pr, 3-ethynyl
4-OMe, 4-F, 4-Cl, 3,5-diCl, 4-CN
Ph
COOH
8a-e
R = H, 3-ethynyl, 4-Cl, 3,5-diCl, 4-CN
Scheme 1. Synthesis of target compounds. Reagents and conditions: a) hydrazine hydrate, methanol, reflux; b)
Na, EtOH, diethyl malonate, reflux; c) Lawesson’s reagent, toluene, reflux; d) chloroacetyl chloride, Et3N,
CH2Cl2; e) K2CO3, KI, N-aryl acetamides, DMF, 50 oC; f) NaOH (1M), EtOH, reflux.
3.2. α-Glucosidase inhibitory activity

The activity of two series of final compounds (7a-i) and (8a-e) were investigated and
the results were depicted in Table 1. All the ester-containing derivatives showed higher
inhibitory activities with IC50 values of 70.1-321.6 µM than the activity of acarbose. The most
potent compound in this series was the unsubstituted derivative 7a with IC50 value of 70.1 µM
which is 10 folds more active than positive control, acarbose, with IC50 value of 750 µM.
Then, various hydrophobic substituents including methyl, isopropyl, methoxy, ethynyl,
and chlorine were examined at 3- and 4-position of phenyl ring. The introduction of a methyl
group at the para position of phenyl ring led to the decreased activity against α-glucosidase
enzyme with IC50 = 161.0 µM. Further enlargement of the methyl group at para position,
isopropyl, retained the inhibitory activity (7b vs 7c). Interestingly, the introduction of a linear
ethynyl group at meta position produced compound (7d, IC50 =91.0 µM) which is 8 times more
potent than positive control. Introducing halogen atoms such as fluoro and chloro to the
benzene ring at para position led to the weakest compounds in this series with IC50s = 321.6,
300.4 µM, respectively. The presence of fluorine, the electronegative atom, led to compound
7f exhibiting decreased activity in comparison with chlorine substituted compound 7g. While,
the presence of two chlorine atom at 3 and 5 positions, 7h, increased the activity (IC50 = 123.3
µM). This addition might be resulted in better interactions of compounds with the active site
of enzyme. Then, the introduction of a linear and polar group, nitrile, yielded compound 7i
which demonstrated two-times more potency in relation to para-substituted fluoro and chloro-
containing compounds.
The conversion of ester to acid led to losses in potency in case of unsubstituted and 3,5-
dichloro substituents. The presence of electron withdrawing groups involving chlorine and
cyano group at para position increased the inhibitory activities compared to unsubstituted
derivative 8a. While, an ethynyl group at meta position resulted in nearly two-fold decrease in
activity in relation to 8c and 8e. In all cases, the comparison between ester and acid analogues
revealed that except 4-chloro derivative (7g vs 8c), the inhibitory activity of acid analogues
decreased.
Table 1. α-Glucosidase inhibitory activity of the target compounds, presented as IC50 (μM). a
Ph
Ph
N
N
H
N
S
X
R
O
X
R
Compound
IC50 (μM)
70.1 ± 0.6
161.0 ± 2.1
COOEt
COOEt
COOEt
COOEt
H
7a
7b
7c
7d
4-Me
4-iPr
166.8 ± 2.3
91.0 ± 1.0
3-Ethynyl

COOEt
COOEt
COOEt
COOEt
COOEt
COOH
COOH
COOH
COOH
4-OMe
4-F
154.5 ± 1.9
321.6 ± 4.4
300.4 ± 4.1
123.3 ± 1.4
140.6 ± 1.7
>750
7e
7f
4-Cl
7g
7h
7i
3,5-diCl
4-CN
H
8a
8b
8c
3-Ethynyl
4-Cl
457.3 ± 5.3
278.7 ± 3.6
>750
3,5-diCl
8d
8e
COOH
4-CN
297.7 ± 3.9
Acarbose
750.0 ± 10.0
aValues are the means of three replicates±standard deviation (SD).
3.3. Kinetic studies
The kinetic studies were utilized to determine the mode of inhibition of the selected
target derivatives. The competitive mode of inhibition was determined for the most active
compound, 7a. The unchanged Vmax value and increased Km value which were determined by
Lineweaver-Burk plot indicated that compound 7a is bound to the active site and competed
with the substrate. Moreover, by drawing the plot of the Km versus different concentrations of
inhibitor, an estimate amount of 70 µM was determined for the inhibition constant, Ki.

Figure 2. Kinetics of α-glucosidase inhibition by compound 7a; (a) The Lineweaver-Burk plot in the absence and
presence of different concentrations of compound 7a; (b) The secondary plot between Km and various
concentrations of compound 7a.
3.4. Cytotoxic studies
The cytotoxic activities of the most active compounds 7a, 7d, 8a, and 8e were evaluated
in relation to normal cell line, Human Dermal Fibroblasts (HDF), using the 3-(4,5-
dimethylthiazole-2-yl)2,5-diphenyltetrazolium bromide (MTT) colorimetic assay. According
to the obtained IC50 values, the results indicated that none of selected compounds were toxic
against the normal cell line [28]. The activity of these compounds is similar to etoposide which
was used as the positive control
Table 2. In vitro antiproliferative effects (IC50, µM) of selected compounds against HDF cell line.a
Compound
IC50 (μM)
>100
7a
7d
8a
>100
>100
8e
>100
Etoposide
>100
aValues were the means of three replicates±standard deviation (SD).
3.5. Docking study
In order to provide insights into the binding interactions of synthesized compounds
in the active site of α-glucosidase, molecular docking studies were utilized with the most active
compound, 7a and its acidic analogue, 8a, using Autodock 4.2.1 package. Since the x-ray
crystallographic structure S. cerevisiae α-glucosidase isn’t accessible, the 3D structure of S.
cerevisiae isomaltase with PDB ID: 3A4A was downloaded from RCSB web site with 84%
similarity to S. cerevisiae α-glucosidase. The docking analysis data including the best scoring
conformation based on binding energy were illustrated by Discovery Studio visualizer 4.5 and
shown in figures 3 and 4.

Figure 3 indicated the interaction of compound 7a with the active site residues.
Compound 7a interacted with the residues present in the active site of enzyme through several
hydrogen bonds and hydrophobic interactions. The amide group established two hydrogen
bonds with Glu 277 and Arg 442 and the nitrogen atom of pyridazine also formed another
hydrogen bond with Gln 279. Likewise, π-anion and π-π T-shaped interactions formed between
phenyl rings and Asp 215, Glu 411, Tyr 72, and Tyr 158, respectively. Two phenyl moieties
attached to the pyridazine ring also interacted with Arg 315 via π-alkyl interactions.
Furthermore, the formation of π-sulfur interaction between the sulfur atom and the phenyl ring
of Phe 303 and some different van der Waals interactions fitted this ligand tightly into the
active site of the enzyme.
The interaction of compound 8a with the active site of enzyme is depicted in figure
4. This compound adopted different binding modes with little resemblance as compared to the
compound 7a (figure 5). In this ligand, formations of hydrogen bonds with Glu 277 and Glu
411 are observed. The phenyl ring adjacent to the amide group and the pyridazine ring formed
π-π stacking with Phe 303 and Tyr 158, respectively. Additionally, this compound is involved
in hydrophobic interactions with several residues such as Asp 352, Arg 442, Gln 279, and Asp
307.
Generally, compound 7a formed an additional hydrogen bond and more favorable
interactions with the enzyme with higher affinity in comparison to compound 8a. These
interactions along with the optimum binding energy (-9.5 kcal/mol for compound 7a vs -6.2
kcal/mol for compound 8a) improved the stability of the ligand-enzyme complex which can be
the reason for better inhibitory activity of compound 7a toward α-glucosidase in biological
results.

Figure 3. 3D binding conformation and 2D binding conformation of compound 7a.

Figure 4. 3D binding conformation and 2D binding conformation of compound 8a.

Figure 5. Molecular docking comparison of compound 7a in blue color with compound 8a in red color.
4. Conclusion
In summary, we described the activity and structural characterization data for two series
of pyridizine based α-glucosidase inhibitors. We performed in-vitro evaluation, cytotoxic,
kinetic, and molecular docking studies. The in-vitro evaluation revealed that compound 7a is
10-times more active than positive control with no cytotoxicity against the normal cell line.
The docking studies rationalized the improved activity of compound 7a compared with 8a. The
results of this study introduced pyridazine as the attractive core to discover potent inhibitors of
α-glucosidase enzyme.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

Design and synthesis of novel pyridazine N-aryl acetamides: In-vitro evaluation of α-
glucosidase inhibition, docking, and kinetic studies
a
b
c
a
Setareh Moghimi , Mahsa Toolabi , Somayeh Salarinejad , Loghman Firoozpour , Seyed
Esmaeil Sadat Ebrahimi c, Fatemeh Safari d, Mohammad Ali Faramarzi e, Alireza Foroumadi
a, c, **
a Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS),
Tehran University of Medical Sciences, Tehran, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran
c
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
d Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran
e Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
Ph
Ph
N
X
N
H
N
S
R
O
X = COOEt, COOH
R = H, Me, i-Pr, ethynyl, OMe, F, Cl, 3,5-diCl, CN

Highlights
A new series of pyridazine-base compounds were synthesized as glucosidase inhibitor.
The most active compound was 10 times more active than acarbose.
Kinetic and molecular docking studies were performed to determine the mode of inhibition.