Synthesis, molecular docking, and biological activities of new cyanopyridine derivatives containing phenylurea

Article abstract of DOI:10.1002/ardp.202000334

A new class of cyanopyridine derivatives (10a–e and 11a–e) containing the phenylurea unit was synthesized and tested against some metabolic enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glycosidase (α-Gly). The new cya

Full text of DOI:10.1002/ardp.202000334

Received: 4 September 2020
DOI: 10.1002/ardp.202000334
Revised: 7 November 2020
Accepted: 13 November 2020
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F U L L P A P E R
Synthesis, molecular docking, and biological activities of new
cyanopyridine derivatives containing phenylurea
Hayreddin Gezegen1
Ümit M. Koçyiğit4
Mustafa Ceylan2
| Meliha B. Gürdere2 | Ayşegül Dinçer2 | Oğuz Özbek3
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| Parham Taslimi5 | Burak Tüzün6 | Yakup Budak2
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1Department of Nutrition and Dietetics,
Faculty of Health Sciences, Cumhuriyet
University, Sivas, Turkey
Abstract
A new class of cyanopyridine derivatives (10a–e and 11a–e) containing the
phenylurea unit was synthesized and tested against some metabolic enzymes
including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and
α‐glycosidase (α‐Gly). The new cyanopyridine derivatives showed Ki values in
the range of 40.73 6.54 to 87.05 16.98 µM against AChE, 29.17 4.88 to
124.03 22.43 µM against BChE, and 3.66 0.93 to 26.33 5.05 µM against
α‐Gly. These inhibition effects were compared with standard enzyme inhibitors
like tacrine (for AChE and BChE) and acarbose (for α‐Gly). Also, these
cyanopyridine derivatives with the best inhibition score were docked into the
active site of the indicated metabolic enzymes. Finally, molecular docking
calculations were made to compare the biological activities of the compounds
against AChE (−8.81 kcal/mol for molecule 11d), BChE (−3.52 kcal/mol for
molecule 11d), and α‐Gly (−2.98 kcal/mol for molecule 11a). After molecular
docking calculations, the ADME/T analysis was performed to examine the
future drug use properties of the new cyanopyridine derivatives containing
phenylurea.
2Department of Chemistry, Faculty of Science
and Arts, Tokat Gaziosmanpaşa University,
Tokat, Turkey
3Science and Technology, Application and
Research Center, Zonguldak Bülent Ecevit
University, Zonguldak, Turkey
4Department of Basic Pharmaceutical
Sciences, Division of Biochemistry, Faculty of
Pharmacy, Sivas Cumhuriyet University,
Sivas, Turkey
5Department of Biotechnology, Faculty of
Science, Bartin University, Bartin, Turkey
6Department of Chemistry, Faculty of Science,
Sivas Cumhuriyet University, Sivas, Turkey
Correspondence
Hayreddin Gezegen, Department of Nutrition
and Dietetics, Faculty of Health Sciences,
Sivas Cumhuriyet University,
58140 Sivas, Turkey.
Email: gezegenh@cumhuriyet.edu.tr
Ümit M. Koçyiğit, Department of Basic
Pharmaceutical Sciences, Faculty of
Pharmacy, Sivas Cumhuriyet University,
58140 Sivas, Turkey.
K E Y W O R D S
acetylcholinesterase, carbonic anhydrase, chalcone, cyanopyridine, molecular docking
Email: ukocyigit@cumhuriyet.edu.tr
Funding information
Sivas Cumhuriyet University,
Grant/Award Number: RGD‐020
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1
INTRODUCTION
activities.[9] In addition, some substituted pyridine derivatives are used
as organic light‐emitting devices,[10] metal ion sensors,[11] liquid crys-
Synthesis of heterocyclic compounds is of importance in organic and
medicinal chemistry.[1] Pyridines and their analogs are a very important
class of heterocyclic compounds.[2] They are known to exhibit various
biological activities such as antimicrobial,[3,4] antiparkinsonian,[5]
anti‐inflammatory,[6] antihypertensive,[7] antiviral,[8] and anticancer
talline polymers,[12] nonlinear optical materials,[13] and chelating
agents.[14] Therefore, the synthesis of pyridines and their analogs has
attracted much attention in organic chemistry.
In addition, diaryl urea derivatives are increasingly used in
medicinal chemistry and drug design. The diaryl urea derivatives
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Arch Pharm. 2020;e2000334.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000334

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GEZEGEN ET AL.
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have diversified biological activities including antimicrobial,[15]
anticonvulsant,[16] antibacterial,[17] antimalarial,[18] antiprolifera-
tive,[19] and anticancer[20] activities.
In light of this information, in this study, we aimed to synthe-
size new compounds containing diaryl urea and cyanopyridine units,
whose pharmacophore properties have been reported in the litera-
ture, and to examine their inhibition activities against some meta-
bolic enzymes. Thus, the synthesis of cyanopyridine derivatives
containing phenylurea (10a–e and 11a–e) was carried out from the
reaction of phenylurea‐substituted chalcone derivatives (7a–e and
8a–e) with malononitrile (9). The structures of all synthesized com-
pounds were elucidated by spectroscopic methods (nuclear magnetic
resonance [NMR], infrared [IR], and elemental analysis).
Alzheimer's disease (AD), the most common cause of
dementia, is characterized by presynaptic cholinergic defi-
ciencies and also decreased levels and function of acetylcholine
(ACh) in the body.[21,22] AD patients have elevated rates of
fracture associated with vitamin D shortage, elevated falls, and
lower average compression of the bone. Acetylcholinesterase
(AChE), expressed in brain tissues and acting in a controlled
manner to characterize ACh functioning in the central neural
system, is a very powerful enzyme for hydrolyzing ACh.[23] α‐
Glycosidase (α‐Gly) as a type of glycoside hydrolase hydrolyzing
the α‐glycosidic bonds in carbohydrate molecules[24] breaks the
α‐1,4‐linked sugar molecular terminals. It also causes carbohy-
drate molecules' breakdown. The α‐Gly inhibition helps to re-
lease the monosaccharide molecules into the bloodstream after a
meal. Diabetes mellitus (DM) condition occurs after the meal due
to the action of insulin resistance. Indeed, to regulate the hy-
perglycemic situation after the meal, α‐Gly inhibitor compounds
are utilized as therapeutic factors.[23]
Then, six of the synthesized compounds (10a, 10c, and 11a–d)
were tested to determine their inhibition properties against some
metabolic enzymes including AChE, butyrylcholinesterase (BChE),
and α‐Gly. In the last stage, for comparison of the obtained activity
results, molecular docking calculations of the new compounds were
performed against the proteins of enzymes, which are AChE (PDB
ID: 4M0E),[29] BChE (PDB ID: 5NN0),[30] and α‐Gly (PDB ID:
1R47).[31] After these calculations, the ADME/T (absorption, dis-
tribution, metabolism, excretion, and toxicity) analysis was per-
formed for these compounds. With this analysis, the potential of
these molecules to be used as a drug was investigated.[32]
Recent studies have shown that there exist theoretical stu-
dies besides many experimental studies. The resulting theoretical
data have enabled more effective and more active molecules to
be synthesized.[25,26] At present, there are many methods and
programs to compare the biological activities of molecules,
whether theoretical or experimental.[27,28] The most widely used
among these is molecular docking. This method gives important
information about the biological activities of the molecules be-
fore the experimental processes. For molecular docking calcula-
tions, numerical values of biological activities against many
enzymes were calculated.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Synthetic routes for the cyanopyridine derivatives (10a–e and
11a–e) are outlined in Scheme 1. First, the starting compounds
phenylurea‐substituted acetophenone derivatives 4 and 5 were
synthesized by refluxing 4‐aminoacetophenone (1) with isocyanate
(2) and 1‐chloro‐4‐isocyanatobenzene (3) according to the published
SCHEME 1 Synthetic routes for the cyanopyridine derivatives (10a–e and 11a–e)

GEZEGEN ET AL.
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procedure.[33] Compounds 4 and 5 were then reacted with benzal-
ii. Finally, for the α‐Gly inhibition, novel cyanopyridine derivatives 10b,
10c, and 11a–d showed Ki values between 3.66 0.93 and
26.33 5.05 µM (Table 2). The results demonstrated that all novel
cyanopyridine derivatives, 10b, 10c, and 11a–d, exhibited effective
α‐Gly inhibition effects as compared with acarbose (Ki:
20.33 4.31 µM) as a standard α‐Gly inhibitor. Also, highly effective
Ki values were calculated for compound 11a (Ki: 3.66 0.93 µM).
dehyde derivatives (6a–e) in
a basic medium to synthesize
phenylurea‐substituted chalcones derivatives (7a–e and 8a–e).[33–36]
At the last stage of the study, the synthesis of cyanopyridine deri-
vatives (10a–e and 11a–e) containing the phenylurea unit, which are
the resultant target compounds, by the reaction of the phenylurea‐
substituted chalcone derivatives (7a–e and 8a–e) with malononitrile
was carried out (Scheme 1 and Table 1).
The 1H NMR spectra of the new cyanopyridine derivatives
(10a–e and 11a–e) were acquired in dimethyl sulfoxide (DMSO)‐d6
solution. The amide H‐atoms of the urea unit, –NHa and –NHb, gave a
singlet at δ 8.62–9.05 and 8.57–8.93 ppm, respectively,[37] whereas
the H–C2 proton on the cyanopyridine ring gave a singlet in the
range of δ 7.47–7.78 ppm (Figure 1). The characteristic doublet of
doublets of the H–C7 protons and the triplet of the H–C8 protons in
the ethyl group were observed. The H–C7 protons resonated in the
range of δ 4.47–4.65 ppm with the coupling constant J = 14.0,
6.8–7.2 Hz, whereas the H–C8 protons were manifested in the range
of δ 1.40–1.46 ppm with the coupling constant J = 6.8–7.6 Hz. In the
13C NMR spectra of 10a–e and 11a–e, C5 of the carbon atoms on
the pyridine ring resonated in the range of δ 164.1–164.6 ppm,
whereas C2 resonated between 118.4 and 110.6 ppm and C4 be-
tween 91.2 and 106.0 ppm. In addition, nitrile carbon C6 and amide
carbonyl carbon C9 in the structure resonated between δ 106.1 and
114.6 ppm and 151.8 and 152.9 ppm, respectively. Other signals
were in harmony with the structure.
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2.3
Molecular docking results
In molecular docking calculations, calculations were performed using the
Maestro Molecular modeling platform (version 12.2) by Schrödinger
program to examine the interactions of ligands and proteins. For these
calculations, proteins were prepared with the protein preparation
module and the molecules were prepared with the LigPrep module.
Finally, with the docking module, molecules and enzymes were made to
interact with each other. Many parameters were calculated from this
interaction. These parameters given in Table 3 are obtained from mo-
lecular docking calculations. It should be noted that the most important
factor affecting the biological activities of molecules is the interactions
between molecules and proteins in enzymes.[41,42] As these interactions
increase, the biological activity of the molecule increases. These inter-
actions include hydrogen bonds, polar and hydrophobic interactions, π–π
interactions, and halogen interactions,[43–49] and they are presented in
Figures 2–4.
The two most important parameters obtained from the mole-
cular docking calculations of the new cyanopyridine derivatives
containing phenylurea are docking score and Glide Emodel para-
meters. The numerical values of these parameters provide important
information about the biological activities of the molecules. The
molecule with a small numerical value of docking score and Glide
Emodel parameters has the highest biological activity against that
enzyme. The high biological activity of molecules against enzymes
shows that the interaction between the molecule and the proteins in
enzymes is very high. It should be noted that as the interactions
between molecules and proteins in enzymes increase, the molecule
attaches more to the enzyme, which increases the biological activity
of the molecule.[32,41,42,50] Another parameter obtained from
molecular docking calculations is Glide ligand efficiency, which is the
numerical value of the effectiveness of new cyanopyridine deriva-
tives containing phenylurea against enzymes. Another parameter is
Glide H‐bond, which is the numerical value of the number of
hydrogen bonds that occur in interactions between molecules and
proteins in enzymes. Another parameter is Glide Evdw, which is the
numerical value of van der Waals interactions between molecules
and proteins in enzymes. Another parameter is Glide Ecoul, which is
the numerical value of Coulomb interactions between molecules and
proteins in enzymes. The last one of the parameters obtained as a
result of these calculations is Glide Einternal, which is the numerical
value of the combination of many parameters obtained.[32,51,52]
The docking results show that the docking score parameter of
the molecules and the experimental results are in great agreement,
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2.2
Enzyme inhibition results
The inhibition of metabolic enzymes was investigated, and their
results are reported as follows:
i. The cholinesterase inhibition effects of novel cyanopyridine deri-
vatives 10b, 10c, and 11a–d were evaluated according to the
spectrophotometric Ellman's method using tacrine (TAC) as a re-
ference compound,[38] as described previously.[39,40] AChE was
obtained from the electric eel (E.C. 3.1.1.7) and BChE from equine
serum (E.C. 3.1.1.8). Both cholinergic enzymes' inhibition results
are reported in Table 2. Novel cyanopyridine derivatives 10b, 10c,
and 11a–d had Ki values ranging from 40.73 6.54 to
87.05 16.98 µM for AChE (Table 2). Also, novel cyanopyridine
derivatives 10b, 10c, and 11a–d had Ki values ranging from
29.17 4.88 to 124.03 22.43 µM for BChE. However, TAC had Ki
values of 116.33 17.45 and 157.13 23.55 µM against indicated
AChE and BChE, respectively. It could be seen from the table that
all novel targets, 10b, 10c, and 11a–d, demonstrated marked in-
hibitory effects against both cholinesterase with Ki values in the
nanomolar range; however, compound 11d showed a perfect in-
hibition effect against AChE and BChE (Ki: 40.73 6.54 µM; Ki TAC
/
K
i 11d: 2.86; Ki: 29.17 4.88 µM; Ki TAC/Ki 11d: 5.38, respectively).
Our results have shown potent results, as represented in the
present study.

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GEZEGEN ET AL.
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TABLE 1 The synthesized compounds 10a–e and 11a–d
Entry
Synthesized compounds
M. p. (°C)
Yield (%)
1
10a
10b
10c
10d
225–228
86
2
3
4
245–248
194–197
215–218
93
91
89
5
6
10e
11a
217–220
268–271
93
89
7
11b
11c
11d
11e
244–247
265–268
263–266
272–274
94
90
86
81
8
9
10

GEZEGEN ET AL.
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interaction between the benzene ring at the center of molecule 11d
and the amino acid TYR124. The 4‐chlorophenyl end of molecule 11d
forms polar interactions with TYR119, GLY120, and GLY121
(Figure 2). Besides, the molecule with the highest biological activity
value against the BChE enzyme is 11d. The polar interaction with
THR300, ASP301, and MET302 occurs with the ethoxy chain in
molecule 11d (Figure 3). Finally, the molecule with the highest
biological activity against the α‐Gly enzyme is 11a. There appears to
be a π–π stacking interaction between the 4‐methylphenyl ring in
molecule 11a and the amino acid TRP19 (Figure 4).
FIGURE 1 The carbon atom numbering of 10a
such that the molecule with the most negative docking score para-
meter against the AChE enzyme is molecule 11d with −8.81 kcal/mol.
Also, the molecule with the most negative docking score para-
meter against the BChE enzyme is molecule 11d with −3.52 kcal/mol.
Finally, the molecule with the most negative docking score para-
meter against the α‐Gly enzyme is molecule 11a with −2.98 kcal/mol.
These results are consistent with experimental results.
After comparing the biological activity of new cyanopyridine
derivatives containing phenylurea against enzymes, the ADME/T
analysis was conducted to theoretically predict the effects and re-
sponses of new cyanopyridine derivatives containing phenylurea in
human metabolism. As a result of this theoretical analysis, many
parameters were obtained, which are presented in Table 4. The first
parameter is the solute molecular weight, which requires the mole-
cule to have a certain molecular weight. Another parameter is PISA,
which is also called solute total solvent‐accessible surface area
(SASA). This parameter is the carbon (carbon and attached hydrogen)
component of the SASA. Another parameter is QP polarizability,
which is the predicted polarizability in cubic angstroms. Another
important parameter is QPlog HERG, which is the numerical value of
the estimated IC50 value when the HERG K channels are blocked.
The next parameter is QPPCaco, which is Caco‐2 cell permeability in
the gut–blood barrier for inactive transport. Another parameter is
QPlog BB, which is the coefficient of the brain–blood barrier of an
orally taken drug. The next parameter is human oral absorption,
which is the predicted qualitative human oral absorption: 1, 2, or 3
for low, medium, or high, respectively.[32,51,52]
Comparing these results with control compounds, it is observed
that although TAC molecule has a numerical value of −7.53 kcal/mol
of docking score parameter against AChE enzyme, the numerical
value of the docking score parameter of molecule 11d is better than
all of them. However, although the docking score parameter of TAC
molecule versus the BChE enzyme has
a numerical value of
−3.95 kcal/mol, the numerical value of the docking score parameter
of molecule 11d is not better than TAC. Finally, although the docking
score parameter of acarbose molecule versus α‐Gly enzyme is
−7.07 kcal/mol, the numerical value of the docking score parameter
of molecule 11a is not better than the acarbose molecule.
If the interactions of molecules with enzymes are examined in
detail, it is observed that there are many interactions between mo-
lecules and enzymes. First, the molecule with the highest biological
activity value against AChE enzyme is 11d. The oxygen atom bound
to the carbonyl carbon in molecule 11d forms a hydrogen bond with
the amino acid TYR337. In addition, there appears to be a π–π
stacking interaction between the N‐(4‐chlorophenyl) group in mole-
cule 11d and TYR337. In addition, there appears to be a π–π stacking
The two most important parameters among all ADME/T para-
meters are the rule of five and the rule of three. The rule of five[53,54]
and rule of three[55] parameters are more important than any other
parameter, and the numerical value of these two parameters is ex-
pected to be zero. The rule of five parameter is also the Lipinski's fifth
TABLE 2 Enzyme inhibition results of the novel compounds against butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and
α‐glycosidase (α‐Gly) enzymes
IC50 (µM)
AChE
Ki (µM)
r2
BChE
88.35
154.30
111.84
89.08
90.43
32.05
178.03
–
r2
α‐Gly
9.03
14.88
2.01
5.83
11.21
21.24
–
r2
AChE
BChE
α‐Gly
Compounds
10b
83.03
102.36
88.20
100.01
74.31
48.04
141.34
–
.9830
.9924
.9411
.9889
.9547
.9483
.9736
–
.9432
.9035
.9880
.9118
.9553
.9523
.9894
–
.9821
.9903
.9942
.9424
.9919
.9430
–
68.04 14.30
83.28 10.44
59.03 11.55
87.05 16.98
66.41 18.32
40.73 6.54
116.33 17.45
–
74.87 17.23
124.03 22.43
97.23 19.02
80.32 13.05
68.45 9.13
29.17 4.88
157.13 23.55
–
13.25 3.51
19.05 3.88
3.66 0.93
7.91 1.05
15.47 3.40
26.33 5.05
–
10c
11a
11b
11c
11d
Tacrinea
Acarboseb
15.32
.9243
20.33 4.31
aUsed as a control for AChE and BChE enzymes.
bUsed as a control for α‐glycosidase enzyme.

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GEZEGEN ET AL.
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TABLE 3 Numerical values of the parameters obtained from the interactions of the studied molecules with some metabolic enzymes
Docking
Molecule score
Glide ligand
efficiency
Glide
Glide
Evdw
Glide
Ecoul
Glide
Glide
Glide
Glide
H‐bond
Emodel
energy
Einternal
posenum
AChE 10a
10b
−7.64
−8.18
−8.18
−7.99
−7.72
−6.91
−8.19
−6.81
−8.81
−7.03
−7.53
−0.23
−0.24
−0.23
−0.24
−0.23
−0.20
−0.23
−0.19
−0.25
−0.20
−0.50
−0.05
−0.45
−0.45
−0.44
−0.25
−0.27
−0.40
−0.45
−0.45
−0.28
−0.16
−35.71
−49.63
−51.13
−49.52
−50.28
−48.48
−54.90
−47.35
−47.35
−48.07
−22.45
−6.67
−3.10
−2.49
−2.35
−2.45
−1.51
−1.51
−1.58
−1.58
−0.88
−10.78
−56.80
−71.59
−70.78
−69.97
−68.79
−62.07
−76.43
−61.29
−73.29
−60.99
−53.38
−42.38
−52.73
−53.62
−51.88
−52.72
−49.99
−56.40
−48.94
−58.94
−48.96
−33.23
9.25
365
238
234
2
10.17
14.08
9.76
10c
10d
10e
13.23
14.09
8.74
2
11a
379
211
328
328
363
358
11b
11c
13.60
13.60
14.92
0.00
11d
11e
Tacrine
BChE 10a
10b
−2.62
−2.74
−3.11
−2.73
−2.20
−2.60
−2.52
−2.52
−3.52
−2.36
−3.95
−0.08
−0.08
−0.09
−0.08
−0.06
−0.07
−0.07
−0.07
−0.07
−0.07
−0.26
0.00
−25.75
−25.77
−27.45
−25.66
−27.69
−27.92
−28.14
−30.36
−25.36
−27.94
−13.58
−2.47
−1.61
−3.79
−3.23
−0.26
−1.80
−1.74
−0.94
−0.94
−0.48
−4.67
−31.19
−30.86
−35.92
−32.46
−30.65
−29.07
−28.59
−34.24
−36.24
−31.60
−23.94
−28.22
−27.38
−31.23
−28.90
−27.96
−29.71
−29.88
−31.30
−32.30
−28.42
−18.25
3.06
2.06
2.31
2.75
2.20
9.11
8.67
3.29
2.29
2.12
0.00
138
63
−0.22
−0.32
−0.53
−0.03
−0.16
−0.14
0.00
10c
28
10d
345
155
56
10e
11a
11b
149
281
281
262
47
11c
11d
0.00
11e
−0.06
0.00
Tacrine
α‐Gly 10a
10b
−2.51
−2.51
−2.73
−2.13
−2.72
−2.98
−2.63
−2.18
−2.18
−2.38
−0.08
−0.07
−0.08
−0.06
−0.08
−0.06
−0.07
−0.06
−0.06
−0.07
−0.16
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
−0.03
−0.47
−29.87
−30.49
−31.28
−27.67
−26.47
−31.78
−31.87
−29.51
−29.51
−29.23
−26.85
−1.69
−1.69
−3.48
−2.96
−3.13
−0.01
−3.60
−2.98
−2.98
−2.77
−25.83
−34.99
−35.43
−39.66
−33.78
−30.75
−41.90
−35.31
−32.01
−32.01
−35.21
−85.08
3.11
3.17
2.25
2.35
8.59
9.16
12.27
9.82
9.82
3.75
−52.68
−34.77
−29.60
−35.47
−32.18
−31.56
−36.00
−32.49
−32.49
−30.62
−29.78
22.11
86
242
29
10c
10d
183
205
355
52
10e
11a
11b
11c
352
352
252
118
11d
11e
Acarbose −7.07
Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; α‐Gly, α‐glycosidase.
rule of Pfizer. The rules are as follows: mol MW < 500, QPlog P
o/w < 5, donorHB ≤ 5, accptHB ≤ 10 (the “five” refers to the limits,
which are multiples of 5). However, the rule of three parameter is
known as the three rules of Jorgensen. The three rules are as follows:
QPlog S > −5.7, QPPCaco > 22 nm/s, and #Primary Metabolites < 7. If
the numerical value of the rule of three parameter is zero, this
molecule can be used orally as a medicine. These two parameters are
among the parameters that must be examined for the molecules to be
theoretical drugs.
823. One of the most important parameters in ADME/T calculations is
the QPPCaco parameter, the numerical value of this parameter is 223
for both. There are many other parameters. The numerical values of
these calculated parameters are within the normal range.
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3
CONCLUSIONS
In conclusion, a series of novel cyanopyridine derivatives (10a–e and
11a–e) containing the phenylurea unit was synthesized in high yields
(81%–94%) and investigated for their inhibition properties against
AChE, BChE, and α‐Gly. Among the compounds whose inhibitory ac-
tivity was examined, compound 11d showed the best activity against
AChE and BChE enzymes, whereas compound 11a showed the best
Experimentally and theoretically, the two molecules with the
highest biological activity are 11a and 11d. When the ADME/T results
of these two molecules were examined in detail, the molecular masses
of the molecules were calculated as 483 and 503, respectively.
However, the numerical value of the total SASA parameter is 837 and

GEZEGEN ET AL.
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|
FIGURE 2 Interaction of molecule 11d with the acetylcholinesterase enzyme
activity against the α‐Gly enzyme. It was observed that the data ob-
tained from molecular docking calculations were in harmony with the
experimental study. The biological activities of molecules against en-
zymes were determined by molecular docking calculations. A com-
parison is made with the numerical values obtained as a result of these
calculations. With the data obtained as a result of these calculations, it
is possible to synthesize more effective and more active molecules.
Afterward, the ADME/T analysis of these molecules was performed
theoretically. As a result of this analysis, these molecules were ex-
amined with many parameters. The analysis of the numerical values of
the parameters obtained may lead to the discovery of new drug
candidates using in vivo and in vitro studies in future.
FIGURE 3 Interaction of molecule 11d with the butyrylcholinesterase enzyme

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GEZEGEN ET AL.
|
FIGURE 4 Interaction of molecule 11a with the α‐glycosidase enzyme
|
|
General method for the synthesis of
cyanopyridine derivatives containing the phenylurea
unit (10a–e and 11a–d)
4
EXPERIMENTAL
Chemistry
4.1.4
|
4.1
|
4.1.1
General
To a solution of chalcones (7a–e or 8a–e; 1 mmol) in 20 ml of ethanol,
malononitrile (9; 4 mmol) and K2CO3 (8 mmol) were added and refluxed
for 4 h. The mixture was neutralized by adding HCl. Then, it was diluted
with CHCl3 (50 ml) and dried over Na2SO4. The solvent was evaporated,
and the crude product was crystallized in CHCl3/ethanol (3:7) mixture.
Their structures were confirmed using IR, 1H NMR, and 13C NMR.
All chemicals and solvents were obtained from Merck (Germany) and
Fluka (Germany). Melting points were measured using an Electro-
thermal 9100 apparatus. The 1H and 13C NMR spectra (see
Supporting Information) were recorded with a Bruker Avance DPX‐
400 instrument. Elemental analyses were obtained from a LECO
CHNS 932 Elemental Analyzer.
1‐[4‐(5‐Cyano‐6‐ethoxy‐4‐phenylpyridin‐2‐yl)phenyl]‐3‐
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
phenylurea (10a)
Yield: 86%, Mp: 225–228°C. IR (KBr, cm−1): 3385, 2966, 2221, 1708,
1634, 1589, 1539, 1493, 1450, 1424, 1377, 1332, 1319, 1233, 1172,
1148, 1035, 827, 752, 698, 642, 511. 1H NMR (400 MHz, DMSO‐d6):
δ = 9.02 (s, 1H), 8.78 (s, 1H), 8.21 (d, J = 8.8 Hz, 2H), 7.73 (s, 1H),
7.64–7.58 (m, 6H), 7.50–7.47 (m, 2H), 7.33–7.29 (m, 3H), 7,02–6.98
(m, 1H), 4.62 (dd, J = 14.0, 7.2 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, DMSO‐d6): δ = 164.43, 157.58, 156.62, 152.77,
142.79, 139.90, 136.57, 132.34, 129.50 (2C), 129.29 (2C), 129.05
(2C), 128.84 (2C), 127.51, 122.58, 118.85 (2C), 118.77 (2C), 116.07,
113.07, 91.64, 63.45, 14.87. Anal. calc. for C27H22N4O2: C, 74.64; H,
5.10; N, 12.89. Found: C, 74.56; H, 5.07; N, 12.80.
|
4.1.2
Synthesis of the phenylurea‐substitued
acetophenone derivatives 4 and 5
The phenylurea‐substituted acetophenone derivatives 4 and 5 were
synthesized in accordance with the published procedures.[33]
|
4.1.3
General method for the synthesis of
phenylurea‐substituted chalcone derivatives 7a–e
and 8a–e
1‐{4‐[5‐Cyano‐6‐ethoxy‐4‐(p‐tolyl)pyridin‐2‐yl]phenyl}‐3‐
phenylurea (10b)
The phenylurea‐substituted chalcone derivatives (7a–e and 8a–e)
were synthesized in accordance with the published procedures.[33,36]
Yield: 93%, Mp: 245–248°C. IR (KBr, cm−1): 3380, 2976, 2220, 1703,
1631, 1582, 1539, 1499, 1444, 1419, 1371, 1339, 1315, 1237, 1176,

GEZEGEN ET AL.
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TABLE 4 ADME properties of the molecules
10a
434
5.9
10b
10c
465
5.3
10d
469
6.1
10e
513
6.9
11a
483
6.2
11b
499
4.8
11c
503
6.2
11d
503
6.2
11e
548
6.6
Reference range
130 to 725
1.0 to 12.5
300 to 1000
0 to 750
Solute molecular weight
Solute dipole moment (D)
SASA
449
5.8
812
194
140
478
0
781
107
140
534
0
809
195
139
475
0
799
107
140
486
66
810
107
140
485
77
837
194
140
430
72
833
195
139
427
72
823
107
140
438
138
1469
2
823
107
140
438
138
1469
2
834
107
140
437
149
1488
2
FOSA
FISA
7 to 330
PISA
0 to 450
WPSA
0 to 175
Solute molecular volume (Å3)
Solute as hydrogen bond donor
Solute as hydrogen bond acceptor
Solute globularity (sphere = 1)
QP polarizability (Å3)
QPlog p for hexadecane/gas
QPlog p for octanol/gas
QPlog p for water/gas
QPlog p for octanol/water
QPlog S aqueous solubility
QPlog S conformation‐independent
QPlog HERG
1391
2
1450
2
1461
2
1425
2
1444
2
1494
2
1506
2
500 to 2000
0 to 6
4.5
4.5
0.8
51.9
16.7
23.2
14.6
5.3
−7.8
−8.2
−6.3
223
−1.5
214
−1.8
9.3
0.8
3
5.25
0.8
4.5
4.5
4.5
5.25
0.8
4.5
4.5
4.5
2.0 to 20.0
0.75 to 0.95
13.0 to 70.0
4.0 to 18.0
8.0 to 35.0
4.0 to 45.0
−2.0 to 6.5
−6.5 to 0.5
0.8
0.8
0.8
0.8
0.8
0.8
0.8
50.1
16.5
22.7
14.9
5.0
51.7
16.8
23.4
15.1
5.0
51.0
17.0
23.2
14.7
5.4
51.8
17.3
23.6
14.7
5.5
53.3
17.4
23.9
14.4
5.8
53.0
17.4
24.1
14.9
5.5
52.3
17.6
23.9
14.4
5.9
52.3
17.6
23.9
14.4
5.9
53.1
17.9
24.3
14.5
6.0
−7.2
−8.0
−6.4
223
−1.4
214
−1.6
9.3
−7.3
−8.3
−6.1
229
−1.5
220
−1.6
9.3
−7.8
−8.7
−6.3
223
−1.3
492
−1.7
9.0
−8.1
−9.6
−6.3
223
−1.3
568
−1.7
9.3
−8.5
−9.0
−6.2
223
−1.3
532
−1.9
9.3
−8.0
−9.0
−6.0
229
−1.3
546
−1.8
9.3
−8.6
−9.4
−6.1
223
−1.1
1221
−1.9
9.0
−8.6
−9.4
−6.1
223
−1.1
1221
−1.9
9.0
−8.8
−10.3
−6.2
223
−1.1
1411
−1.9
9.4
−6.5 to 0.5
a
b
QPPCaco (nm/s)
QPlog BB
−3.0 to 1.2
b
QPPMDCK (nm/s)
QPlog Kp
Kp in cm/h
7.9 to 10.5
−0.9 to 1.7
1 to 8
IP (eV)
EA (eV)
0.8
0.9
0.8
0.9
0.8
0.9
0.8
0.8
0.9
#metab
2
3
2
2
2
2
1
1
1
QPlog Khsa
0.8
1.0
1
0.8
0.9
1.0
1.1
0.9
1.0
1.0
1.1
−1.5 to 1.5
Human oral absorption
Percent human oral absorption
PSA
1
1
1
1
1
1
1
1
1
–
c
100
93
87
86
87
75
90
89
77
77
78
93
102
1
93
93
93
102
1
93
93
93
7 to 200
Rule of five
0
1
1
2
1
2
2
2
Maximum is 4
Rule of three
1
1
1
1
1
1
1
1
1
1
Maximum is 3
–
Jm
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
aCorcern below −5.
b<25 is poor and >500 is great.
c>80% is high.
1142, 1032, 821, 748, 691, 640, 509. 1H NMR (400 MHz, DMSO‐d6):
δ = 8.99 (s, 1H), 8.75 (s, 1H), 8.20 (d, J = 8.8 Hz, 2H), 7.69 (s, 1H), 7.63
(t, J = 8.0 Hz, 4H), 7.47 (d, J = 7.6 Hz, 2H), 7.40 (d, J = 7.6 Hz, 2H), 7.30
(t, J = 8.0 Hz, 2H), 6.99 (t, J = 7.6 Hz, 1H), 4.64 (dd, J = 14.0, 7.2 Hz,
2H), 2.42 (s, 3H), 1.45 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
DMSO‐d6): δ = 164.48, 157.43, 156.30, 152.67, 142.30, 139.92,
139.48, 133.67, 129.56, 128.86 (2C), 128.36 (2C), 128.18 (3C),
122.34 (2C), 118.66 (2C), 118.16 (2C), 115.87, 112.33, 91.61, 63.08,
21.36, 14.63. Anal. calc. for C28H24N4O2: C, 74.98; H, 5.39; N, 12.49.
Found: C, 74.86; H, 5.21; N, 12.37.

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|
1‐{4‐[5‐Cyano‐6‐ethoxy‐4‐(4‐methoxyphenyl)pyridin‐2‐yl]phenyl}‐3‐
phenylurea (10c)
138.93, 136.56, 130.51, 130.42, 129.29 (2C), 129.12 (2C), 129.03
(2C), 128.82 (2C), 126.38, 121.47 (2C), 118.52 (2C), 116.34, 114.65,
91.90, 67.04, 14.86. Anal. calc. for C27H21ClN4O2: C, 69.15; H, 4.51;
N, 11.95. Found: C, 69.03; H, 4.38; N, 11.82.
Yield: 91%, Mp: 194–197°C. IR (KBr, cm−1): 3400, 2218, 1725, 1584,
1538, 1442, 1375, 1339, 1314, 1233, 1174, 1024, 823, 748, 682,
634, 574. 1H NMR (400 MHz, DMSO‐d6): δ = 8.98 (s, 1H), 8.74 (s,
1H), 8.19 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.68 (s, 1H), 7.62
(d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.2 Hz, 2H), 7.30 (t, J = 8.0 Hz, 2H), 7.14
(d, J = 8.4 Hz, 2H), 7.00 (t, J = 7.2 Hz, 1H), 4.63 (dd, J = 14.0, 7.2 Hz,
2H), 3.86 (s, 3H), 1.45 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz,
DMSO‐d6): δ = 164.56, 161.21, 157.38, 156.19, 152.77, 142.69,
139.90, 130.60 (2C), 130.46, 129.26 (2C), 128.74 (2C), 128.66,
122.56, 118.87 (2C), 118.38 (2C), 116.30, 114.74 (2C), 112.79,
91.26, 63.33, 55.89, 14.87. Anal. calc. for C28H24N4O3: C, 72.40; H,
5.21; N, 12.06. Found: C, 72.37; H, 5.18; N, 12.01.
1‐(4‐Chlorophenyl)‐3‐{4‐[5‐cyano‐6‐ethoxy‐4‐(p‐tolyl)pyridin‐2‐
yl]phenyl}urea (11b)
Yield: 94%, Mp: 244–247°C. IR (KBr, cm−1): 3338, 2227, 1694, 1694,
1581, 1535, 1492, 1444, 1397, 1377, 1338, 1310, 1237, 1214, 1177,
1145, 1089, 1034, 1011, 819, 616, 508. 1H NMR (400 MHz,
DMSO‐d6): δ = 9.03 (s, 1H), 8.90 (s, 1H), 8.20 (d, J = 8.0 Hz, 2H), 7.70
(s, 1H), 7.65–7.61 (m, 4H), 7.51 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz,
2H), 7.34 (d, J = 8.4 Hz, 2H), 4.63 (dd, J = 14.0, 7.2 Hz, 2H), 2.48 (s,
3H), 1.44 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO‐d6):
δ = 164.50, 157.48, 156.62, 152.69, 142.55, 140.31, 138.93, 133.68,
130.58, 129.85 (2C), 129.13 (2C), 128.95 (2C), 128.79, 127.32,
126.12, 120.41 (2C), 118.53 (2C), 116.11, 112.97, 91.58, 63.40,
21.36, 14.86. Anal. calc. for C28H23ClN4O2: C, 69.63; H, 4.80; N,
11.60. Found: C, 69.59; H, 4.78; N, 11.53.
1‐{4‐[4‐(4‐Chlorophenyl)‐5‐cyano‐6‐ethoxypyridin‐2‐yl]phenyl}‐3‐
phenylurea (10d)
Yield: 89%, Mp: 215–218°C. IR (KBr, cm−1): 3316, 2979, 2357, 2222,
1649, 1597, 1544, 1495, 1443, 1424, 1379, 1342, 1314, 1234, 1206,
1093, 1035, 1013, 821, 745, 693, 647, 509, 484. 1H NMR (400 MHz,
DMSO‐d6): δ = 8.89 (s, 1H), 8.73 (s, 1H), 8.14 (d, J = 8.8 Hz, 2H), 7.93
(d, J = 8.4 Hz, 2H), 7.78 (s, 1H), 7.60–7.57 (m, 4H), 7.47 (d, J = 7.6 Hz,
2H), 7.30 (t, J = 8.4 Hz, 2H), 6.99 (t, J = 7.6 Hz, 1H), 4.49 (dd, J = 14.0,
7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO‐d6):
δ = 164.20, 154.89, 152.88, 150.39, 141.28, 140.04, 136.89, 134.51,
132.08, 130.98, 129.45 (2C), 129.37 (2C), 129.27 (2C), 127.79 (2C),
122.44, 118.78 (2C), 118.40 (2C), 110.52, 106.03, 61.69, 15.07. Anal.
calc. for C27H21ClN4O2: C, 69.15; H, 4.51; N, 11.95. Found: C, 69.10;
H, 4.48; N, 11.86.
1‐(4‐Chlorophenyl)‐3‐{4‐[5‐cyano‐6‐ethoxy‐4‐(4‐methoxyphenyl)-
pyridin‐2‐yl]phenyl}urea (11c)
Yield: 90%, Mp: 265–268°C. IR (KBr, cm−1): 3381, 1722, 1607, 1578,
1535, 1494, 1445, 1315, 1295, 1259, 1236, 1174, 1024, 870, 827,
637, 517, 501. 1H NMR (400 MHz, DMSO‐d6): δ = 8.77 (s, 1H), 8.64
(s, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.53 (d,
J = 8.4 Hz, 2H), 7.47 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.4 Hz,
2H), 7.02 (d, J = 8.4 Hz, 2H), 4.57 (dd, J = 14.0, 7.2 Hz, 2H), 3.81 (s,
3H), 1.43 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO‐d6):
δ = 164.51, 161.00, 157.26, 155.93, 152.52, 142.22, 138.59, 130.62,
130.14 (2C), 128.75 (2C), 128.65 (2C), 128.31, 126.35, 120.02 (2C),
118.28 (2C), 116.01, 114.42 (2C), 112.39, 91.36, 63.06, 55.55, 14.72.
Anal. calc. for C28H23ClN4O3: C, 67.40; H, 4.65; N, 11.23. Found: C,
67.34; H, 4.59; N, 11.18.
1‐{4‐[4‐(4‐Bromophenyl)‐5‐cyano‐6‐ethoxypyridin‐2‐yl]phenyl}‐3‐
phenylurea (10e)
Yield: 93%, Mp: 217–220°C. IR (KBr, cm−1): 3416, 2923, 1645, 1597,
1542, 1496, 1444, 1377, 1341, 1314, 1236, 1209, 820, 745, 693. 1H
NMR (400 MHz, DMSO‐d6): δ = 8.90 (s, 1H), 8.74 (s, 1H), 8.14 (d,
J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.77 (s, 1H), 7.71 (d, J = 8.4 Hz,
2H), 7.59 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 8.0 Hz,
2H), 6.97 (t, J = 6.8 Hz, 1H), 4.47 (dd, J = 14.0, 6.8 Hz, 2H), 1.40 (t,
J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO‐d6): δ = 164.22, 154.93,
152.89, 150.46, 141.35, 140.10, 136.99, 132.37 (2C), 132.24, 131.18,
129.64 (2C), 129.26 (2C), 127.78 (2C), 123.19, 122.43, 118.79 (2C),
118.42 (2C), 110.46, 105.98, 61.69, 15.07. Anal. calc. for C27H21BrN4O2:
C, 63.17; H, 4.12; N, 10.91. Found: C, 63.08; H, 4.09; N, 10.81.
1‐(4‐Chlorophenyl)‐3‐{4‐[4‐(4‐chlorophenyl)‐5‐cyano‐6‐
ethoxypyridin‐2‐yl]phenyl}urea (11d)
Yield: 86%, Mp: 266–263°C. IR (KBr, cm−1): 3304, 2975, 1644, 1595,
1546, 1491, 1427, 1398, 1379, 1344, 1303, 1236, 1207, 1090, 1012,
821, 654, 515, 482. 1H NMR (400 MHz, DMSO‐d6): δ = 8.62 (s, 1H),
8.57 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.50 (s,
1H), 7.49 (d, J = 8.4 Hz, 2H), 7.41 (m, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.02
(d, J = 8.4 Hz, 2H), 4.57 (dd, J = 14.0, 7.2 Hz, 2H), 1.43 (t, J = 7.2 Hz,
3H). 13C NMR (100 MHz, DMSO‐d6): δ = 164.08, 154.77, 152.65,
150.35, 140.77, 138.66, 137.07, 134.60, 132.44, 129.17 (2C), 128.71
(2C), 128.68 (2C), 127.42 (2C), 126.28, 119.92 (2C), 118.31 (2C),
110.25, 106.07, 92.44, 61.50, 14.89. Anal. calc. for C27H20Cl2N4O2:
C, 64.42; H, 4.00; N, 11.13. Found: C, 64.38; H, 3.89; N, 11.10.
1‐(4‐Chlorophenyl)‐3‐[4‐(5‐cyano‐6‐ethoxy‐4‐phenylpyridin‐2‐
yl)phenyl]urea (11a)
Yield: 89%, Mp: 268–271°C. IR (KBr, cm−1): 3410, 2222, 1585, 1540,
1491, 1445, 1399, 1373, 1339, 1309, 1234, 1175, 828, 762, 698,
607, 505. 1H NMR (400 MHz, DMSO‐d6): δ = 9.05 (s, 1H), 8.92 (s,
1H), 8.22 (d, J = 8.8 Hz, 2H), 7.75–7.73 (m, 3H), 7.62 (d, J = 8.8 Hz,
2H), 7.60–7.58 (m, 3H), 7.51 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz,
2H), 4.65 (dd, J = 14.0, 6.8 Hz, 2H), 1.46 (t, J = 6.8 Hz, 3H). 13C NMR
(100 MHz, DMSO‐d6): δ = 164.44, 157.57, 156.66, 152.69, 142.60,
1‐(4‐Chlorophenyl)‐3‐{4‐[4‐(4‐bromophenyl)‐5‐cyano‐6‐
ethoxypyridin‐2‐yl]phenyl}urea (11e)
Yield: 81%, Mp: 272–274°C. IR (KBr, cm−1): 3300, 2968, 1621, 1585,
1541, 1487, 1426, 1389, 1374, 1338, 1300, 1236, 1201, 1089, 1009,

GEZEGEN ET AL.
11 of 12
|
820, 659, 505, 474. 1H NMR (400 MHz, DMSO‐d6): δ = 8.98 (s, 1H),
8.93 (s, 1H), 8.14 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.78
(s, 1H), 7.60–7.57 (m, 4H), 7.51 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz,
2H), 4.51 (dd, J = 14.0, 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, DMSO‐d6): δ = 164.6, 155.7, 154.5, 151.8, 140.6, 138.66,
137.07, 134.60, 132.44, 129.17 (2C), 128.71 (2C), 128.68 (2C),
127.42 (2C), 126.28, 119.92 (2C), 118.31 (2C), 110.25, 106.07,
92.44, 61.50, 14.89. Anal. calc. for C27H20ClBrN4O2: C, 59.20; H,
3.68; N, 10.23. Found: C, 59.18; H, 3.61; N, 10.20.
Calculations were performed to find high‐energy isomers with
physiological pH values of new cyanopyridine derivatives containing
phenylurea 3D structures and the correct protonation conditions. In
the next step, the prepared protein and molecules were docked with
each other. The Glide ligand docking module[62] was used for this
step. In this module, the OPLS3e method was used in all calculations
for docking calculations of molecule and proteins. The numerical
value of many parameters obtained as a result of molecular docking
calculations using this module is used. After the docking calculations,
the ADME/T analysis was performed to examine the molecule's
ability to be used as a drug in the future. The Qik‐prop module[63] of
the Schrödinger software was used for the ADME/T analysis.
|
4.2
Enzymes studies
The inhibitory effect of novel cyanopyridine derivatives on AChE and
BChE activities was examined according to the spectrophotometric
method of Ellman,[38] as described previously.[39] The α‐Gly inhibition
effect of the novel cyanopyridine derivatives was evaluated ac-
cording to the method of Tao et al.[40] The absorbance of samples
was recorded at 405 nm.[32]
ACKNOWLEDGMENTS
This study is supported by the Scientific Research Project Fund of
Sivas Cumhuriyet University under project number RGD‐020. This
study was made possible by TUBITAK ULAKBIM, High Performance
and Grid Computing Center (TR‐Grid e‐Infrastructure).
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
|
4.3
Molecular docking method
ORCID
Theoretical calculations provide good assistance to experimental
studies. By theoretical calculations, biological activities of molecules
against enzymes are compared. There are many programs and
methods in theoretical calculations. The most commonly used among
these is molecular docking. The numerical values of many parameters
obtained from molecular docking calculations provide important
information about the biological activities of molecules. These nu-
merical values can be used for the discovery of new drugs.
Hayreddin Gezegen
https://orcid.org/0000-0003-3602-7400
https://orcid.org/0000-0001-8710-2912
Ümit M. Koçyiğit
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
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How to cite this article: Gezegen H, Gürdere MB, Dinçer A,
et al. Synthesis, molecular docking, and biological activities of
new cyanopyridine derivatives containing phenylurea.
Arch Pharm. 2020;e2000334.
https://doi.org/10.1002/ardp.202000334