Synthesis and molecular modeling of novel non-sulfonylureas as hypoglycemic agents and selective ALR2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.06.024

Novel non-sulfonylureas derivatives bearing an acetamide linker between a spirohydantoin scaffold and a phenyl ring were prepared and their hypoglycemic activity was estimated in vivo. Their abilities to discriminate in vitro between aldehyde reductase (A

Full text of DOI:10.1016/j.bmc.2019.06.024

Accepted Manuscript
Synthesis and molecular modeling of novel non-sulfonylureas as hypoglycemic
agents and selective ALR2 inhibitors
Manar G. Salem, Yasmine M. Abdel Aziz, Marwa Elewa, Hosam A. Elshihawy,
Mohamed M. Said
PII:
S0968-0896(19)30301-3
https://doi.org/10.1016/j.bmc.2019.06.024
BMC 14961
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 February 2019
11 June 2019
12 June 2019
Please cite this article as: Salem, M.G., Abdel Aziz, Y.M., Elewa, M., Elshihawy, H.A., Said, M.M., Synthesis and
molecular modeling of novel non-sulfonylureas as hypoglycemic agents and selective ALR2 inhibitors, Bioorganic
& Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.06.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and molecular modeling of novel non-
sulfonylureas as hypoglycemic agents and selective
ALR2 inhibitors
Manar G. Salem, Yasmine M. Abdel Aziz, Marwa Elewa, Hosam A. Elshihawy and Mohamed M.
Said
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Suez Canal University,
P.O. 41522, Ismailia, Egypt
Keywords
Non-sulfonylureas, Meglitinides, Aldose reductase, Hypoglycemic agents.
Corresponding Author
Prof. Dr. Mohamed Mokhtar Said, Telephone: 064-3230741, Fax: 002-064-3230741, Mobile:
002-011110012098, E-mail: msaid123eg@yahoo.com
1

Abstract
Novel non-sulfonylureas derivatives bearing an acetamide linker between a spirohydantoin
scaffold and a phenyl ring were prepared and their hypoglycemic activity was estimated in vivo.
Their abilities to discriminate in vitro between aldehyde reductase (ALR1) and aldose reductase
(ALR2) were determined. The molecular docking and the in silico prediction studies were
performed to rationalize the obtained biological results and to predict the physicochemical
properties and drug-likeness scores of the new compounds. N-(2,4-Dichlorophenyl)-2-(2',4'-
dioxospiro[fluorene-9,5'-imidazolidine]-3'-yl)acetamide (3e) displayed an 84% reduction in
blood glucose level superior to that of repaglinide 66% and showed an IC50 value of 0.37 μM
against ALR2 that is superior to that of sorbinil 3.14 µM. Compound (3e) was selective 96 fold
towards ALR2 which is closely related to serious diabetic complications. Based on the
identification of this hit candidate, a new generation of safe and effective antidiabetic agents
could be designed.
2

1. Introduction
Meglitinides are non-sulfonylureas antidiabetic agents that help the pancreatic beta cells to
release insulin properly.1,2 One major example of these agents is repaglinide, which is mainly
metabolized in the liver rather than the kidney. Therefore, repaglinide can be used safely in the
treatment of patients with renal insufficiency.1-3 Repaglinide is characterized by a rapid onset of
action, a short duration of action and a low risk of hypoglycemia.4,5 It binds to site B of
sulfonylurea receptor 1 (SUR1) in the pancreatic beta cells (Figure 1). On the other hand,
sulfonylureas, such as tolazamide, are known to bind to site A (Figure 1). They are actually
believed to develop hypoglycemia and to accelerate pancreatic beta cells exhaustion and
apoptosis.6 Thus, non-sulfonylureas have many advantages superior to sulfonylureas antidiabetic
agents. It is well known that the minimum pharmacophoric requirements for enhancing the
antidiabetic activity of non-sulfonylureas include the presence of an amide bridge in between
two lipophilic moieties (Figure 2).7
Unfortunately, despite of the availability of many sophisticated antidiabetic agents, the patients
still suffer from serious complications that are responsible for a high rate of mortality.8 These
complications are hypoglycemia, hyperglycemic crises, diabetes ketoacidosis (DKA) and
9
hyperglycemic hyperosmolar state (HHS). It is worth to mention that ALR2 is responsible for
these diabetic complications while ALR1 is responsible for an essential detoxification
mechanism in liver.10,11 Aldose reductase ALR2 inhibitors are known to overcome these
complications.12 Therefore, selective inhibition of ALR2 is essential. A large number of
structurally diverse compounds have been observed to inhibit ALR2, such as sorbinil (Figure 2)
9. All ALR2 inhibitors interact at a common site, known as ‘inhibitor site’.13 Most notably, the
hydantoin ring of sorbinil is responsible for essential hydrogen bonding interactions with the key
3

amino acids in ALR2 active site (Figure 2).14 Kador and coauthors proposed that the minimum
requirements for enhancing the selective inhibition of ALR2 include a primary lipophilic region
and a carbonyl group.14,15 In addition, the introduction of a secondary lipophilic moiety coplanar
with the primary lipophilic one enhances binding with ALR2 specificity pocket (Figure 2). We
previously published the synthesis, ALR2 inhibition as well as hypoglycemic activity of
sulfonylureas derivatives.16 In the present study, our tactic is to synthesize a new generation of
non-sulfonylureas analogues keeping their advantages as hypoglycemic agents and to modulate
their aldose reductase inhibitory activity to overcome the diabetic complications. The design of
our target molecules is based on the presence of an acetamide linker between a spirohydantoin
scaffold and a phenyl ring (Figure 2). These molecules are expected to fulfill all the minimum
requirements for enhancing both hypoglycemic activity and selective ALR2 inhibition according
to the mixed pharmacophore theory.15,17,18 In attempt to improve the physicochemical properties
of our target molecules, two diversity points were generated (Figure 2). The first point was the
incorporation of various substituents on the phenyl ring. The second point was based on
modulating the lipophilic ring attached to the spirohydantoin scaffold.
4

Figure 1. The structures of sulfonylurea and non-sulfonylurea derivatives.
Figure 2. The design of novel non-sulfonylurea derivatives as hypoglycemic agents and aldose
reductase inhibitors.
5

2. Results and discussion
2.1. Chemistry
The synthesis of different acetamide derivatives is shown in scheme 1. 2-Chloro-N-
phenylacetamide derivatives (2a-e) were prepared through reaction of the appropriate aniline
(4-chloro, 4-bromo, 4-methyl, 4-methoxy or 2,4-dichloroaniline) with chloroacetyl chloride (1)
in glacial acetic acid.19,20 Spiro-imidazolidinedione derivatives (spirohydantoin or
cyclohexanonyl moieties) were prepared as previously reported.15,21 Reaction of (2a-e) with
spiro-imidazolidinediones in the presence of sodium bicarbonate (NaHCO3) and sodium iodide
(NaI) resulted in the formation of the newly synthesized target compounds (3a-e) and (4a-e).22
The structures of the synthesized compounds (3a-e) and (4a-e) were approved with elemental
1
analyses and spectral data (IR, H-NMR,13C-NMR and EI-MS). IR spectra showed collectively
secondary amine signals at 3200-3400 cm-1 that indicated the coupling step. Besides, the
3
1
appearance of (C-H sp ) signals at around 2850-3000 cm-1. In the H NMR spectra of these
compounds, the appearance of one upfield NH signal in the range the range 5.70-6.00 ppm
confirmed the nucleophilic substitution reaction that occurred at the desheilded NH of
spiroimidazolidine-2,4-dione nucleus. Another singlet signal appeared downfield at 9.76-10.10
that referred to NH group of the secondary aromatic amine. Moreover, a characteristic singlet
peak for CH2 protons appeared at 3.82-4.25 ppm. The aromatic protons of the fluorenonyl ring
were displayed downfield as multiplet signals at 7.15-8.08 ppm. The aliphatic protons of the
cyclohexanonyl ring were displayed upfield as multiplet signals at 1.39-2.08 ppm. The 13C-NMR
spectra showed signals for fluorenonyl, cyclohexanyl, imidazolidinedionyl, phenyl rings as well
as acetamide moiety at the expected regions.
6

Scheme 1. Synthesis of acetamide derivatives of spiro-imidazolidinediones. Reagents and
conditions: (i) aniline derivatives (4-chloro, 4-bromo, 4-methyl, 4-methoxy or
2,4-dichloroaniline), glacial acetic acid, 100 °C; (ii) NaHCO3, NaI and spiro[fluorene-9,5’-
imidazolidine]-2',4’-dione (iii) NaHCO3, NaI and spiro[cyclohexane-1,5'-imidazolidine]-2',4'-
dione.
7

2.2.
Biological activity
The antidiabetic activities of the newly synthesized compounds were tested in vivo. Moreover,
their inhibitory activities against aldehyde reductase (ALR1, EC 1.1.1.2) and aldose reductase
(ALR2, EC 1.1.1.21) were evaluated in vitro. The selectivity indexes of the new compounds
were determined.
2.2.1. In vivo hypoglycemic activity
Diabetic BALB/c mice were used to perform in vivo study. The blood glucose levels were
measured by collecting the blood samples at 0 h followed by 2, 4, 6 and 8 h.15 Most of the new
compounds were found to be active hypoglycemic agents (Table 1). Compounds (3e and 4b)
were the most active compounds with 84.3 and 85.1% reduction in blood glucose, respectively.
They were more potent than the standard drug repaglinide with 66.1% reduction. Both
compounds have a halogen atom (Cl or Br) at the phenyl ring. It was observed that the presence
of two halogen atoms at the ortho and para positions on the phenyl ring increases the activity of
the compound. Compounds (3a, 3b and 4b) possess moderate activities (80.4%, 81.1% and
79.2% reduction, respectively) but they are still more potent than the standard drug repaglinide.
On the other hand, compound (3d) did not lower the plasma glucose level.
8

Comp.
Blood
glucose
Blood
glucose
Blood
glucose
Blood
glucose
Blood
glucose
Inhibition
after
concentration concentration concentration concentration concentration
8 h (%)
± SEMa
± SEMa
± SEMa
± SEMa
± SEMa
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
basal
After 2h
After 4h
After 6h
After 8h
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
460 ± 3.56
452 ± 4.87
358 ± 6.98
320 ± 5.67
398 ± 3.81
406 ± 5.98
459 ± 3.88
387 ± 7.43
370 ± 2.94
384 ± 7.71
350 ± 5.53
331 ± 4.52
314 ±6.84
258 ± 3.98
290 ± 5.67
347 ± 7.62
386 ± 7.01
305 ± 5.74
322 ± 4.88
319 ± 7.43
313 ± 5.69
234 ± 4.76
218 ± 3.82
283 ± 7.11
237 ± 5.54
223 ± 4.61
242 ± 6.56
276 ± 4.54
268 ± 7.71
299 ± 6.53
269 ± 5.44
259 ± 3.81
189 ± 6.78
173 ± 6.33
211 ± 4.71
279 ± 5.67
168 ± 4.89
170 ± 2.75
182 ± 7.22
197 ± 7.21
231 ± 5.74
217 ± 6.32
160 ± 4.22
90 ± 2.87
85 ± 2.87
167 ± 5.45
370 ± 3.91
63 ± 3.45
85 ± 5.63
69 ± 4.77
115 ± 5.67
163 ± 6.63
121 ± 4.42
94 ± 1.76
80.4
81.1
53.4
≥ 200
84.3
79.2
85.1
70.3
56.1
68.5
66.1
Repaglinide 392 ± 6.87
a: number of trials = 3
Table 1. Blood glucose concentration (mg/dl) in diabetic mice treated with the new compounds.
2.2.2. ALR1 and ALR2 activity
The decrease in the UV absorption of NADPH at 340 nm - using UV spectrophotometer - was
used as indication of the inhibitory activities of the new compounds.23 Valproic acid was used as
a positive control to evaluate ALR1 inhibitory activity.15 On the other side, sorbinil was the
positive control in case of ALR2 inhibitory activity.24
Compounds (3e and 4b) (with IC50 ± SEM values of 0.37 ± 0.05 μM and 0.72 ± 0.06 μM,
respectively), were found more potent inhibitors for ALR2 than the standard inhibitor, sorbinil
9

with IC50 value 3.14 ± 0.02 μM.25 This may be assigned to the presence of chlorine atom at the
ortho and para positions of the phenyl ring of compound (3e) and bromo atom at the para
position of the phenyl ring of compound (4b). These small lipophilic halogen atoms are expected
to enhance the binding character of these compounds. The presence of para methoxy group in
compounds (3d and 4d) instead, resulted in a remarkable decrease in the activity. Moreover, the
new compounds exhibited moderate inhibitory activities against ALR1 and were more potent
than valproic acid with IC50 value 57.40 ± 0.89 μM.23 This may be due to their relative lipophilic
structures compared to valproic acid that contains 2-propylpentanoic acid. IC50 values against
ALR1 versus ALR2 was used to calculate the selectivity of the synthesized inhibitors. The
selectivity index displayed that compound (3e) was 96 fold more selective towards ALR2 versus
ALR1. In addition, compound (4b) was 43 fold more selective towards ALR2 (Table 2).
S.E a
Comp.
ALR1
ALR2
IC50[ALR1]/ IC50[ALR2]
IC50 ± SEM (µM)b/(% Inhibition)c
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
22.2 ± 1.72
30.31 ±1.42
10.92 ±1.87
24.25 ±1.03
35.52 ± 1.70
20.67 ±2.51
31.20 ± 0.29
6.43 ± 1.13
25.80 ± 0.68
32.90 ± 0.54
1.03 ± 0.33
0.93 ± 0.03
6.21 ± 0.77
2.87 ± 0.18
0.37 ± 0.05
1.13 ± 0.86
0.72 ± 0.06
10.2 ±2.48
2.76 ± 0.31
1.95 ± 1.06
21.60
32.60
1.76
8.45
96.00
18.30
43.40
0.63
9.36
16.90
10

sorbinil
3.14 ± 0.0225
-
-
Valproic acid 57.40 ± 0.8923
a:
S.E
=
Selectivity index
b: number of trials = 3
c: % Inhibition at 0.6 µM inhibitor concentration
Table 2. In vitro ALR1 and ALR2 activities of the new compounds.
2.3. Molecular docking study
The most active compounds (3e and 4b), compounds (3a and 4e) with moderate activities and
the least active compounds (3c and 4d) were selected for performing the molecular docking
studies. Our aim is to create a correlation between the found in vitro biological results and the
probable binding modes of these compounds together with the reference drugs repaglinide,
sorbinil and the native ligand 37v within the active site of ALR2 (Figures S1a,b, S2a,b, S3a,b
and S4a-c, supplementary information). The X-ray crystal structure of ALR2 complexed with
NADPH
(PDB
ID,
4QR6-ALR2)
co-crystalized
with
(2-[(1,3-benzothiazol-2-
ylmethyl)carbamoyl]-5-fluorophenoxy)acetic acid (37V) was downloaded from the Protein Data
Bank (PDB). The molecular docking simulations were performed by MOE dock tool of the
modeling software Molecular Operating Environment (MOE2016. 8).26 The binding affinity
between the inhibitor and the enzyme is optimum when the value of the binding score is low.
The molecular docking studies revealed that the acetamide linkers of the new compounds were
extended plausibly in the active site of ALR2. These linkers orient the phenyl rings deep in the
specificity pocket created by TYR 48, HIS 110, TRP 111, TRP 20, PHE 115 and SER 210 for
optimum fitting. Moreover, the polar atoms of the hydantoin rings and the acetamide linkers
played crucial roles in the creation of 3-6 major hydrogen bonding interactions with the key
amino acids CYS 298, TRP 111, HIS 110 and TYR 48 (Figures S1a,b, S2a,b and S3a,b,
11

supplementary information). On the other hand, the reference drug repaglinide formed only
one hydrogen bond interaction with Cys298 and displayed a relatively high binding score -5.69
(Figure S4a, supplementary information). This might be explained by the absence of the
hydantoin scaffold in repaglinide structure. Moreover, sorbinil formed only 2 hydrogen bonding
interactions with HIS 110 and TYR 48 (Figure S4b, supplementary information). It showed
high binding score -6.92. This might be attributed to the lack of acetamide linkers in sorbinil
structure. The native ligand 37v formed 3 hydrogen bonding interactions with the key amino
acids HIS 110, TYR 48 and TRP 111, it displayed a binding score -7.02 (Figure S4c,
supplementary information). These findings indicate the importance of both of the hydantoin
rings and the acetamide linkers in enhancing the inhibitory activity.
The molecular docking of the most active compounds (3e and 4b) with IC50 values 0.37 μM
and 0.72 μM, respectively, showed optimum binding modes with low binding scores -7.69 and -
7.63, respectively (Figures 3a,b). The docking studies of the compounds (3a and 4e) with
moderate activities and IC50 values 1.03 μM and 1.95 μM, respectively, revealed plausible
binding modes with binding scores -7.02 and -6.87, respectively (Figures S2a,b, supplementary
information). It is worth noting that the phenyl rings of all the previous compounds (3a, 3e, 4b
and 4e) have small lipophilic halogen atoms. While their analogues (3c and 4d) were the least
active compounds with IC50 values 6.21 μM and 2.76 μM and high binding scores -6.11 and -
6.68, respectively (Figures S3a,b, supplementary information). This might be attributed to the
steric clashes produced by the bulky methyl and methoxy groups of compounds (3c and 4d)
respectively.
It was observed that the hydantoin ring of standard ALR2 inhibitor (sorbinil) was
superimposed with that of compound (3e), with RMSD value 1.5 (Figure 4). Moreover, the
12

acetamide linkers lying between the two lipophilic moieties of standard hypoglycemic agent
(repaglinide) and compound (3e) were almost aligned, with RMSD value 1.9 (Figure 4).
Furthermore, the lipophilic rings attached to the spirohydantoin scaffold of compound (3e) and
sorbinil were aligned with the lipophilic rings of repaglinide (Figure 4). Thus, compound (3e) is
expected to fulfill all the minimum pharmacophoric requirements essential for enhancing both
hypoglycemic activity and selective ALR2 inhibition.
Figures 3a,b. 3D Docking the most active compounds (3e and 4b) with target 4QR6
cocrystalized with ligand 37V, compounds (3e and 4b) (Pink), ligand 37V (Green) and NADP
(yellow).
Figure 4. The alignment of the most active compound 3e (red), sorbinil (green) and repaglinide
(blue).
13

2.4.
In silico prediction of physicochemical properties and drug-likeness
properties.
The results are presented in (Table S1, Supplementary information). Candidate drugs that
obey the Lipinski's "rule of five" (Ro5) are expected to be promising future drugs.27-29
Topological polar surface area TPSA values should be ≤ 140 Å2 for prevailing drug absorption
through intestine and ≤ 90 Å2 for penetrating the blood brain barrier.30,31 All the tested compounds
showed good permeability and absorption. They possessed 2 hydrogen bond donors and 3-4
hydrogen bond acceptors (Table S1, Supplementary information). In addition, all tested
compounds showed good permeability through cell membranes (log P ranges from 2.12 – 4.81).
Veber and co-authors revealed that for controlling conformational changes and for having good
oral bioavailability, the number of rotatable bond (nrotb) should be ≤ 10.27,32 All the tested
compounds had 3-4 nrotb, while repaglinide had 10 nrotb. In addition, drug-likeness scores are
presented in (Table S2, Supplementary information). Compounds which showed positive
values should be considered as drug-like molecules. All the investigated compounds displayed
positive values except compounds (3c and 4e) as well as the reference standard sorbinil.
Moreover, compounds (4b, 4d, and 3e) showed higher drug-likeness scores 1.24, 1.01 and 1.07
respectively, than the reference drug repaglinide 0.92 (Figure S5, Supplementary
information).
3. Experimental
3.1.
Materials and methods for synthesis and analytical characterization
The commercial chemicals and solvents for the synthesis were reagent grade and used without
1
further purification. Varian Mercury VX-300 NMR spectrometer or Jeol LA (400 MHz for H-
1
NMR, 100 MHz for 13C-NMR) were used to measure H- and 13C-NMR Spectra. Electron
impact mass spectra (EI-MS) were recorded on Shimadzu GCMS-QP 5050A gas chromatograph
14

mass spectrometer (70 eV). Elemental analysis was performed at the Microanalytical Center of
Cairo University, Egypt. Melting points (m.p.) were determined in open capillaries using
Gallenkemp melting point apparatus and are uncorrected. The progress of reactions as well as
purity of all compounds were monitored through thin layer chromatography (TLC) using Merck
0.2 mm pre-coated DF-Aluminium sheets 60F254. Infrared spectra (IR) spectra were recorded on
a Shimadzu FT-IR 8101 PC IR spectrophotometer (KBr pellets).
General procedure for the synthesis of 2-chloro-N-arylacetamide derivatives (2a-e)
The appropriate aniline (4-chloro, 4-bromo, 4-methyl, 4-methoxy or 2,4-dichloroaniline) (24.4
mmol) was dissolved in glacial acetic acid (15 mL). Chloroacetyl chloride (25.1 mmol) was
added dropwise. The reaction mixture was stirred at 100 ºC for 1 h. The mixture was cooled to
room temperature. Sodium acetate solution (0.4 M) was added to give a heavy precipitate. The
mixture was stirred in an ice bath for 5 min. The precipitate was collected after filtration.19
Melting points (m.p.) of the synthesized compounds were determined and were comparable with
the reported values.22,33,34
General procedure for the synthesis of N-aryl-2-(2',4'-dioxospiro[fluorene-9,5'-
imidazolidine]-3'-yl)acetamide derivatives (3a-e)
Spiro[fluorene-9,5’-imidazolidine]-2',4’-dione (2.36 mmol) and the appropriate 2-chloro-N-
arylacetamide derivatives (2a-e) (2.36 mmol) were dissolved in DMF (30 mL).21 NaHCO3 (2.36
mmol) and NaI (2.36 mmol) were added. The reaction mixture was stirred at room temperature
for 36 h. The mixture was then added to water (50 mL) to afford the desired product. The
precipitate was filtered and left to dry. The solid residue was recrystallized from absolute ethanol
to give the corresponding acetamide derivatives.22
N-(p-Chlorophenyl)-2-(2',4'-dioxospiro[fluorene-9,5'-imidazolidine]-3'-yl)acetamide (3a)
15

Yield: 64%. m.p. (273-275) ºC. IR (KBr, cm-1): 3390 (N-H), 1667 (amide & lactam), 1559
(C=C sp2), 2956 (C-H sp3). EI-MS (m/z, %): 417 (M+), 418 (M++1), 291, 249, 207, 179, 126. 1H-
NMR (400MHz) (DMSO-d6): δ 10.10 (s, 1H, NH-Ph), 8.11 (d, 2H20, 22 phenyl), 8.09 (d, 2H19, 23
phenyl), 7.96 (m, 8H, Ar-H), 5.79 (s, 1H, NH), 3.82 (s, 2H, CH2). 13C-NMR (DMSO-d6): δ 49.8,
76.2, 120.6, 123.4, 127.8, 129.6, 131.7, 133.4, 138.8, 141.6, 142.2, 143.1, 150.8, 169.4, 172.8.
Anal.Calcd. for : C23H16ClN3O3: C, 66.11; H, 3.86; N,10.06. Found: C,66.31; H, 4.15; N, 9.88.
N-(p-Bromophenyl)-2-(2',4'-dioxospiro[fluorene-9,5'-imidazolidine]-3'-yl)acetamide (3b)
Yield: 61%. m.p. (283-285) ºC. IR (KBr, cm-1): 3331 (N-H), 1658 (amide & lactam) , 1571
(C=C sp2), 2963 (C-H sp3). EI-MS (m/z, %): 461 (M+), 463 (M++2), 291, 249, 207, 179, 169.
1H-NMR (400MHz) (DMSO-d6): δ 9.92 (s, 1H, NH-Ph ), 8.06 (d, 2H20, 22 phenyl),7.92 (d, 2H19,
13
phenyl), 7.27 (m, 8H, Ar-H), 5.69 (s, 1H, NH at position 1'), 3.92 (s, 2H, CH2). C-NMR
23
(DMSO-d6): δ 49.3, 77.1, 121.2, 123.9, 126.3, 129.3, 130.5, 131.7, 133.4, 137.1, 140.4, 143.1,
151.4, 168.9, 172.1. Anal.Calcd. for C23H16BrN3O3: 59.76; H, 3.49; N, 9.09. Found: C,59.53; H,
3.77; N, 8.82.
N-(p-Tolyl)-2-(2',4'-dioxospiro[fluorene-9,5'-imidazolidine]-3'-yl)acetamide (3c)
Yield: 59%. m.p. (226-228) ºC. IR (KBr, cm-1): 3244 (N-H), 1653 (amide & lactam), 1561
1
(C=C sp2), 2946 (C-H sp3). EI-MS (m/z, %): 397 (M+), 291, 249, 207, 179, 106. H-NMR
(400MHz) (DMSO-d6): δ 10.03 (s, 1H, NH-Ph ), 7.94 (d, 2H20, 22 tolyl), 7.91 (d, 2H19, 23 tolyl),
7.77 (m, 8H, Ar-H), 5.69 (s, 1H, NH at position 1'), 4.01 (s, 2H, CH2), 2.32 (s, 3H, CH3).
Anal.Calcd. for C24H19N3O3: 72.53; H, 4.82; N, 10.57. Found: C,72.81; H, 4.64; N, 10.81.
N-(p-Methoxyphenyl)-2-(2',4'-dioxospiro[fluorene-9,5'-imidazolidine]-3'-yl)acetamide (3d)
Yield: 70%. m.p. (135-137) ºC. IR (KBr, cm-1): 3361(N-H), 1659 (amide & lactam), 1533
1
(C=C sp2), 2921 (C-H sp3). EI-MS (m/z, %): 413 (M+), 357, 291, 249, 207, 179, 122. H-NMR
16

(400MHz) (DMSO-d6): δ 9.76 (s, 1H, NH-Ph ), 8.09 (d, 2H20, 22 phenyl),7.96 (d, 2H19, 23
phenyl), 7.30 (m, 8H, Ar-H), 5.81 (s, 1H, NH at position 1'), 4.25 (s, 2H, CH2), 3.83 (s, 3H,
CH3). 13C-NMR (DMSO-d6): δ 49.8, 57.1, 76.8, 121.4, 125.1, 129.1, 130.9, 132.4, 132.9, 136.5,
140.8, 143.7, 150.6, 163, 168.9, 172.1. Anal.Calcd. for C24H19N3O4: C, 69.72; H, 4.63; N, 10.16.
Found: C,70.04; H, 4.87; N, 9.97.
N-(2,4-Dichlorophenyl)-2-(2',4'-dioxospiro[fluorene-9,5'-imidazolidine]-3'-yl)acetamide
(3e)
Yield: 56%. m.p. (210-212) ºC. IR (KBr, cm-1): 3342 (N-H), 1657 (amide & lactam), 1518
(C=C sp2), 2895 (C-H sp3). EI-MS (m/z, %): 451 (M+), 453 (M++2), 291, 249, 207, 179, 159.
1H-NMR (400MHz) (DMSO-d6): δ 9.78 (s, 1H, NH-Ph), 8.25 (s, 1H20 phenyl), 8.08 (d, 1H22
phenyl), 7.94 (d, 1H23 phenyl), 7.25 (m, 8H, Ar-H), 5.87 (s, 1H, NH at position 1'),4.25 (s, 2H,
CH2). Anal.Calcd. for : C23H15Cl2N3O3: C, 61.08; H, 3.34; N, 9.29. Found: C,60.77; H, 3.62; N,
9.06.
General procedure for the synthesis of N-aryl-2-(2',4'-dioxospiro[cyclohexane-1,5'-
imidazolidine]-3'-yl)acetamide derivatives (4a-e)
Spiro[cyclohexane-1,5'-imidazolidine]-2',4'-dione was used to prepare compounds (4a-e)
applying the same procedure used for the synthesis of compounds (3a-e).21,22
N-(p-Chlorophenyl)-2-(2',4'-dioxospiro[cyclohexane-1,5'-imidazolidine]-3'-yl)acetamide
(4a)
Yield: 75%. m.p. (223-225) ºC. IR (KBr, cm-1): 3335 (N-H), 1659 (amide & lactam), 1598
(C=C sp2), 2876 (C-H sp3). EI-MS (m/z, %): 335 (M+), 336 (M++1), 249, 209, 167, 97, 85.1H-
NMR (400MHz) (DMSO-d6): δ 9.72 (s, 1H, NH- Ph ), 7.72 (d, 2H17, 19 phenyl),7.50 (d, 2H16, 20
phenyl), 6.00 (s, 1H, NH at position 1'), 4.24 (s, 2H, CH2), 2.08 (m, 4H, cyclohexanonyl), 1.89
17

(m, 6H, cyclohexanonyl). 13C-NMR (DMSO-d6): δ 26.2, 27.6, 29.2, 48.5, 76.8, 126.8, 130.7,
137.9, 142.7, 152.1, 170.4, 173.1. Anal.Calcd. for : C16H18ClN3O3: C, 57.23; H, 5.40; N, 12.51.
Found: C,57.47; H, 5.21; N, 12.29.
N-(p-Bromophenyl)-2-(2',4'-dioxospiro[cyclohexane-1,5'-imidazolidine]-3'-yl)acetamide
(4b)
Yield: 63%. m.p. (252-254) ºC. IR (KBr, cm-1): 3295 (N-H), 1652 (amide & lactam), 1543
(C=C sp2), 2919 (C-H sp3). EI-MS (m/z, %): 379 (M+), 381 (M++2), 249, 209, 167, 97, 129. 1H-
NMR
(400MHz)
(DMSO-d6):
δ
10.14
(s,
1H,
NH-Ph),
8.05
phenyl), 6.00 (s, 1H, NH at position 1'), 4.20 (s, 2H, CH2), 2.02 (m,4H, cyclohexanonyl), 1.38
(m, 6H, cyclohexanonyl). Anal.Calcd. for : C16H18BrN3O3: C, 50.54; H, 4.77; N, 11.05. Found:
C,50.28; H, 5.06; N, 11.30.
N-(p-Tolyl)-2-(2',4'-dioxospiro[cyclohexane-1,5'-imidazolidine]-3'-yl)acetamide (4c)
Yield: 61%. m.p. (259-260) ºC. IR (KBr, cm-1): 3315 (N-H), 1657 (amide & lactam), 1566
1
(C=C sp2), 2930(C-H sp3). EI-MS (m/z, %): 315 (M+), 275, 209, 167, 97, 40. H-NMR
(400MHz) (DMSO-d6): δ 9.84 (s, 1H, NH-Ph ), 7.70 (d, 2H17, 19 tolyl), 7.51 (d, 2H16, 20 tolyl),
6.10 (s, 1H, NH at position 1'), 3.92 (s, 2H, CH2), 2.30 (s, 3H, CH3), 1.89 (m, 4H,
cyclohexanonyl), 1.43 (m,6H, cyclohexanonyl). Anal. Calcd. for: C17H21N3O3: C, 64.74; H, 6.71;
N, 13.32. Found: C,64.81; H, 6.53; N, 13.06.
N-(p-Methoxyphenyl)-2-(2',4'-dioxospiro[cyclohexane-1,5'-imidazolidine]-3'-yl)acetamide
(4d)
Yield: 73%. m.p. (127-129) ºC. IR (KBr, cm-1): 3308 (N-H), 1649 (amide & lactam), 1533
1
(C=C, sp2), 2911(C-H, sp3). EI-MS (m/z, %): 331 (M+), 234, 209, 167, 97, 82. H-NMR
(400MHz) (DMSO-d6): δ 9.86 (s, 1H, NH- Ph ), 7.78, (d, 2H17, 19 phenyl), 7.64 (d, 2H16, 20
18

phenyl), 5.74 (s, 1H, NH at position 1'), 4.29 (s, 2H, CH2), 4.05 (s, 3H, OCH3), 2.06 (m, 4H,
cyclohexanonyl), 1.74 (m, 6H, cyclohexanonyl). Anal.Calcd. for : C17H21N3O4: C, 61.62; H,
6.39; N, 12.68. Found: C,61.38; H, 6.63; N, 12.37.
N-(2,4-Dichlorophenyl)-2-(2',4'-dioxospiro[cyclohexane-1,5'-imidazolidine]-3'-yl)acetamide
(4e)
Yield: 76%. m.p. (187-188) ºC. IR (KBr, cm-1): 3346 (N-H), 1657 (amide & lactam), 1533
1
(C=C sp2), 2956 (C-H sp3). EI-MS (m/z, %): 369 (M+), 371 (M++2), 283, 209, 167, 97, 85. H-
NMR (400MHz) (DMSO-d6): δ 9.95 (s, 1H, NH-Ph ), 8.35 (s, 1H17 phenyl), 8.05 (d, 1H19
phenyl),
7.86
(d,
1H20
phenyl),
5.90
(s,
1H,
NH
at
positi
cyclohexanonyl), 1.77 (m, 6H, cyclohexanonyl). 13C-NMR (DMSO-d6): δ 25.7, 27.9, 28.3, 49.4,
76.7, 130, 131.6, 133.7, 136.2, 137.4, 142.4, 153.2, 169.4, 172.1. Anal.Calcd. for :
C16H17Cl2N3O3: C, 51.91; H, 4.63; N, 11.35. Found: C,52.16; H, 4.52; N, 11.59.
3.2.
Biological evaluation
We previously published methods used for biological evaluation including inhibition assay of
aldose and aldehyde reductase in vitro, induction of diabetes in experimental animals, in addition
to the blood glucose level determination.16 Both ALR1 and ALR2 were obtained from
BioVision, Incorporated.35 The Ethics Committee of the Suez Canal University approved the
experimental protocols in the present study.
3.3.
Molecular docking study
Molecular Operating Environment (MOE®, 8) version 2016 was used to perform Molecular
docking simulation studies.36 Target compounds optimization as well as optimization of the
enzymes active site mainly followed our published procedures.16
3.4.
Bioinformatics study
19

In silico molecular properties and bioactivity prediction of the new compounds were calculated
using MolSoft software.37,38
4. Conclusion
Hydantoin core and acetamide moiety were used to design a novel series of non-sulfonylureas.
The target compounds were tested for their hypoglycemic activities in vivo and their inhibitory
activities against both ALR1 and ALR2 in vitro. The most active compound (3e) displayed an
IC50 ± SEM value 0.37 ± 0.05 µM. The selectivity index showed that compound (3e) is 96 fold
more selective towards ALR2. In vivo hypoglycemic study showed that compound (3e) was more
potent than the commercial drug repaglinide. Docking studies indicate the importance of using
both of the hydantoin ring and the acetamide moiety together in the design of target compounds.
This finding could help in the discovery of a new generation of clinically useful hypoglycemic
agents.
5. Acknowledgment
We would like to thank Dr. Waleed Aly (Biochemistry Department, Faculty of Pharmacy,
Cairo University) for biological testing and Prof. Dr. Nadia Mahfouz (Pharmaceutical Chemistry
Department, Faculty of Pharmacy, 6th October University) for molecular modeling study. This
research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors. The authors declare no potential conflicts of interest.
20

6. References
(1) Uzu, T.; Harada, T.; Sakaguchi, M.; Kanasaki, M.; Isshiki, K.; Araki, S.; Sugiomoto, T.;
Koya, D.; Haneda, M.; Kashiwagi, A. Glucocorticoid-induced diabetes mellitus: prevalence and
risk factors in primary renal diseases. Nephron Clinical Practice 2007, 105, c54-c57.
(2)
Guardado-Mendoza, R.; Prioletta, A.; Jiménez-Ceja, L. M.; Sosale, A.; Folli, F. The role
of nateglinide and repaglinide, derivatives of meglitinide, in the treatment of type 2 diabetes
mellitus. Archives of medical science: AMS 2013, 9, 936.
(3)
DeRuiter, J. Endocrine Pharmacotherapy Module Overview of the antidiabetic agents.
Spring 2003, 1, 1-33.
(4)
Han, X.; Chiang, C.; Leonard, C. E.; Bilker, W. B.; Brensinger, C. M.; Li, L.; Hennessy,
S. Biomedical Informatics Approaches to Identifying Drug–Drug Interactions: Application to
Insulin Secretagogues. Epidemiology 2017, 28, 459-468.
(5)
Anesthesia; Elsevier: 2019, p 708-731.
(6) Del Prato, S.; Pulizzi, N. The place of sulfonylureas in the therapy for type 2 diabetes
mellitus. Metabolism-Clinical and Experimental 2006, 55, S20-S27.
(7) Brown, G. R.; Foubister, A. J. Receptor binding sites of hypoglycemic sulfonylureas and
related [(acylamino) alkyl] benzoic acids. Journal of medicinal chemistry 1984, 27, 79-81.
(8) Kador, P. F. The role of aldose reductase in the development of diabetic complications.
Medicinal research reviews 1988, 8, 325-352.
(9) Gosmanov, A. R.; Gosmanova, E. O.; Kitabchi, A. E. Hyperglycemic crises: diabetic
Keegan, M. T. Endocrine pharmacology. In Pharmacology and Physiology for
ketoacidosis (DKA), and hyperglycemic hyperosmolar state (HHS). In Endotext [Internet];
MDText. com, Inc.: 2018.
21

(10) Vander Jagt, D. L.; Hunsaker, L. A. Methylglyoxal metabolism and diabetic
complications: roles of aldose reductase, glyoxalase-I, betaine aldehyde dehydrogenase and 2-
oxoaldehyde dehydrogenase. Chemico-biological interactions 2003, 143, 341-351.
(11) Bachur, N. R. Cytoplasmic aldo-keto reductases: a class of drug metabolizing enzymes.
Science 1976, 193, 595-597.
(12) Sun, W.; Gerhardinger, C.; Dagher, Z.; Hoehn, T.; Lorenzi, M. Aspirin at Low-
Intermediate Concentrations Protects Retinal Vessels in Experimental Diabetic Retinopathy
Through Non–Platelet-Mediated Effects. Diabetes 2005, 54, 3418-3426.
(13) Kador, P. F.; Kinoshita, J. H.; Sharpless, N. E. Aldose reductase inhibitors: a potential
new class of agents for the pharmacological control of certain diabetic complications. Journal of
medicinal chemistry 1985, 28, 841-849.
(14) Lee, Y. S.; Chen, Z.; Kador, P. F. Molecular modeling studies of the binding modes of
aldose reductase inhibitors at the active site of human aldose reductase. Bioorganic & medicinal
chemistry 1998, 6, 1811-1819.
(15) Iqbal, Z.; Hameed, S.; Ali, S.; Tehseen, Y.; Shahid, M.; Iqbal, J. Synthesis,
characterization, hypoglycemic and aldose reductase inhibition activity of arylsulfonylspiro
[fluorene-9, 5′-imidazolidine]-2′, 4′-diones. European journal of medicinal chemistry 2015, 98,
127-138.
(16) Salem, M. G.; Aziz, Y. M. A.; Elewa, M.; Elshihawy, H. A.; Said, M. M. Molecular
modelling and synthesis of spiroimidazolidine-2, 4-diones with dual activities as hypoglycemic
agents and selective inhibitors of aldose reductase. Bioorganic chemistry 2018, 79, 131-144.
22

(17) Viegas-Junior, C.; Danuello, A.; da Silva Bolzani, V.; Barreiro, E. J.; Fraga, C. A. M.
Molecular hybridization: a useful tool in the design of new drug prototypes. Current medicinal
chemistry 2007, 14, 1829-1852.
(18) Lang, V.; Light, P. E. The molecular mechanisms and pharmacotherapy of ATP-sensitive
potassium channel gene mutations underlying neonatal diabetes. Pharmacogenomics and
personalized medicine 2010, 3, 145.
(19) Reilly, T. J. The preparation of lidocaine. J. Chem. Educ 1999, 76, 1557.
(20) Aziz, Y. M. A.; Said, M. M.; El Shihawy, H. A.; Abouzid, K. A. Discovery of novel
tricyclic pyrido [3′, 2′: 4, 5] thieno [3, 2-d] pyrimidin-4-amine derivatives as VEGFR-2
inhibitors. Bioorganic chemistry 2015, 60, 1-12.
(21) Mahmoodi, N. O.; Khodaee, Z. Evaluating the one-pot synthesis of hydantoins. Arkivoc
2007, 3, 29-36.
(22) Qandil, A. M.; Fakhouri, L. I. a-Anilinoketones, Esters and Amides: A Chemical Study.
Pharmaceuticals 2012, 5, 591-612.
(23) Stefek, M.; Snirc, V.; Djoubissie, P.-O.; Majekova, M.; Demopoulos, V.; Rackova, L.;
Bezakova, Z.; Karasu, C.; Carbone, V.; El-Kabbani, O. Carboxymethylated pyridoindole
antioxidants as aldose reductase inhibitors: Synthesis, activity, partitioning, and molecular
modeling. Bioorganic & medicinal chemistry 2008, 16, 4908-4920.
(24) Miyamoto, S. Recent advances in aldose reductase inhibitors: potential agents for the
treatment of diabetic complications. Expert Opinion on Therapeutic Patents 2002, 12, 621-631.
(25) Rakowitz, D.; Maccari, R.; Ottanà, R.; Vigorita, M. G. In vitro aldose reductase
inhibitory activity of 5-benzyl-2, 4-thiazolidinediones. Bioorganic & medicinal chemistry 2006,
14, 567-574.
23

(26) Takashita, E.; Fujisaki, S.; Shirakura, M.; Nakamura, K.; Kishida, N.; Kuwahara, T.;
Shimazu, Y.; Shimomura, T.; Watanabe, S.; Odagiri, T. Influenza A (H1N1) pdm09 virus
exhibiting enhanced cross-resistance to oseltamivir and peramivir due to a dual H275Y/G147R
substitution, Japan, March 2016. Eurosurveillance 2016, 21.
(27) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and
computational approaches to estimate solubility and permeability in drug discovery and
development settings1. Advanced drug delivery reviews 2001, 46, 3-26.
(28) Lipinski, C. A. Lead-and drug-like compounds: the rule-of-five revolution. Drug
Discovery Today: Technologies 2004, 1, 337-341.
(29) Leeson, P. D.; Springthorpe, B. The influence of drug-like concepts on decision-making
in medicinal chemistry. Nature reviews. Drug discovery 2007, 6, 881.
(30) Clark, D. E.; Pickett, S. D. Computational methods for the prediction of ‘drug-likeness’.
Drug discovery today 2000, 5, 49-58.
(31) Ghose, A. K.; Herbertz, T.; Hudkins, R. L.; Dorsey, B. D.; Mallamo, J. P. Knowledge-
based, central nervous system (CNS) lead selection and lead optimization for CNS drug
discovery. ACS chemical neuroscience 2011, 3, 50-68.
(32) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
Molecular properties that influence the oral bioavailability of drug candidates. Journal of
medicinal chemistry 2002, 45, 2615-2623.
(33) Aktürk, Z.; Kılıç, F.; Erol, K.; Pabuçcuoğlu, V. Synthesis and anticonvulsant activity of
some ω-(1H-1-imidazolyl)-N-phenylalkanoic acid amide derivatives. Il Farmaco 2002, 57, 201-
206.
24

(34) Ma, L.; Li, S.; Zheng, H.; Chen, J.; Lin, L.; Ye, X.; Chen, Z.; Xu, Q.; Chen, T.; Yang, J.
Synthesis and biological activity of novel barbituric and thiobarbituric acid derivatives against
non-alcoholic fatty liver disease. European journal of medicinal chemistry 2011, 46, 2003-2010.
(35) BioVision, I. EZDetect™ Aldo-keto Reductase Activity Assay Kit (Colorimetric).
(36) Chemical Computing Group Inc. MO Canada. Molecualr Operating Environment (MOE).
2016
(37) Koch, M. A.; Schuffenhauer, A.; Scheck, M.; Wetzel, S.; Casaulta, M.; Odermatt, A.;
Ertl, P.; Waldmann, H. Charting biologically relevant chemical space: a structural classification
of natural products (SCONP). Proceedings of the National Academy of Sciences of the United
States of America 2005, 102, 17272-17277.
(38) Agnihotri, S.; Narula, R.; Joshi, K.; Rana, S.; Singh, M. In silico modeling of ligand
molecule for non structural 3 (NS3) protein target of flaviviruses. Bioinformation 2012, 8, 123.
Graphical abstract
25

Compound (3e) exhibited 96 folds selective inhibitory activity against ALR2 versus ALR1, with
an IC50 ± SEM value 0.37 ± 0.05 µM.
26