Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of novel quinoxaline derivatives as potential PPARγ and SUR agonists

Article abstract of DOI:10.1016/j.bmc.2017.01.015

In our effort to develop potent anti-hyperglycemic agents with potential agonistic activities toward PPARγ and SUR, three novel series of quinoxaline derivatives bearing sulfonylurea or sulfonylthiourea moieties with different linkers were designed and synthesized. Some of the newly synthesized compounds were evaluated in vivo for their anti-hyperglycemic activities in STZ-induced hyperglycemic rats. Compounds 15_a, 15_e, 19_band 24_aexhibited the highest anti-hyperglycemic activities with % reduction in blood glucose level of (50.58, 43.84, 45.10 and 49.62, respectively). Additionally, eight compounds revealed potent anti-hyperglycemic activities were further evaluated in vitro for their PPARγ binding affinity and insulin-secreting ability as potential mechanisms for anti-hyperglycemic activity. Four compounds (15_a, 15_b, 15_dand 15_e) significantly bound to PPARγ with IC₅₀values of 0.482, 0.491, 0.350 and 0.369 μM, respectively. Moreover, Compounds 15_aand 15_bhave demonstrated induction of insulin-secretion with EC₅₀values of 0.92 and 0.98 μM, respectively. Furthermore, molecular docking and pharmacophore generation techniques were carried out to investigate binding patterns and fit values of the designed compounds with PPARγ and SUR, respectively.

Full text of DOI:10.1016/j.bmc.2017.01.015

Accepted Manuscript
Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of
novel quinoxaline derivatives as potential PPARγ and SURs agonists
Mohamed.K. Ibrahim, Ibrahim H. Eissa, AbdallahE. Abdallah, Ahmed M.
Metwaly, M.M. and M.A. Radwan ElSohly
PII:
S0968-0896(16)31420-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.015
BMC 13494
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
14 December 2016
6 January 2017
11 January 2017
Please cite this article as: Ibrahim, Mohamed.K., Eissa, I.H., Abdallah, AbdallahE., Metwaly, A.M., Radwan
ElSohly, M.M.a., Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of novel quinoxaline
derivatives as potential PPARγ and SURs agonists, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.01.015
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Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of novel
quinoxaline derivatives as potential PPARγ and SURs agonists
Mohamed. K. Ibrahima, Ibrahim H. Eissa* a, Abdallah E. Abdallah a, Ahmed M. Metwalyb,, M.
M. Radwanc and M. A. ElSohly*c
a
Pharmaceutical Chemistry departments, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo, 11884, Egypt
b Pharmacognosy department, Faculty of Pharmacy(Boys), University of Al-Azhar, Cairo, Egypt
c
National Center for Natural Products Research, University of Mississippi, University, MS
38677, USA
* Corresponding author:
Ibrahim H. Eissa
Pharmaceutical Chemistry departments, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo, 11884, Egypt
Email: Ibrahimeissa@azhar.edu.eg
M. A. ElSohly
National Center for Natural Products Research, University of Mississippi, University, MS
38677, USA
Email: Msohly@olemiss.edu

Abstract
In our effort to develop potent anti-hyperglycemic agents with potential agonistic activities
toward PPARγ and SURs, three novel series of quinoxaline derivatives bearing sulfonylurea or
sulfonylthiourea moieties with different linkers were designed and synthesized. Some of the
newly synthesized compounds were evaluated in vivo for their anti-hyperglycemic activities in
STZ-induced hyperglycemic rats. Compounds 15a, 15e, 19b and 24a exhibited the highest anti-
hyperglycemic activities with % reduction in blood glucose level of (50.58, 43.84, 45.10 and
49.62, respectively). Additionally, eight compounds revealed potent anti-hyperglycemic
activities were further evaluated in vitro for their PPARγ binding affinity and insulin-secreting
ability as potential mechanisms for anti-hyperglycemic activity. Four compounds (15a, 15b, 15d
and 15e) significantly bound to PPARγ with IC50 values of 0.482, 0.491, 0.350 and 0.369 µM,
respectively. Moreover, Compounds 15a and 15b have demonstrated induction of insulin-
secretion with EC50 values of 0.92 and 0.98 µM, respectively. Furthermore, molecular docking
and pharmacophore generation techniques were carried out to investigate binding patterns and fit
values of the designed compounds with PPARγ and SUR, respectively.
Key words: Quinoxaline, PPARγ, Sulfonylurea, Sulfonylthiourea, Docking, Pharmacophore,
Anti-hyperglycemic
1. Introduction
Type-2 diabetes mellitus (T2DM) is a disease often associated with a cluster of metabolic
disturbances and cardiovascular risk factors such as obesity, insulin resistance, hypertension,
atherosclerosis, dyslipidemia, and endothelial dysfunction [1]. So that, beyond glucose control,
management of lipid metabolism have to be considered in the treatment of T2DM [2] .
The most important receptors involved in treatment of type II diabetes mellitus are
peroxisome proliferator-activated receptors (PPARs) [3] and sulfonylurea receptors (SURs) [4] .
PPARs are ligand activated transcription factors belonging to the nuclear hormone receptor super
family. Three PPAR isotypes have been identified, PPAR α, δ and γ which are involved in the
regulation of glucose and lipid homeostasis [5, 6]. PPARγ has an essential role in glucose
metabolism and fatty acid storage, so that it is a crucial target for the development of insulin
sensitizing anti-hyperglycemic agents [7].

The basic structural requirements for binding to PPARγ are an acidic head, a linker
attached to an aromatic scaffold (spacer group), a linker, and a hetero-aromatic lipophilic tail [8,
9] (Fig. 1A). The most famous class of compounds reported as high-affinity PPARγ agonists are
known as ‘glitazones’, or thiazolidinediones (TZDs), on the basis of their common 2,4
thiazolidinedione structural motif [10, 11]. A wide variety of lipophilic ‘tails’ have been reported
to confer good anti-diabetic activity in TZDs and subsequently have been shown to bind and
activate PPARγ [12]. The presence of a heterocyclic nitrogen atom in this region such as
rosiglitazone 1, pioglitasone 2, darglitazone 3 [13], and imidazopyridine 4 [14] has been shown
to confer excellent PPARγ potency. This nitrogen atom may serve as a hydrogen-bond acceptor
to either a water molecule or a specific residue within this portion of the ligand binding domain
(LBD)[15]. The lipophilic tail has usually been attached to the central ‘spacer group’ via an alkyl
ether linkage [16]. The 2,4-thiazolidinedione ‘head group’ is usually linked to the ‘spacer group’
via a single methylene group. Shortening or lengthening of this linker , leads to a reduction of
PPARγ agonistic activity [17]. Many groups have been reported on cyclic bioisosteric
replacements of the 2, 4 thiazolidinedione head group as oxazolidinedione [18],
oxadiazolidinedione [19] and isoxazolidine-3,5-diones [20]. Several acyclic TZD as N-
hydroxyureas and carbonylated hydroxyureas have been reported as TZD replacements [19].
Unfortunately, the use of TZDs has been beset with adverse side effects such as
congestive heart failure, myocardial infarction, weight gain and risk of death from cardiovascular
causes [21, 22]. Therefore, demand for new PPARγ ligands other than TZD could be of special
interest.
The second receptors are sulfonylurea receptors present in pancreatic β-cells.
Sulfonylureas (SUs) stimulate insulin secretion via binding to sulfonylurea receptors [23]. The
reported pharmacophoric features of sulfonylurea acting drugs are three lipophilic centers
separated by an amide and an anionic linker (Fig. 1B). First generation sulfonylureas, such as
tolbutamide, contain only two lipophilic centers and were proposed to interact with only A site of
SURs. On the other hand, 2nd and 3rd generation sulfonylureas such as glipizide 7 and
glimepiride 8 comprise amido (-CONH-) group which is required for interaction with B site of
SURs [24]. Moreover, it was reported that some of 2nd and 3rd generation sulfonylureas exert
their clinical efficacy by acting through both SURs and PPARγ [25-27]

1.1 Rationale of drug design
The great similarity between the basic structural requirements of PPARγ and SUR agonists drove
us to design some novel compounds comprising both requirements (Fig. 2). The acidic head
required to PPARγ agonistic activity, has been replaced by sulfonylurea or sulfonylthiourea
moieties. Sulfonylurea and sulfonylthiourea moieties were incorporated into our molecules with
the goal of providing PPARγ and SUR agonistic activities. They form hydrogen bonds with
influencing residues of the protein active site (Ser289, His323, His449 and Tyr473) [28].
Aromatic and aliphatic substitutions on sulfonylurea and sulfonylthiourea moieties act as a
lipophlic center required to SUR agonists. Moreover, NH group of sulfamoyl moiety is acidic
(pKa = 4.9 – 6.5) and there for completely ionized at physiological pH [29]. This ionization
produces an anionic linker which contributes significantly to SUR agonistic affinity [24].
Sulfonyl (SO2) group acts as one atom spacer between an acidic head and aromatic group. This is
considered as optimal length for PPARγ agonistic activity. Para-disubstituted phenyl group acts
as an aromatic scaffold (spacer group) which is required for optimal PPARγ agonism [30], in
addition to its role as lipophilic center for SUR agonists. Several linkers between lipophilic tail
and an aromatic scaffold (spacer group) have been synthesized to study SAR of the new
compounds. These linkers are essential for PPARγ agonistic activity, on the other hand, they
contain amido(-CONH-) group which is required to interact with B site of SURs [24]. Finally, 3-
methylquinoxalin-2(1H)-one and 3,4-dihydroquinoxalin-2(1H)-one nuclei, act as lipophilic tail
and lipophlic center required for PPARγ and SURs agonistic activities, respectively.
The choice of quinoxaline nucleus was based on some bioisoeteric considerations. First,
the bicyclic structure of quinoxaline core is convenient to the large size of the hydrophobic
entrance of PPARγ active site [28]. Second, the heterocyclic nitrogen serves as a hydrogen-bond
acceptor conferring excellent PPARγ potency.
Based on the above considerations, and in an attempt to obtain new multi-target
antihyperglycemic agents, new scaffolds of quinoxaline-sulfonylurea hybrids have been designed
and synthesized (Fig. 2).

Fig. 1: (A): The basic structural features of PPARγ agonists and some reported compounds.
(B): The basic structural features of sulfonylurea acting compounds and some reported
compounds.

Fig. 2: Rationale of molecular design showing the basic structural requirements for PPARγ and
SURs agonists.

2. Results and discussion
2.1.
Chemistry
The sequence of reactions that was used in the synthesis of the target compounds is
illustrated in Schemes 1–3. Synthesis was initiated by reaction of o-phenylenediamine 9 with
sodium pyruvate 10 in glacial acetic acid to furnish 3-methylquinoxalin-2(1H)-one 11 [31], and
subsequent treatment with alcoholic potassium hydroxide to afford the corresponding potassium
salt 12 [32]. Heating of the obtained potassium salt with 2-chloro-N-(4-
sulfamoylphenyl)acetamide 13 [33] in dry DMF afforded the key intermediate 14 in a good
yield (76%). The reaction of compound 14 with the appropriate iso(thio)cyanates namely,
cyclohexyl isocyanate, butyl isocyanate, phenyl isocyanate, cyclohexyl isothiocyanate, butyl
isothiocyanate, and phenyl isothiocyanate in refluxing dry acetone in the presence of anhydrous
potassium carbonate as a base afforded the target compounds 15a-f, respectively (Scheme 1).
Next, 3,4-dihydroquinoxalin-2(1H)-one 17 ]43[ was prepared via the reaction of
o-phenylenediamine 9 with chloroacetic acid 16 in aqueous ammonia. Reaction of compound 17
with 2-chloro-N-(4-sulfamoylphenyl)acetamide 13 in dry DMF afforded the key intermediate 18
in a good yield (72.63%). Upon the reaction of compound 18 with the appropriate
iso(thio)cyanates namely, cyclohexyl isocyanate, butyl isocyanate, phenyl isocyanate, cyclohexyl
isothiocyanate, butyl isothiocyanate, and phenyl isothiocyanate in refluxing dry acetone with
presence of anhydrous potassium carbonate as base, the target compounds 19a-f were produced
(Scheme 2).
Finally, reaction of 3,4-dihydroquinoxalin-2(1H)-one 17 with chloroacetyl chloride in dry
DMF afforded 4-(2-chloroacetyl)-3,4-dihydroquinoxalin-2(1H)-one 21 ]43[ and subsequent
treatment with commercially available sulfanilamide afforded the intermediate compound 23.
The target compounds 24a-c were synthesized through the reaction of compound 23 with
appropriate isocyanates namely, cyclohexyl isocyanate, butyl isocyanate and phenyl isocyanate,
in refluxing dry acetone with presence of anhydrous potassium carbonate as a base. Structures of
the target compounds were characterized by 1H NMR, 13C NMR, DEPT, IR and EI mass spectral
data.
The IR spectra of the key compounds 14, 18, and 23 revealed the presence of two
characteristic NH and NH2 absorption bands of 4-aminobenzenesulfonamide moiety around 3382
- 3234 cm-1, in addition to absorption bands due to C=O groups of quinoxaline and amide
moieties around 1681-1657 cm-1. Moreover, the two characteristic absorption bands of SO2

group appeared around 1325 cm-1 and 1148 cm-1. The IR spectra of compounds 15a-f, 19a-f, and
24a-c showed sharp absorption bands of 3NH groups of 4-aminobenzene sulfonyl(thio)urea
moiety in the region 3383-3192 cm-1.
The 1H NMR spectra of the key compounds 14, 18, and 23 revealed the presence of D2O
exchangeable singlet peak of sulfonamide NH2 at about 7.2 ppm, and a singlet peak for the
1
methylene group flanked between C=O and quinoxaline moieties at about 5.1 ppm. The H
NMR spectra of compounds 15a-f, 19a-f, and 24a-c showed disappearance of sulfonamide NH2
peak and appearance of more deshielded singlet peak for NH of sulfonylurea.
The aromatic area of compound 14 showed a characteristic para-disubstituted benzene ring
pattern at 7.51 (d, J = 6.0 Hz, 2H) and 7.73 (d, J = 6.0 Hz, 2H).
Scheme 1: General procedure for preparation of target compounds 15a-f

Scheme 2: General procedure for preparation of target compounds 19a-f
Scheme 3: General procedure for preparation of target compounds 24a-c

2.2. Biological testing
2.2.1. In vivo anti-hyperglycemic activity
The in vivo anti-hyperglycemic activity of the newly prepared compounds 15a-f, 19a-f and 24a-c
was examined against streptozotocin-induced hyperglycemic rats using anti-hyperglycemic assay
as described by Ramsey et al [36]. Rosiglitazone and glimepiride were included in the
experiment as positive controls. The results were expressed as % reduction in blood glucose
level (Table 1). Most of the synthesized compounds showed excellent to modest anti-
hyperglycemic activity against streptozotocin-induced hyperglycemic rats. Compound 15a was
found to be the most potent derivatives (% reduction in blood glucose level of50.58). Besides,
compounds 24a, 19b and 15e possessed strong anti-hyperglycemic activities (% reduction in
blood glucose level of 49.62, and 45.10, 43.84, respectively). Several compounds showed good
anti-hyperglycemic activity as 15b, 24b and 19a (% reduction in blood glucose level of 40.55,
40.55 and 39.81, respectively). Moreover, compounds 15c, 15d, 15f, 19c, 24c and 19e were
moderately active with % reduction in blood glucose level values ranging from 33.23 to 20.54.
On the other hand, Compounds 19d and 19f were found to the weakest anti-hyperglycemic agents
(% reduction in blood glucose level of 13.08, and 2.88, respectively).
2.2.2. In vitro PPARγ- ligand binding assay
Compounds with strong in vivo anti-hyperglycemic activities (15a, 15b, 15d, 15e, 19a, 19b,
24a, and 24b) were further evaluated to determine their in vitro binding affinity to PPARγ. The
ability of these compounds to bind with the PPARγ-LBD have been assessed through
Fluorescence Polarization Assay technique [37]. Rosiglitazone was used as a positive control.
Table 1 shows a comparison of IC50 values for the test compounds. Compounds 15a, 15b, 15d
and 15e significantly bound to PPARγ with IC50 values of 0.482, 0.491, 0.350 and 0.369 µM,
respectively. The results were not significant as rosiglitazone. Rosiglitazone PPARγ binding
affinity was 1.6, 1.7, 1.2 and 1.3 folds stronger than 15a, 15b, 15d and 15e, respectively. On the
other hand, compounds 19a, 19b, 24a and 24b showed moderate PPARγ binding affinities with
IC50 values ranging from 1.141 to 1.320 µM.

2.2.3. In vitro insulin assay
Compounds (15a, 15b, 15d, 15e, 19a, 19b, 24a, and 24b) were further evaluated to determine their
in vitro insulin-secreting activities against isolated pancreatic islets of rats via quantitative
sandwich enzyme immunoassay technique [38]. Glimiperide was used as a positive control. The
results were reported as the EC50 values (Table 1).
The examined compounds exhibited good to moderate insulin-secreting activities with
EC50 values ranging from 0.92 to 2.82 µM. Glimiperide showed EC50 of 0.73 µM. Compounds
15a and 15b have demonstrated potent insulin-secreting activities with EC50 values of 0.92 and
0.98 µM, respectively. Compounds 19a, 19b, 24a, and 24b exhibited moderate activities with IC50
values of 1.54, 1.69, 1.89 and 1.95 µM, respectively. Moreover, compounds 15d and 15e
displayed weak insulin-secreting activities with IC50 values of 2.69 and 2.82 µM, respectively.

Table 1: In vivo reduction in blood glucose levels, in vitro PPARγ binding affinities and in vitro
insulin secreting activities of the newly synthesized compounds and reference drugs
In vivoa
in vitroa
Insulin Secreting
Comp.
Reduction in blood
glucose level (%)
PPARγ binding
affinity IC50 (µM)b
activity
EC50 (µM)c
50.58
40.55
25.15
33.23
43.84
20.54
39.81
45.10
21.89
13.08
30.75
2.88
0.482
0.491
NTd
0.92
0.98
NTd
2.69
2.82
NTd
1.54
1.69
NTd
NTd
NTd
NTd
1.89
1.95
NTd
15a
15b
15c
15d
15e
15f
19a
19b
19c
19d
19e
19f
24a
24b
24c
0.350
0.369
NTd
1.150
1.293
NTd
NTd
NTd
NTd
49.62
40.55
22.01
1.141
1.320
NTd

32.21
26.53
0.292
NTd
NTd
0.73
Rosiglitazone
Glimiperide
a All data within 10 % (n = 3)
b Concentration of the test compound required to displace 50% of titrated ligand
c Concentration required to produce 50% of maximal effect.
d NT: Compounds not tested
2.3. Molecular modeling
Many crystal structures of PPARγ receptor are available in the Protein Data Bank (PDB). So
that, receptor based drug design (docking) technique [39] was selected to investigate the binding
pattern of the synthesized compounds with the prospective target, PPARγ. Since the crystal
structure for SUR is not available, ligand based drug design (pharmacophore) technique [40]
was used as a guide to predict the sulfonylurea agonistic activities of the synthesized compounds
to give an idea on the interaction that would exist between the ligand and the receptor.
2.3.1. Docking studies
To predict the PPARγ agonistic activity of the synthesized compounds, docking studies were
carried out using Discovery studio software program [41]. The PPARγ cavity is Y-shaped and
consists of an entrance, arm I and arm II. Arm I contains four polar residues, Ser289, His323,
His449 and Tyr473. These residues involved in hydrogen bond network between the receptor
and ligands. Arm II is formed from Ile 341, Leu353, Ile281 and Val339 while the entrance
consists of Leu330, Leu333, Ser342 and Arg288 [28] (Fig.3).

Fig. 3: PPARγ cavity composed of 3 main parts arm I, arm II and entrance [28]
The proposed binding mode of the ligand, rosiglitazone, revealed an affinity value of -48.74
kcal/mol. It demonstrated the important interactions with the residues at the active site of
PPARγ. The acidic polar thiazolidinedione head is oriented in the polar arm I of the receptor
where the acidic hydrogen of the imide group formed a hydrogen bond with a distance of 2.38 A˚
with Tyr473 (OH). The hydrophobic tail occupied the hydrophobic pocket (arm II and the
hydrophobic part of the entrance),These results were found to be identical with the reported data
[28] (Fig. 4).
The obtained results indicated that all studied ligands showed a similar position and
orientation inside the putative binding site of PPARγ. All designed molecules exhibited good
binding energies ranging from – 36.62 to -55.52 kcal/mol (Table 2). The proposed binding mode
of compound 15e (affinity value of -50.81 kcal/mol with four hydrogen bonds) was virtually the
same like that of rosiglitazone (Fig. 5). The acidic polar head (sulfonylthiourea moiety) was
oriented in the polar arm I and formed two hydrogen bonds with the polar residues Tyr327 and
His449. The acidic hydrogen of the sulfamoyl group formed a hydrogen bond with a distance of
2.07 A˚ with Tyr327 (OH) and the carbamothioyl group formed a hydrogen bond with a distance

of 2.02 A˚ with His449 (nitrogen of imidazole ring ). The heterocyclic quinoxaline moiety was
oriented in the hydrophobic entrance. The nitrogen heteroatom of quinoxaline moiety formed a
hydrogen bond and a cationic pi bond with a distance of 2.39 and 4.90 A˚ with Ser342 (HN) and
Arg288, residues of entrance, respectively.
The proposed binding mode of compound 15b (affinity value of -55.52 kcal/mol with two
hydrogen bonds) was virtually the same as that of rosiglitazone, where its acidic polar head
(sulfonylurea moiety) occupied the polar arm I. It formed two hydrogen bonds; the sulfamoyl
group formed a hydrogen bond via its acidic hydrogen with a distance of 2.43 A˚ with Tyr473
(OH). The carbamoyl group formed a hydrogen bond with a distance of 2.47 A˚ with His323
(nitrogen 2 of imidazole ring) (Fig. 6).
Table 2: The docking binding free energies of the synthesized compounds
Binding free
energy (kcal/mol)
- 46.31
Binding free energy
(kcal/mol)
Comp. No.
15a
Comp. No
- 46.52
19c
- 55.52
- 47.33
- 36.62
- 50.81
- 40.23
- 52.43
- 52.23
- 45.31
- 46.73
- 53.43
- 52.91
- 50.44
- 47.92
- 48.74
15b
19d
15c
19e
15d
19f
15e
24a
24b
15f
19a
24c
19b
Rosiglitazone

Fig. 4: Rosiglitazone docked into the active site forming hydrogen bond (green dot) between its
polar head and tyr473
Fig. 5: Compound 15e docked into the active site, and its polar head formed hydrogen bonds
(green) with the polar residues (Tyr327 & His449) in arm I. Quinoxaline moiety formed
hydrogen bond with Ser342 residue of entrance.

Fig. 6: Compound 15b docked into the active site, and its polar head formed hydrogen bonds
(green) with the influencing polar residues Tyr473 and His323 in arm I
2.3.2. Pharmacophore study
2.3.2.1. Generation of 3D-pharmacophore model
The main objective of this technique was to generate 3D pharmacophore models based on the
known SU agonists, which can correctly reflect the SAR of the existing SU agonists. Then, this
generated model was used as 3D search query for evaluating the synthesized compounds and
identify SU agonistic activities. Ten hypotheses were generated (Table 3). All of them have cost
difference more than 60 bits and correlation coefficient values higher than 0.87. Cost difference
is the difference between the total cost of the null hypothesis and total cost of the generated
hypothesis. In general if the cost difference higher than 60 bits, there is an excellent chance
(more than 90 %) the model represents a true correlation. Hypo 1 total cost is the closet to the
fixed cost, RMS is the smallest and the correlation coefficient is the highest so that it was
selected as the best hypothesis and employed for further analyses. The null cost, fixed cost and
hypo 1 total cost values are 141.54, 48.60 and 68.09 bits respectively. Hypo 1 cost difference is
73.45, RMS is 1.88 and correlation coefficient is 0.91. All hypotheses except the tenth one
comprised the following features; two hydrogen bond acceptors (HBA) (some of them
comprised one hydrogen bond acceptor (HBA) and hydrogen bond acceptor (HBF)),
hydrophobic aliphatic (HA) and ring aromatic (RA) (Table 3).

Table 3. Information of statistical significance and predictive power of top 10 hypotheses as a
result of applying of HypoGen pharmacophore generation to the training set.
Cost
difference a
Hypo no. Total cost
RMSb
1.88
1.91
1.99
2.10
2.13
2.16
2.13
2.17
Correlation
0.91
Featuresc
Max. fit
8.36
7.74
8.43
8.34
7.31
8.78
6.65
8.17
HBA, HBA, HA,
RA
68.09
68.67
70.64
72.84
73.66
74.19
74.28
74.42
73.45
Hypo 1
Hypo 2
Hypo 3
Hypo 4
Hypo 5
Hypo 6
Hypo 7
Hypo 8
HBA, HBA, HA,
RA
72.87
70.90
68.70
67.88
67.35
67.26
67.12
0.91
HBA, HBA,
HBA, HA
HBA, HBA,
HBD, HA
HBA, HBA, HA,
RA
0.90
0.89
0.89
HBA, HBD, HA,
RA
0.88
HBA, HBA,
HBD, HA
HBA, HBA,
HBA, HA
HBA, HBA, HA,
RA
0.89
0.88
74.43
74.78
67.11
66.76
2.17
2.16
0.88
0.88
8.03
5.06
Hypo 9
HBD, HA, RA
Hypo 10
a Cost difference is the difference between null cost and total cost. Null cost = 141.54, fixed cost
= 48.60, configuration cost = 21.44, cost unit is bit.
b
RMS, root mean square deviation, the deviation of log estimated activities from log
experimental activities normalized by log uncertainty.
c Abbreviation used for features: HBA, hydrogen bond acceptor; HBD, hydrogen bond donor;
HA, hydrophobic aliphatic; RA, ring aromatic.

Hypo 1 consists of hydrophobic aliphatic (HA), ring aromatic (RA) and two hydrogen
bond acceptors (HBA) (Fig. 7). The spatial arrangement and the distances between the
pharmacophore features of hypo 1 are demonstrated in Fig. 8. The results of this study
confirmed the reliability of the generated pharmacophore model.
Fig. 7: The best hypothetical 3D-pharmacophore geometry (Hypo 1) with four features; two H-
bond acceptors (green color), hydrophobic aliphatic (blue), aromatic ring (brown).

Fig. 8: 3D spatial relationship and geometric parameters of Hypo1. The distance between
Pharmacophore features is reported in angstroms.
The Hypo1 predictability was internally evaluated by using compounds of training set
divided on the basis of their activity scale in to; highly active (+++, 1/C < 10 µM), moderately
active (++, 10 µM ≤ 1/C < 100 µM) and inactive (+, 1/C ≥ 100 µM). The validation results
indicated that there is no inconsistency between experimental and predicted activity values for all
compounds. Table 4 shows the experimental and estimated 1/C values of training set molecules
with error values, fit values and matching degrees with the pharmacophore Hypo-1.
Table 4. Experimental and estimated activities of the training set compounds based on
pharmacophore model hypo 1.
Comp.
25a
25b
25c
Exp.a (µM) Estb (µM)
Exp. scalec Est. scalec
Fit valued
Errore
1.60
12.50
3.40
2.36
11.40
2.34
+++
++
+++
+++
+++
++
+
+++
++
+++
+++
+++
+
7.77
1.47
7.09
-1.10
-1.46
-1.32
1.17
7.78
3.10
2.34
7.78
25d
25e
2.40
2.81
7.70
33.30
100.00
1000.00
33.30
33.30
136.73
131.07
139.44
30.91
45.36
6.01
4.11
26a
26b
26c
+
6.03
1.31
+
+
6.00
-7.17
-1.08
1.36
++
++
++
++
6.66
26d
26e
6.49
a Experimental activity value
b Estimated activity value.
c
Activity scale: 1/C < 10 µM = +++ (highly active); 10 µM ≤ 1/C > 100 µM = ++ (moderately
active); 1/C ≥ 100 µM = + (low active).
d
Fit value calculated by geometry fitting between the hypothesis and the compound; the higher
value, the better fit.

e
Error factor calculated as the ratio of the measured activity to the estimated activity or the
inverse if the estimated activity is greater than the measured activity. If measured activity is
greater than the estimated activity, the error factor is negative and vice versa.
2.3.2.2. Validation of Hypo 1 pharmacophore model
This step was carried out in order to examine the ability of the developed model to identify
active structures and predict their activities precisely. Two methods were applied to validate the
hypo 1, test set activity prediction and mapping of glimepiride as an external reference of the
generated pharmacophore.
2.3.2.2.1. Test set activity prediction
Test set of seven compounds was prepared using the same protocol as training set, then used to
verify the ability of the selected pharmacophore model (hypo 1) to predict the activities of the
new chemical compounds. Results demonstrated that hypo 1 is able to predict activities and
differentiate between the active and inactive compounds correctly. Values of the experimental
and estimated activities of the test set compounds are listed in Table 5.
Table 5: Experimental and estimated activities of the test set compounds based on
pharmacophore model hypo 1.
Comp.
25f
Exp.a (µM)
20.00
7.14
Est.b(µM)
2.30
Fit valuec
7.78
3.36
7.62
25g
25h
26f
2.04
5.32
7.42
NAd
128.93
134.90
134.03
4.31
6.03
NAd
6.02
26g
26h
27
NAd
6.02
4.00
7.51
a Experimental activity value
b Estimated activity value.
c Fit value indicates how well the features in the pharmacophore overlap the chemical features in
the molecule.
d NA: not active

2.3.2.2. 2. Mapping of glimepiride
Mapping of glimepiride as a reported external reference of the generated pharmacophore was
demonstrated in fig. 9. It was found that the Fit value of glimepiride according to hypo 1 is high
(7.51).
Fig. 9: Mapping of glimepiride on the generated high ranked pharmacophore (Hypo1)
2.3.2.3. Estimation of designed molecules
The valid 3D pharmacophore was used to estimate the activities of the designed molecules to
choose the best fitted compounds. The designed molecules were prepared and their activities
were estimated according to the fitting with Hypo1. Estimated activities and fit values of the
designed compounds were listed in Table 6. The result revealed that most of the designed
compounds are promising as SUR agonists, and the most promising compounds are arranged in
descending order as; 15b, 19a, 15a, 15d, 19b, 19e, 19d, 24a and 15e. Mapping of four designed
compounds (15b, 15a, 19d, and 24a) on the generated pharmacophore was demonstrated in Fig. 10,
11, 12 and 13, respectively.

Table 6: Expected activities and fit values of designed compounds based on the Hypo 1
pharmacophore model
Comp.
15a
Est.a (µM)
Fit valueb
7.61
Comp.
19c
Est.a (µM)
184.34
7.46
Fit valueb
5.88
3.42
2.55
7.74
7.28
15b
15c
19d
19e
126.34
3.42
6.05
5.57
7.40
7.61
184.70
7.68
5.88
15d
15e
19f
9.92
7.15
7.26
24a
102.67
2.82
6.14
118.91
184.58
6.07
15f
24b
24c
7.70
5.88
19a
4.84
7.46
19b
a Estimated activity value.
b Fit value indicates how well the features in the pharmacophore overlap the chemical features in
the molecule.
Fig. 10: Mapping of compound 15b (fit value = 7.74) on the generated high ranked
pharmacophore (Hypo1)

Fig. 11: Mapping of compound 15a (fit value = 7.61) on the generated high ranked
pharmacophore (Hypo1)
Fig. 12: Mapping of compound 19d (fit value = 7.28) on the generated high ranked
pharmacophore (Hypo1)

Fig. 13: Mapping of compound 24a (fit value = 7.26) on the generated high ranked
pharmacophore (Hypo1)
2.4. Structure Activity Relationship (SAR)
The newly synthesized compounds were designed to comprise the basic structural
requirements of PPARγ and SUR agonists. Observing biological results, we could deduce
valuable data about the structure activity relationships.
Firstly, the substitution on the sulfonylurea or sulfonylthiourea moieties was explored.
The increased % reduction in blood glucose level values in addition to decreased both IC50 and
EC50 of compounds 15a, 19a and 24a, with incorporated cyclohexyl moiety, than those of their
corresponding members 15c, 19c and 24c with incorporated phenyl moiety, indicated that
substitution with alicyclic moiety is advantageous. Moreover, substitution of butyl group (open
chain aliphatic) resulted in slight decrease of activity comparable to alicyclic substitution, as for
compounds 15b, 19b and 24b.
Comparing the anti-hyperglycemic activities of sulfonylurea and sulfonylthiourea
derivatives showed that, sulfonylureas were more active than sulfonylthioureas. In addition,
sulfonylurea derivatives showed insulin-secreting activities higher than that of sulfonylthioureas.
Interestingly, sulfonylthioureas were more active than sulfonylureas as PPARγ binders.
The increased % reduction in blood glucose level values of compounds 15a-f, with
incorporated 3-methylquinoxalin-2(1H)-one moiety, than those of their corresponding members
19a-f with incorporated 3,4-dihydroquinoxalin-2(1H)-one moiety, indicated that substitution with
3-methylquinoxalin-2(1H)-one moiety is more preferred biologically.
Finally, the effect of structural modification on the linker between lipophilic tail and an
aromatic scaffold (spacer group) has been investigated. Comparing the anti-hyperglycemic
activities of compounds 19a-c with that of compounds 24a-c, it was found that changing the
position of carbonyl group of the linker did not affect the anti-hyperglycemic activity.

3. Conclusion
In summary, fifteen novel quinoxaline-based derivatives have been designed and synthesized
incorporating the substantial sulfonylurea and sulfonylthiourea moieties with the aim of targeting
the PPARγ and SURs. The synthesized compounds were evaluated in-vivo for their anti-
hyperglycemic activities against STZ induced hyperglycemic rats. It was found that many
compounds showed good anti-hyperglycemic activities. Two additional in-vitro studies were
carried out for the most potent anti-hyperglycemic agents to estimate PPARγ binding affinity and
insulin-secreting ability as potential mechanisms for anti-hyperglycemic activity. Some of the
synthesized compounds (15a, 15b, 15d and 15e) significantly bound to PPARγ with IC50 values
ranging from 0.350 to 0.491 µM. Moreover, Compounds 15a and 15b have exhibited potent
insulin-secreting activities with EC50 values of0.92 and 0.98 µM, respectively. Docking studies
was carried out to investigate their binding pattern with the prospective target, PPARγ.
Pharmacophore studies were performed to justify the biological activities of the synthesized
compounds against SURs. The obtained data from molecular modeling studies showed that most
of the synthesized compounds had considerable high affinity towards PPARγ and SURs. The
most active candidates may serve as useful lead compounds in search for powerful and selective
anti-hyperglycemic agents.
4. Materials and methods
4.1. Chemistry
All melting points were measured on a Gallenkamp melting point apparatus and were
uncorrected. The IR spectra were recorded on a Pye-Unicam SP-3-300 infrared
1
spectrophotometer (potassium bromide dicks) and expressed in wave number (cm-1). HNMR
spectra were run at 400 MHz, on a Varian Mercury VX-300 and Bruker Avance III NMR
13
spectrometer respectively, while C NMR spectra were run at 100 MHz. TMS was used as an
internal standard in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts (δ) are quoted
in ppm. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. All coupling
constant (J) values are given in hertz. The mass spectra were recorded on Shimadzu GCMS-QP-
1000EX mass spectrometer at70 eV. Elemental analyses were performed on CHN analyzer and
all compounds were within ± 0.4 of the theoretical values. The reactions were monitored by thin-
layer chromatography (TLC) using TLC sheets coated with UV fluorescent silica gel Merck 60
F254 plates and were visualized using UV lamp and different solvents as mobile phases. All

reagents and solvents were purified and dried by standard techniques. All the newly synthesized
compounds gave satisfactory elemental analysis. Compounds 11, 12, 13, 17, 21 were prepared
according to reported methods [31, 32, 34, 35].
4.1.1. 2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)-N-(4-sulfamoylphenyl) acetamide (14)
A mixture of the potassium salt of 3-methylquinoxalin-2(1H)-one 12 (4.00 g, 20.17 mmol),
2-Chloro-N-(4-sulfamoylphenyl)acetamide 13 (5.00g, 20.17 mmol) and KI (0.10 g, 0.60 mmol)
in DMF (40 ml) was heated on a water bath for 20 h. After cooling, the reaction mixture was
poured on crushed ice. The formed precipitate was collected by filtration, dried and washed by
boiled absolute ethanol to afford compound 14.
o
Yellow powder (yield 76 %, 5.7 g); m.p. 290-293 C; IR (KBr, υ cm-1): 3327, 3258 (NH2), 3118
(C-H aromatic), 1657 (C=O amide), 1601 (C=N quinoxaline), 1534 (amide II band), 1306, 1152
1
(SO2); H NMR (DMSO-d6, 400 MHz) δ (ppm): 2.47 (s, 3H, CH3- quinoxaline), 5.15 (s, 2H,
CH2), 7.24 (s, 2H, D2O exchangeable, NH2), 7.33-7.80(m, 4H, Ar-H quinoxaline), 7.51 (d, J =
6.0 Hz, 2H, Ar-H phenyl),7.73 (d, J = 6.0 Hz, 2H, Ar-H phenyl), 10.77 (s, 1H, D2O
exchangeable, NH); 13C NMR (DMSO-d6, 100MHz) δ(ppm): 21.55 (CH3), 45.76 (CH2), 115.14,
119.22, 119.31, 123.91, 127.25, 129.25, 130.14, 132.42, 133.40, 139.16, 141.90, 154.81, 157.92,
165.92; DEPT spectrum differentiated 13C signals to be; one CH3, one CH2 and six CH; MS
(m/z): 372 (M+, 1.81 %), 202 (11.37 %), 201 (100 %, base peak), 173 (23.89 %), 146 (15.84 %),
145 (72.83), 92 (11.30 %), 77 (14.77 %); Anal. Calcd. for C17H16N4O4S (372.40): C, 54.83; H,
4.33, N, 15.05; S: 8.61; Found: C, 55.06; H, 4.37; N, 15.27; S, 8.68.
4.1.2. General procedure for synthesis of compounds (15a-f)
A mixture of of 2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)-N-(4-sulfamoy lphenyl)acetamide 14
(0.75g, 2.00 mmol) and anhydrous potassium carbonate (0.55 g, 4.00 mmol) in dry acetone (150
ml) was stirred at refluxing temperature for 1.5 h then cooled to room temperature. Next, the
appropriate isocyanates or isothiocyanates namely, cyclohexyl isocyanate, butyl isocyanate,
phenyl isocyanate, cyclohexyl isothiocyanate, butyl isothiocyanate, and phenyl isothiocyanate
(2.20 mmol) was added to the reaction mixture. Refluxing with stirring was continued for 24 h.
Acetone was removed by evaporation under reduced pressure, and then water (200 ml) was
added to dissolve the resulting residue. The solution was acidified with 1.0 N aqueous

hydrochloric acid (10 ml) and the resulted residue was collected by filtration. Crystallization of
the filter cake from 90% aqueous ethanol yielded purified corresponding sulfonyl(thio)urea
derivatives 15a-f respectively.
4.1.2.1. N-{4-[N-(Cyclohexylcarbamoyl)sulfamoyl]phenyl}2-(3-methyl-2-oxoquinoxalin-1(2H)-
yl) acetamide (15a)
White crystals (yield 69.85 %); m.p. 172-175 ˚C; IR (KBr, υ cm-1): 3281, 3205 (3NH,
overlapped), 3075 (C-H aromatic), 2932, 2861 (C-H aliphatic), 1676 (C=O amide), 1602 (C=N
quinoxaline), 1535 (amide II band) and 1332, 1161 (SO2);1H NMR (DMSO-d6, 400 MHz) δ
(ppm): 1.03-1.55 (m, 10H, cyclohexyl), 2.47 (s, 3H, CH3- quinoxaline), 3.42 (m, 1H, CH
cyclohexyl), 5.16 (s, 2H, COCH2), 6.33 (d, J = 7.5 Hz, 1H, D2O exchangeable, NH-cyclohexyl),
7.33 (dd, J = 7.8, 7.2 Hz, 1H, Ar-H- quinoxaline),7.48 (d, J = 7.8 Hz, 1H, Ar-H- quinoxaline),
7.52 (dd, J = 8.1, 7.2 Hz, 1H, Ar-H quinoxaline), 7.77 (d, J = 8.1 Hz, 1H, Ar-H quinoxaline),
7.73 (d, J = 8.7 Hz, 2H, Ar-H phenyl), 7.82 (d, J = 8.7 Hz, 2H, Ar-H phenyl), 10.26 (s, 1H, D2O
exchangeable, SO2NH), 10.89 (s, 1H, D2O exchangeable, NH amide);13C NMR (DMSO-d6, 100
MHz) δ (ppm): 21.55, 24.59, 25.43, 32.70, 45.77, 48.46, 115.11, 119.09, 123.91, 129.08, 129.25,
13
130.17, 132.42, 133.37, 134.82, 143.08, 150.93, 154.79, 157.92, 166.08; DEPT classified C
signals into; four CH2, one CH3 and seven CH; Anal. Calcd. for C24H27N5O5S (497.57): C,
57.93; H, 5.47; N, 14.08; S, 6.44; Found: C, 58.17; H, 5.54; N, 14.26; S, 6.51.
4.1.2.2. N-{4-[N-(Butylcarbamoyl)sulfamoyl]phenyl}2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)
acetamide (15b)
White crystals (yield 68.45 %); m.p. 188-190 ˚C; IR (KBr, υ cm-1): 3274, 3196 (3NH,
overlapped), 3105 (C-H aromatic), 2948, 2876 (C-H aliphatic), 1633 (C=O amide), 1601 (C=N
1
quinoxaline), 1535 (amide II band) and 1334, 1160 (SO2); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 0.81 (t, J = 7.5 Hz, 3H, CH3 butyl), 1.19 (m, 2H, CH2CH3), 1.30 (m, 2H, CH2CH2CH3),
2.92 (dt,J = 6.0, 6.3 Hz, 2H, CH2CH2CH2CH3), 2.48 (s, 3H, CH3- quinoxaline), 5.16 (s, 2H,
COCH2), 6.32 (s, 1H, D2O exchangeable, NH-butyl),7.33 - 7.77 (m, 4H, Ar-H quinoxaline), 7.71
(d, J = 8.7 Hz, 2H, Ar-H), 7.82 (d, J = 8.7 Hz, 2H, Ar-H), 10.81 (s, 1H, D2O exchangeable, NH
amide).13C NMR (DMSO-d6, 150 MHz) δ (ppm): 13.97, 18.75, 21.52, 31.75, 39.27, 45.72,
114.96, 119.15, 123.96, 129.00, 129.25, 130.18, 132.40, 133.24, 135.06, 142.98, 152.08, 154.81,

157.93, 166.03; DEPT showed four CH2, two CH3 and six CH; Anal. Calcd. for C22H25N5O5S
(471.53): C, 56.04; H, 5.34; N, 14.85; S, 6.80; Found: C, 56.32; H, 5.37; N, 14.98; S, 6.87.
4.1.2.3. N-{4-[N-(Phenylcarbamoyl)sulfamoyl]phenyl}2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)
acetamide (15c)
White crystals (yield 60.61 %); m.p. 229-231 ˚C; IR (KBr, υ cm-1): 3332 (br, 3NH, overlapped),
3107 (C-H aromatic), 1652 (C=O amide), 1604 (C=N quinoxaline), 1538 (amide II band) and
1327, 1160 (SO2);1H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.47 (s, 3H, CH3- quinoxaline), 5.16
(s, 2H, COCH2), 6.57-7.93 (m, 13H, Ar-H), 7.22 (s, 1H, D2O exchangeable, NH-phenyl), 8.91 (s,
1H, D2O exchangeable, SO2NH), 10.91 (s, 1H, D2O exchangeable, NH amide).13C NMR
(DMSO-d6, 150 MHz) δ (ppm): 21.54, 45.73, 119.17, 119.33, 123.68, 123.99, 129.24, 129.29,
129.34, 130.24, 132.38, 133.26, 134.45, 138.42, 143.27, 149.84, 154.81, 157.95, 166.09; DEPT
revealed one CH2, one CH3 and nine CH; Anal. Calcd. for C24H21N5O5S (491.52): C, 58.65; H,
4.31; N, 14.25; S, 6.52; Found: C, 58.82; H, 4.29; N, 14.37; S, 6.60.
4.1.2.4. N-{4-[N-(Cyclohexylcarbamothioyl)sulfamoyl]phenyl}2-(3-methyl-2-oxoquinoxalin-
1(2H)-yl)acetamide (15d)
Yellow crystals (yield 62.84 %); m.p. 160-162 ˚C; IR (KBr, υ cm-1): 3484, 3322, 3236 (3NH),
3047 (C-H aromatic), 2931, 2863 (C-H aliphatic), 1670 (CO amide), 1605 (C=N quinoxaline),
1527 (amide II band) and 1316, 1124 (SO2);1H NMR (DMSO-d6, 300 MHz) δ (ppm): 1.23-1.88
(m, 10H, cyclohexyl), 2.48 (s, 3H, CH3- quinoxaline), 3.94 (m, 1H, CH cyclohexyl), 5.17 (s, 2H,
COCH2), 7.33-7.77 (m, 4H, Ar-H quinoxaline),7.76-7.86 (m, 4H, Ar-H phenyl), 8.23 (d, J = 6.9
Hz, 1H, D2O exchangeable, NH-cyclohexyl), 10.93 (s, 1H, D2O exchangeable, NH amide), 11.26
(s, 1H, D2O exchangeable, SO2NH); Anal. Calcd. for C24H27N5O4S2 (513.63): C, 56.12; H, 5.30;
N, 13.64; S, 12.48; Found: C, 56.31; H, 5.36; N, 13.80; S, 12.56.
4.1.2.5. N-{4-[N-(butylcarbamothioyl)sulfamoyl]phenyl}2-(3-methyl-2-oxoquinoxalin-1(2H)-
yl)acetamide (15e)
Yellow powder (yield 61.10 %); m.p. 140-143 ˚C; IR (KBr, υ cm-1): 3321, 3192 (3NH,
overlapped), 3103 (C-H aromatic), 2947 (C-H aliphatic), 1653 (C=O amide), 1597 (C=N
quinoxaline), 1535 (amide II band) and 1393, 1158 (SO2);1H NMR (DMSO-d6, 400MHz) δ
(ppm): 0.80 (t, J = 7.2 Hz, 3H, CH3 butyl), 1.15 (m, 2H, CH2CH3), 1.41 (m, 2H, CH2CH2CH3),
2.46 (s, 3H, CH3- quinoxaline), 3.39 (dt,2H, CH2CH2CH2CH3), 5.17 (s, 2H, COCH2), 7.34-7.79
(m, 4H, Ar-H quinoxaline), 7.77 (d, J = 8.4 Hz, 2H, Ar-H phenyl),7.86 (d, J = 8.4 Hz, 2H, Ar-H