Anti-glycemic potential of benzophenone thio/semicarbazone derivatives: synthesis, enzyme inhibition and ligand docking studies

Article abstract of DOI:10.1080/07391102.2021.1897045

Inhibition of dipeptidyl peptidase-IV (DPP-IV) has been identified as a promising approach for the treatment of type 2 diabetes mellitus (T2DM). Therefore, development of DPP-IV inhibitors with new chemical scaffold is of utmost importance to medicinal chemistry. In the present study, we identified benzophenone thio- and semicarbazone scaffolds as novel DPP-IV inhibitors. For that purpose, benzophenone thio- and semicarbazone were synthesized through a 2-step reaction. These newly synthetic derivatives were characterized by different spectroscopic techniques, including HREI-MS and NMR. whereas stereochemistry of the iminic bond was predicted by NOESY experiments. Thio- and semicarbazones derivatives were evaluated for their DPP-IV inhibitory potential and found to exhibit a good to moderate enzyme inhibitory activity. Most active and non-cytotoxic derivatives were further evaluated for their DPP-IV inhibitory potential in in cellulo model. The binding sites as well as affinity of active compounds for DPP- IV enzyme were predicted by in silico studies, and compared to a standard drug, sitagliptin. Pharmacophore studies of thio- and semicarbazones derivatives 1–29 suggest that substitution of aryl group, particularly a lipophilic substituents at C-4″ of benzene ring, and a hydroxyl at C-4′ strongly influenced the DPP-IV inhibitory activity. Compound 9 showed the highest inhibitory activity (IC₅₀ = 15.0 ± 0.6 μM), whereas compounds 10, 17, 12, 14 and 23 showed a moderate activity with IC₅₀ values in the range of 28.9–39.2 μM. This study identifies thio- and semicarbazones as new classes of DPP-IV inhibitors which may translate into safe and effective therapeutics for a better management of type 2 diabetes. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2021.1897045

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Anti-glycemic potential of benzophenone thio/
semicarbazone derivatives: synthesis, enzyme
inhibition and ligand docking studies
Arshia, Sharmeen Fayyaz, Muniza Shaikh, Khalid Mohammed Khan & M.
Iqbal Choudhary
To cite this article: Arshia, Sharmeen Fayyaz, Muniza Shaikh, Khalid Mohammed Khan & M.
Iqbal Choudhary (2021): Anti-glycemic potential of benzophenone thio/semicarbazone derivatives:
synthesis, enzyme inhibition and ligand docking studies, Journal of Biomolecular Structure and
Dynamics, DOI: 10.1080/07391102.2021.1897045
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2021.1897045
Anti-glycemic potential of benzophenone thio/semicarbazone derivatives:
synthesis, enzyme inhibition and ligand docking studies
a#
b#
b
a,c
a,b,d
Arshia , Sharmeen Fayyaz , Muniza Shaikh , Khalid Mohammed Khan and M. Iqbal Choudhary
a
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan;
b
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of
c
Karachi, Karachi, Pakistan; Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman
Bin Faisal University, Dammam, Saudi Arabia; Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga,
d
Surabaya, Indonesia
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Inhibition of dipeptidyl peptidase-IV (DPP-IV) has been identified as a promising approach for the
treatment of type 2 diabetes mellitus (T2DM). Therefore, development of DPP-IV inhibitors with new
chemical scaffold is of utmost importance to medicinal chemistry. In the present study, we identified
benzophenone thio- and semicarbazone scaffolds as novel DPP-IV inhibitors. For that purpose, benzo-
phenone thio- and semicarbazone were synthesized through a 2-step reaction. These newly synthetic
derivatives were characterized by different spectroscopic techniques, including HREI-MS and NMR.
whereas stereochemistry of the iminic bond was predicted by NOESY experiments. Thio- and semicar-
bazones derivatives were evaluated for their DPP-IV inhibitory potential and found to exhibit a good
to moderate enzyme inhibitory activity. Most active and non-cytotoxic derivatives were further eval-
uated for their DPP-IV inhibitory potential in in cellulo model. The binding sites as well as affinity of
active compounds for DPP- IV enzyme were predicted by in silico studies, and compared to a standard
drug, sitagliptin. Pharmacophore studies of thio- and semicarbazones derivatives 1–29 suggest that
Received 26 November 2020
Accepted 24 February 2021
KEYWORDS
Type 2 diabetes mellitus;
dipeptidyl peptidase-IV
(
DPP-IV); thiosemicarbazone;
benzophenone; Caco-2
cell line
00
substitution of aryl group, particularly a lipophilic substituents at C-4 of benzene ring, and a hydroxyl
0
at C-4 strongly influenced the DPP-IV inhibitory activity. Compound 9 showed the highest inhibitory
activity (IC50 ¼ 15.0 ± 0.6 mM), whereas compounds 10, 17, 12, 14 and 23 showed a moderate activity
with IC50 values in the range of 28.9–39.2 mM. This study identifies thio- and semicarbazones as new
classes of DPP-IV inhibitors which may translate into safe and effective therapeutics for a better man-
agement of type 2 diabetes.
1
. Introduction
et al., 2018). DPP-IV is, therefore, an important metabolic
enzyme, involved in the regulation of glucose levels in
serum. It cleaves incretin hormones to their inactive forms
which results in an increased level of serum glucose. DPP-IV
inhibitors control the glucose level in blood by increasing
insulin secretion. Inhibition of DPP-IV, is therefore, an effect-
ive strategy for the control of hyperglycemia in T2DM (Xie
et al., 2017). Given the increasing prevalence of type 2 dia-
betes and the substantial social and financial costs associated
with disease management, every effort should be made for
the prevention and treatment. Over the past years, several
DPP-IV inhibitors, such as vildagliptin, sitagliptin, saxagliptin,
etc. have been developed. Current DPP-IV inhibitors belong
to different chemical classes, however these inhibitors pos-
Type 2 diabetes mellitus (T2DM) is a complex chronic meta-
bolic disease that is progressive in nature. This disease has
acquired an epidemic proportion worldwide in recent years.
According to an estimate of World Health Organization
(
WHO), approximately 422 million people in the world are
suffering from diabetes and this number will rise up to 592
million by 2035 (Song et al., 2017). In view of the increasing
burden of diabetes and health risks associated with the dis-
ease, this is imperative to develop effective, and safe treat-
ments of type 2 diabetes. During the past decade different
drugs for the management of type 2 diabetes have been
developed, such as inhibition of a-glucosidase enzyme, of
anti-glycation drugs, and incretin hormones targeting drugs.
Incretins are gut hormones secreted in response to meal sess primary amine moiety which is responsible for potent
ingestion and triggers glucose-dependent insulin secretion. DPP-IV inhibition. Unfortunately, these inhibitors suffer some
However, these hormones are instantly degraded by an disadvantages, such as short biological half-life (vildagliptin)
endogenous enzyme, dipeptidyl peptidase-IV (DPP-IV) (Kato or a need of larger doses (sitagliptin). Occurrence of
CONTACT M. Iqbal Choudhary
Sciences, University of Karachi, Karachi 75270, Pakistan
These authors contribute equally in this work and shared first authorship.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2021.1897045
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
iqbal.choudhary@iccs.edu
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
#

2
ARSHIA ET AL.
Scheme 1. Synthesis of benzophenone thio- and semicarbazones.
pancreatitis and pancreatic cancer is also observed with
NMR spectra were recorded on Avance Bruker AM 300
administration of DPP-IV inhibitors (Baig et al., 2019; and 400 MHz spectrometers. EI-MS were run on a Finnigan
Prabahar et al., 2020). This encouraged investigation of the MAT-311A (Germany) mass spectrometer. Melting points of
V
R
chemical space for the identification of lead molecules the compounds were recorded on Stuart SMP10 melting
against this clinically important enzyme with lower side point apparatus. IR spectra (KBr discs) were recorded on a
effects, and increased inhibitory potential.
FTS 3000 MX, Bio-RAD Merlin (Excalibur Model) spectropho-
tometer. Thin layer chromatography (TLC) analyses were per-
formed on pre-coated silica gel aluminum plates (Kieselgel
The cost effective synthesis of thio- and semicarbazones
derivatives and their importance in drug designing have
attracted the attention of many researchers. These are
known to possess many important biological activities, such
as antimicrobial, antimalarial (Kpomah, 2017), antifungal, anti-
cancer (Divar et al., 2017), anti-inflammatory, antioxidant,
antitumor (Kalaiarasi et al., 2017), antituberculosis (Kaplancı
klı et al., 2016), antiepileptic (Rajak et al., 2017), antiprolifera-
tive (Zaltariov et al., 2017), antiglycating, antioxidant, (Arshia
et al., 2020) and antileishmanial properties (Silva & Silva,
6
0, 254, E. Merck, Germany). TLC chromatograms were visual-
ized under ultraviolet lamp at 254 and 365 nm.
2
.2. Synthesis of thio- and semicarbazone derivatives
Synthesis and characterization of compounds 1–8, 15–23,
25–27 and 29 are reported previously by Arshia et al. (2016),
and same methodology is used for the synthesis of com-
pounds 9–14, 24 and 28. Briefly, benzophenone semicarba-
zones and thiosemicarbazones were synthesized by refluxing
benzophenone hydrazones and substituted aryl isocyanates
or isothiocyanates in acetonitrile for 24 h (Scheme 1).
Completion of reaction was confirmed by TLC analysis.
Synthetic mixtures were then left at room temperature for
precipitation. The precipitates were filtered and dried under
2
017). There are several studies demonstrating the anti-dia-
betic effect of thiosemcarbazones as aldose reductase and
glycogen phosphorylase inhibitors (Alexacou et al., 2010;
Kulkarni et al., 2012; Shehzad et al., 2019). Recently, Sever
et al. (2020) reported the DPP-IV inhibitory effect of pyrazole
incorporated thiosemicarbazone derivatives.
In the present study, we identified benzophenone-based
thio- and semicarbazone as DPP-IV inhibitors by high
throughput screening. In situ cellular assay using Caco-2 cell
line further indicates the potential of identified leads in com-
plex physiological environment. Inhibitors identified during
this research can serve as leads for further research in this
important field.
ꢀ
vacuum at 40 C to afford good yields of the desired com-
pounds. The compounds, were crystallized from ethanol.
2
2
.2.1. Characteristics spectral features of synthesized
derivatives
.2.1.1. (E)-N-(2-Chlorophenyl)-2-((4-hydroxyphenyl)(phe-
nyl)methylene)hydrazinecarbothioamide (9). Yield: 75%;
2
. Experimental
ꢀ
1
m.p. 177–179 C; R : 0.35 (ethyl acetate/hexanes, 2:8); H NMR
f
2.1. Material and methods
(
400 MHz, DMSO-d ): d 10.34 (d, 1H, NH)), 10.04 (d, 1H, NH),
6 H
9
.09 (d, 1H, OH), 7.71 (d, 2H, J
¼ 7.6 Hz, H-2, H-6), 7.66
¼ 7.6 Hz, H-3, H-5),
2
,3/6,5
Acetic acid, hydrazine hydrates, benzophenones and different
aryl isocyanates/isothiocyantes were purchased from Sigma
Aldrich, USA. Acetonitrile was dried by charging it with 3 Å
molecular sieves. Recombinant human DPP-IV (EC 3. 4. 14. 5)
was purchased from Prof. Dr. Mark D. Gorrell, Sydney,
Australia. Sitagliptin phosphate monohydrate (S4002) was
purchased from Selleck Chemicals (TX, USA). Gly-pro-pNA
00
(m, 2H, H-4, H-5 ), 7.56 (t, 2H, J
3(2,4)/5(4,6)
0
0
0
0
7
.40 (m, 4H, H-2 , H-3 , H-5 , H-6 ), 7.21 (d, 1H, J
¼ 6.8 Hz,
4(3,5)
00
H-4), 7.02 (d, 1H,
J
00 00 ¼ 8.8 Hz, H-6 ), 6.77 (d, 1H,
6 ,5
0
0
þ
J
00 00 ¼ 8.8 Hz,
H-3 );
FAB-MS
m/z
382
[M þ H] ,
3 ,4
þ
3
84 [M þ H þ 2] .
was obtained from LeapChem Ltd. (Hangzhou, China). 2.2.1.2. (E)-N-(3-Chlorophenyl)-2-((4-hydroxyphenyl)(phe-
Dulbecco’s modified eagle medium (DMEM) was purchased nyl)methylene)hydrazine carbothioamide (10). Yield: 72%;
ꢀ
1
from Cassion Labs, USA. All solvents (DMSO and ethanol) m.p. 144–146 C; R
were of HPLC grade. (300 MHz, DMSO-d ): d 10.42 (d, 1H, NH), 10.06 (d, 1H, NH),
: 0.34 (ethyl acetate/hexanes, 2:8); H NMR
f
6
H

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
9
.04 (d, 1H, NH), 7.73 (d, 2H, J2,3/6,5 ¼ 7.6 Hz, H-2, H-6), 7.65
(
d, 1H, J
¼ 6.8 Hz, H-4), 7.57 (m, 2H, H-3, H-5), 7.41 (m,
4
(3,5)
0 0 0 0 00
4
H, H-2 , H-3 , H-5 , H-6 ), 7.28 (m, 1H, H-2 ), 7.20 (d, 1H,
00
00
J500 00 00 ¼ 9.0 Hz, H-5 ), 7.02 (d, 1H, J 00 00 ¼ 9.0 Hz, H-6 ), 6.78
(4 ,6 )
6 ,5
0
0
(
[
(
(
d, 1H, J 00 00 ¼ 9.0 Hz, H-4 ); EI-MS: m/z (rel. abund.%) 383
4
,5
þ þ
M þ 2] (15.5), 381 [M] (38.0), 348 (5.4), 304 (6.8), 254
100.0), 211 (55.3), 196 (100.0), 169 (8.0), 152 (13.7), 127
87.2), 92 (13.1), 77 (42.5), 51 (9.2); HREI-MS: m/z
þ
C H ClN OS [M] 381.0703, found 381.0702.
2
0
16
3
0
0
2
.2.1.3. (E)-N-(4 -Chlorophenyl)-2-((4-hydroxyphenyl)(phe-
Figure 1. Structural features of newly synthesized thio- and semi-carbazones
nyl)methylene)hydrazinecarbothioamide (11). Yield: 79%; derivatives.
ꢀ
1
m.p. 198–200 C; R : 0.33 (ethyl acetate/hexanes, 2:8); H NMR
f
(
400 MHz, DMSO-d ): d 10.39 (d, 1H, NH), 10.05 (d, 1H, OH), 2.2.1.7. N-(4-Chloro-2-(trifluoromethyl)phenyl)-2-(diphenyl-
6
H
8
.97 (d, 1H, NH), 7.72 (d, 1H, J
¼ 6.8 Hz, H-4), 7.65 (m, 4H, methylene)hydrazinecarboxamide (24). Yield: 69%; m.p.
4
(3,5)
0
0
00
0
0
ꢀ
1
H-2, H-6, H-6 , H-2 ), 7.48 (m, 3H, H-6 , H-3 , H-5 ), 7.38 (t, 2H, 185–187 C; R : 0.38 (ethyl acetate/hexanes, 2:8); H NMR
f
00
J3(2,4)/5(4,6) ¼ 8.8 Hz, H-3, H-5), 7.20 (d, 1H, J 00 00 ¼ 8.0 Hz, H-5 ), (300 MHz, DMSO-d ): d 9.85 (s, 1H, NH), 9.37 (s, 1H, NH),
5 ,6
6
H
0
0
00
7
2
.02(d, 1H, J 00 00 ¼ 8.4 Hz, H-3 ), 6.77 (d, 1H, J 00 00 ¼ 8.4 Hz, H- 9.15 (s, 1H, OH), 8.15 (s, 1H, H-3 ), 7.76 (d, 2H,
3
,5
2 ,3
0
0
þ
þ
0
0
); EI-MS: m/z (rel. abund.%), 383 [M þ 2] (3.3), 381 [M]
J2,3 ¼ J ¼ 9.0 Hz, H-2, H-6), 7.56 (m, 5H, H-3, H-5, H-4, H-2 ,
6,5
0
0
(
5.5), 254 (35.0), 211 (11.3), 196 (100.0), 181 (20.8),169 (22.9), H-6 ), 7.29 (m, 1H, H-5), 7.15 (d, 2H, J
0
0
¼ J
0 0
5 ,6
¼ 8.7 Hz, H-3 ,
3
,2
00
1
27 (90.8), 106 (64.0), 77 (32.2), 65.0 (27.9); HREI-MS: m/z H-5 ), 6.95 (d, 1H
J
00 00 ¼ 8.4 Hz, H-6 ), 6.77 (d, 1H
6
,5
þ
13
00
Calcd C H ClN OS [M] 381.0703, found 381.0702.
J
1
00 00 ¼ 8.7 Hz, H-5 );
C NMR: (100 MHz, DMSO-d ): d
C
2
0
16
3
5 ,6
6
58.45, 153.00, 152.20, 149.71, 137.69, 135.18, 135.16, 133.20,
00
0
2
.2.1.4. (E)-N-(2 -Fluorophenyl)-2-((4 -hydroxyphenyl)(phe- 132.82, 130.13, 129.38, 128,74, 128.35, 127.55, 127.09, 125.98,
nyl)methylene)hydrazinecarbothioamide (12). Yield: 77%; 122.28, 116.12, 115.23, 115.20, 110.91; EI-MS: m/z (rel.
ꢀ
1
þ
þ
m.p. 120–122 C; R : 0.34 (ethyl acetate/hexanes, 2:8); H NMR abund.%): [M þ 2] 419 (3.3), [M] 417 (13.0), 221 (29.3), 211
f
(
400 MHz, DMSO-d ): d 10.23 (d, 1H, NH), 10.03 (d, 1H, NH), (56.7), 195 (100.0), 175 (63.3), 43 (74.4); HREI-MS: m/z Calcd
6 H
þ
9
.02 (d, 1H, OH), 7.73 (d, 1H, J4(3,5) ¼ 6.8 Hz, H-4), 7.66 (m, 2H, for C20
H16ClN O [M] 433.0805, found 433.0808; IR (KBr,
3 2
0
0
ꢁ1
H-2, H-6), 7.55 (d, 2H, J
0
0 0 0
,3 /6 ,5
¼ 8.8 Hz, H-2 , H-6 ), 7.41 (m, cm ): 3344 (O–H), 3278 (N–H), 1691 (C ¼ O), 1589 (C ¼ N),
2
0
0
00
4
7
H, H-3, H-5, H-3 , H-5 ), 7.26 (d, 1H, J 00 00 00 ¼ 8.8 Hz, H-5 ), 1589 (C ¼ C), 1311 (C–N), 839 (C–F).
5
(4 ,6 )
00
.02 (d, 1H, J 00 00 ¼ 8.8 Hz, H-6 ), 6.76 (d, 1H, J 00 00 ¼ 8.8 Hz,
6
,5
3 ,4
00
þ
00
00
0
H-3 ); EI-MS: m/z (rel. abund.%), 365 [M] (89.2), 332 (15.9), 2.2.1.8. (E)-N-[4 -Chloro-2 -(trifluoromethyl)phenyl]-2-[(4 -
2
88 (12.0), 254 (93.0), 211 (8.9),196 (100.0), 181 (22.5), 111 hydroxyphenyl)(phenyl)methylene] hydrazinecarboxamide
þ
ꢀ
(
59.5), 77 (29.0); HREI-MS: m/z Calcd C H FN OS [M]
(28). Yield: 69%; White solid; m.p. 172–174 C; R : 0.37 (ethyl-
2
0
16
3
f
1
3
65.0998, found 365.0997.
acetate/hexanes, 2:8); H NMR (300 MHz, DMSO-d ): d 9.85
6
H
(
s, 1H, NH), 9.37 (s, 1H, NH), 9.15 (s, 1H, OH), 8.15 (s, 1H, H-
0
0
00
2
.2.1.5. (E)-N-(3 -Fluorophenyl)-2-((4-hydroxyphenyl)(phe- 3 ), 7.76 (d, 2H, J2,3 ¼ J6,5 ¼ 9.0 Hz, H-2, H-6), 7.56 (m, 5H, H-3,
0
0
nyl)methylene)hydrazinecarbothioamide (13). Yield: 75%; H-5, H-4, H-2 , H-6 ), 7.29 (m, 1H, H-5), 7.15 (d, 2H,
ꢀ
1
0
0
m.p. 144–146 C; R : 0.34 (ethyl acetate/hexanes, 2:8); H NMR
J
3
0
0
,2
0
¼ J
5
0
,6
0
¼ 8.7 Hz, H-3 , H-5 ), 6.95 (d, 1H J
6
00,500 ¼ 8.4 Hz, H-
f
0
00
13
(
400 MHz, DMSO-d ): d 10.40 (d, 1H, NH), 10.04 (d, 1H, NH), 6 ), 6.77 (d, 1H J
.76 (d, OH), 7.72 (m, 3H, H-3, H-4, H-5), 7.55 (d, 2H, DMSO-d
5
00,600 ¼ 8.7 Hz, H-5 ); C NMR: (100 MHz,
6
H
0
8
J
2
6
): d
C
158.4 (C ¼ O), 152.2 (C ¼ N), 149.7 (C-4 ), 137.6
00
00
00
00
¼ J ¼ 6.6 Hz, H-2, H-6), 7.42 (d, 2H, J
0 0 0 0
¼ 6.3 Hz, H- (C-4 ), 135.1 (C-2 ), 133.2 (C-1 ), 130.1 (C-3 ), 129.3 (C-1),
2
0
,3
6,5
0
2 ,3 /6 ,5
00
00
00
00
, H-6 ), 7.36 (m, 2H, H-2 , H-5 ), 7.20 (d, 1H, J 00 00 ¼ 6.3 Hz, 128.7 (C-5 ), 128.5 (C-6 ), 128.3 (C-2, C-6), 127.5 (C-3, C-5),
4
,5
00
0
0
0
0
0
0
H-4 ), 7.06 (m, 2H, H-3 , H-5 ), 6.78 (d, 1H, J 00 00 ¼ 6.6 Hz, H- 127.0 (C-4), 125.9 (C-2 , C-6 ), 122.2 (C-1 ), 116.1 (2 -CF
),
6
,5
3
0
0
þ
0
0
þ
6
); EI-MS: m/z (rel. abund.%), 365 [M] (42.0), 288 (9.6), 254 115.2 (C-3 , C-5 ); EI-MS: m/z (rel. abund.%): [M þ 2] 435
þ
(
(
3
65.1), 211 (65.2), 196 (100.0), 152 (53.7), 120 (32.5), 111 (3.2), 433 [M] (9.2), 416 (1.4), 221 (29.3), 211 (56.7), 195
þ
94.3), 77 (96.8); HREI-MS: m/z Calcd C H FN OS [M]
65.0998, found 365.0997.
(100.0), 175 (63.3), 43 (74.4); HREI-MS: m/z Calcd for
C
2
0
16
3
þ
ꢁ1
H16ClN
O [M] 433.0805, found 433.0808; IR (KBr, cm ):
3 2
20
3
344 (O–H), 3278 (N–H), 1691(C ¼ O), 1589 (C ¼ N), 1589
1
00
.2.1.6. (E)-N-(2 -Bromophenyl)-2-((4-hydroxyphenyl)(phe-
(C ¼ C), 1311 (C–N), 839 (C–F). H NMR spectra of newly syn-
2
thetic derivatives are given in Supplementary Materials.
nyl)methylene)hydrazinecarbothioamide (14). Yield: 77%;
ꢀ
1
m.p. 158–160 C; R : 0.35 (ethyl acetate/hexanes, 2:8); H NMR
f
(
9
400 MHz, DMSO-d ): d 10.32 (d, 1H NH), 10.03 (d, 1H, OH),
6 H
.08 (d, 1H, NH), 7.71 (d, 2H, J2,3/6,5 ¼ 6.8 Hz, H-2, H-6), 7.66
2
.3. DPP-IV Inhibitory assay
(
m, 2H, H-3, H-5), 7.54 (d, 1H, J
¼ 8.4 Hz, H-4), 7.43 (m, DPP-IV inhibitory activity of compounds 1–29 was evaluated
4
(3,5)
0
0
0
0
00
4
H, H-2 , H-3 , H-5 , H-6 ), 7.25 (m, 2H, H-4, H-5 ), 7.02 (d, 1H, using gly-pro-pNA as chromogenic substrate. Release of p-
0
0
00
J600 00 ¼ 8.4 Hz, H-6 ), 6.77 (d, 1H, J 00 00 ¼ 8.4 Hz, H-3 ), 2.06 (s, nitroanilide, a light yellow colored compound, due to cleav-
,5
3 ,4
0
0
00
þ
þ
3
H, 2 -CH ); FAB-MS m/z 426 [M þ H] , 428 [M þ H þ 2] .
age of substrate by the DPP-IV enzyme was monitored.
3

4
ARSHIA ET AL.
Table 1. DPP-IV inhibitory potential of thio/semicarbazone derivatives 1–29.
was changed after two days, until cells reached confluence
Brandt et al., 2005).
(
2
.5. In situ Caco-2 cell DPP-IV inhibition assay
DPP-IV inhibition assay using Caco-2 cellular model was per-
formed with slight modifications, as described by Sadir et al.
(
2004). Caco-2 cells were used as a source of DPP-IV enzyme.
5
Caco-2 cells were seeded at a density of 1 ꢂ 10 cells per
well in a 96-well flat-bottom plate in 200 mL of Dulbecco’s
modified eagle medium. At confluency, cell culture medium
was removed and cells were washed with sterile phosphate
buffer, followed by the addition of 50 mL of test sample pre-
pared in HEPES buffer (20 mM, pH 7.4). Cells were incubated
a
Compounds
Structure
IC50 ± SEM (mM)
00
R
X
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
b
1
2
3
4
5
6
7
8
9
Phenyl
NA
0
0
3 -Chloro
NA
NA
NA
NA
NA
NA
0
0
00
2 ,5 -Dichloro
ꢀ
further at 37 C for 4 h, prior to the addition of the substrate
0
0
00
2 ,3 - Dichloro
0
0
00
Gly-Pro-pNA (0.2 mM). Enzymatic activity was determined by
monitoring the release of pNA in the supernatant with spec-
trophotometry at 400 nm using Spectramax M5 (Molecular
Devices, CA, USA) (Sadir et al., 2004).
3 ,4 -Dichloro
0
0
4 -Bromo
0
0
3 -Bromo
H
69.9 ± 1.01
15.0 ± 0.6
38.27 ± 1.84
79.0 ± 1.3
28.9 ± 0.32
73.0 ± 0.27
34.0 ± 2.06
49.6 ± 0.07
161.7 ± 6.47
39.2 ± 1.55
NA
00
2 -Chloro
00
3 -Chloro
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
00
4 -Chloro
00
2 -Fluoro
2
.6. Molecular docking studies
00
3 -Fluoro
00
2 -Bromo
For docking analysis, the X-ray crystal structure of human
DPP-IV (PDB ID 1 ꢂ 70), complexed with sitagliptin, was
retrieved from PDB. The crystal structure of the protein is a
homodimer of subunits A and B. The subunit A was retained
for protein preparation by Protein Preparation Wizard in
Maestro Schrodinger (2019). The co-crystallized water mole-
cules were deleted and the missing hydrogens were added.
Assignment of partial charges was carried out via OPLS3e
force field. Restrained minimization was carried out in order
to optimize the heavy atoms and hydrogens in the refine tab
of preparation wizard.
Compounds 9 and 14 were sketched in ChemDraw and
then converted to their respective 3D structures through the
Lig Prep module of Maestro (Schrodinger, 2019a). The com-
pounds were further corrected for their protonation and ion-
ization states in the same module by using OPLS3e
force field.
00
3 -Bromo
00
4 -Bromo
00 00
2 ,5 -Dichloro
S
S
S
00 00
2 ,3 -Dichloro
0
0
00
2 ,4 -Dichloro
H
NA
43.5 ± 0.44
NA
NA
O
O
O
O
O
O
O
O
O
O
00
H
H
H
H
3 -Chloro
3 -Trifluoromethyl
0
0
0
0
4 -Trifluoromethyl
37.46 ± 0.97
NA
66.04 ± 0.77
NA
87.5 ± 0.7
136.4 ± 2.89
59.37 ± 0.17
0.033 ± 0.4
0
0
0
0
2 -Trifluoromethyl-4 -chloro
0
0
00
H
2 -Chloro-5 -trifluoromethyl
00
OH
OH
OH
OH
3 -Chloro
4 -Trifluoromethyl
0
0
0
0
00
2 -Trifluoromethyl-4 -chloro
0
0
00
2 -Chloro-5 -trifluoromethyl
Sitagliptin (standard)
a
SEM, standard error of mean of three experiments.
NA, not active at 0.2 mM.
b
Compounds were dissolved in DMSO and diluted in assay
buffer (Tris-HCl, pH 8.0). Sitagliptin was used as the reference
drug (Hsu et al., 2015; Tanwar et al., 2014). Reaction mixture
contain 35 mL of test samples along with 15 mL of enzyme
0.025 units/mL). After 20 min incubation at 37 C, 50 mL of
Prior to docking, the receptor grid was generated by
selecting the co-crystallized sitagliptin molecule as centroid
to define the volume of the docking grid. The docking was
performed in Glide extra precision (XP) mode based on
ꢀ
(
0
OPLS3e force field (Friesner et al., 2004, 2006; Halgren et al.,
.2 mM substrate was added and changes in absorbance was
€
2
004; SchrOdinger, 2019b), where the protein was kept rigid
recorded continuously for 60 min at 400 nm using
Spectramax M5 (Molecular Devices, CA, USA). Experiments
were carried out in triplicates (Nongonierma et al., 2013).
and ligands were kept flexible. The docking protocol was fur-
ther validated by docking the sitagliptin in the binding site
of DPP-IV. The docking reproduced the pose of co-crystal-
lized sitagliptin (Figure S1(a)). The molecular interactions
were also reproduced after docking of sitagliptin
2
.4. Human colorectal adenocarcinoma cell (Caco-2)
(
Figure S1(b)).
culture and maintenance
Caco-2 cell line (ATCC-HTB37) was purchased from American
Type Culture Collection (ATCC). DMEM supplemented with
2
.7. Statistical analysis
1
1
% non-essential amino acids (Sigma-Aldrich, Germany) and All the experiments were carried out in triplicate and proc-
0% fetal bovine serum was used as a growth medium. Cells essed using SoftMax Pro 4.8 software (Molecular Devices, CA,
ꢀ
were cultured at 37 C and 5% CO atmosphere. Medium USA). Results are expressed as mean ± standard error of
2

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
1
13
Figure 2. H (a), C NMR (b) chemical shifts of compound 9.
Figure 3. Distinctive NOESY interaction.
1
3
mean. IC50 values were calculated using EZ-FIT enzyme kinet-
ics software (Perrella Scientific Inc., NH, USA).
In broad-band C NMR spectrum of compound 9, sixteen
(16) signals are appeared. Out of these seven (7) are due to
ꢀ
ꢀ
quaternary carbons. Nevertheless, DEPT-135 and -90 experi-
ments indicated the presence of nine methines and the
absence of a methylene group. The most down-field signal
3
. Results and discussion
was of thioamidic carbon at d 176.5. The iminic carbon res-
3
.1. Spectroscopic studies of compound (E)-N-
C
onated at d 159.4. Aromatic carbon with OH substituent (C-
(2-chlorophenyl)-2-((4-hydroxyphenyl)
C
0
4
) resonated at d 150.4. Aromatic carbon with chloro sub-
C
stituents (C-2 ) resonated at d 129.3. Remaining aromatic
(phenyl)methylene)hydrazinecarbothioamide (9)
00
C
Detailed characterization of structures of synthetic benzophe-
none thio- and semicarbazone derivatives was accomplished.
The structure elucidation of compound 9 is presented here
as a representative example, since it is most active com-
pound of synthetic series. Important structural features and
binding sites of of all other derivatives are shown in
Figure 1.
carbons resonated with their respected frequency in the aro-
matic region (Figure 2(b)).
Mass of compound 9 was calculated by FAB-MS techniques
þ
þ
which showed [M þ H] at m/z 382, and [M+H+2] 384.
Fourier transform IR (FT-IR) spectrum of compound 9 dis-
played vibrational frequencies at 3349 cm which showed
the presence of hydroxyl functionality in the compound.
Absorbance at 3269 cm
Aromatic (C ¼ C) functionalities vibrated at 1539 cm
However, C–N and C–Cl bonds vibrated at 1367 and
ꢁ
1
ꢁ
1
NMR spectroscopy was performed to elucidate the struc-
showed NH functionality.
1
13
ꢁ1
ture 9. H and C NMR spectra were recorded in deuterated
DMSO on Bruker-Avance AM 400 and 500 MHz instruments,
.
1
ꢁ
1
respectively. In H NMR, two NH protons and one phenolic OH
8
21 cm , respectively.
protons resonate at dH 10.34, 10.04 and 9.09, respectively.
NOESY (nuclear Overhauser enhancement spectroscopy)
However, protons of non-hydroxybenzene ring of benzophe- has been performed on most active compound 9 for the
none resonated as a doublet at d 7.71 (J2,3 ¼ J ¼ 7.6 Hz, H- verification of the stereochemical configuration of Schiff base
H
6,5
2
7
, H-6) and the other ring protons resonated as a triplet at d
(iminic) double bond. Many interactions were observed in
H
0
.38 (t, 3H, J3(4,5)/4(3,5)/5(4,6) ¼ 7.6 Hz. H-3, H-4, H-5). 4 -Hydroxy the NOESY spectrum. Strong interaction was observed
benzophenone ring also resonated as a doublet at d 7.66 between iminic NH and H-2 of benzophenone ring which
H
0
0
(J3
0
0
/
0
0
¼ 6.8 Hz, H-3 , H-5 ) and at d 7.56 (J
0 0 0 0
/
¼ 6.8 Hz, can only be seen in case of E-isomer. In the same manner
,2
0
5 ,6
H
2 ,3 6 ,5
0
00
0
H-2 , H-6 ). In addition, H-5 resonated as triplet at d 7.33 absence of NOESY interaction between iminic NH and H-2
H
00
00
00
00
(
J500 00 00 ¼ 7.6 Hz, H-5 ) and H-4 , H-6 , H-3 resonated as further validate the E-stereochemistry of resulting isomer.
(6 ,4 )
00
0
0
doublets at d 7.21 (J 00 00 ¼ 8.4 Hz, H-4 ), 7.02 (J 00 00 ¼ 8.4 Hz, Other interactions, such as interactions of H-2/H-2 and H-2 /
H
4 ,3
6 ,5
00
00
00
H-6 were also observed (Figure 3).
H-6 ), 6.77 (J 00 00 ¼ 8.8 Hz, H-3 ), respectively (Figure 2(a)).
3
,5

6
ARSHIA ET AL.
3
.2. Enzyme inhibitory studies
the DPP-IV inhibitory activity of these compounds. Strong
electron donating effect of –OH may involve in the activation
of benzophenone ring system for p–p interactions with the
aromatic amino acids in the active site of enzyme. Arshia
In the current study, we evaluated 29 semicarbazones and
thiosemicarbazones (Table 1) for their dipeptidyl peptidase-IV
(
DPP-IV) inhibitory potential. Results showed that thiosemi-
0
et al. have also reported that hydroxyl group at C-4 of thio-
0
carbazones having hydroxyl group at C-4 of benzophenone
ring were more active as compared to their un-substituted
analogues 1–7. This suggests the role of hydroxyl group in
semicarbazones is vital for enzyme inhibitory activity against
jack bean (Canavalia ensiformis (L.)) urease. It was also noted
that thiosemicarbazone showed stronger DPP-IV inhibitory
activity than semicarbazones.
Thiosemicarbazone 8 with a hydroxyl group at C-4 and
un-substituted phenyl ring showed only a weak inhibitory
activity (IC50 ¼ 69.9 ± 1.01 mM) as compared to standard drug
(
IC50 ¼ 0.033 ± 0.4 mM). Whereas compound 1, which lacks the
hydroxyl group, was found inactive (Figure 4a).
Substitution of halogens at different positions of phenyl
ring resulted in altered enzyme inhibitory activity of thiose-
micarbazone derivatives. Compound 9 with a chloro group
00
0
at C-2 and hydroxyl group at C-4 showed an IC value of
5
0
Figure 4a. Structure–activity relationship of thiosemicarbazone analogs with
non-substituted substituted aryl part.
1
5.0 ± 0.6 mM and was found to be the most active member
of the series. However, compounds 10 and 11 having chloro
Figure 4b. Structure–activity relationship of thiosemicarbazone analogs with chloro substituted aryl part.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 4c. Structure–activity relationship of thiosemicarbazone analogs with bromo and fluoro substituted aryl part.
0
0
00
group at C-3 and C-4 , respectively, showed a decreased and 7 showed no inhibition of DPP-IV enzyme, irrespective
DPP-IV inhibitory (IC50 ¼ 38.27 ± 1.84 and 79.0 ± 1.3 mM, of the position of bromo group, which might be attributed
0
respectively) activity, as compared to the compound 9. to the absence of hydroxyl group at C-4 (Figure 4c). These
00
Thiosemicarbazone (2) which has chloro group at 3 -position results suggested that halogen substituent at ortho position
0
and having no hydroxyl group at 4 -position was found to are more active than those with meta substituted derivatives,
be completely inactive. Compound 17 having chloro sub- and they are more active than their analogues at para pos-
0
0
00
stituents at 2 - and 5 -positions and having hydroxyl group ition. Halogens at ortho position might increase the electro-
0
at 4 -position showed only a weak DPP-IV inhibitory activity philicity of the thioamide group for possible interaction with
(
IC50 ¼ 39.2 ± 1.55 mM). Whereas, compounds 18 and 19 the hydroxyl group of serine in the active site of DPP-IV
which only differ with chloro group positions were found to enzyme, which may have resulted in an increased enzyme
be completely inactive. Increased steric bulk in di-chlorinated inhibitory activity.
derivatives 17, 18 and 19 might be responsible for the weak
Moderate to weak enzyme inhibition was observed in
or inactivity of these compounds against DPP-IV enzyme semicarbazone derivatives. Interestingly, inhibitory potential
(
Figure 4b).
of semicarbazone derivatives depend on the position of tri-
Thiosemicarbazone (12) having fluoro group at C-2 and a fluoromethyl group rather than the hydroxyl group at C-4 .
00
0
0
hydroxyl at C-4 (IC ¼ 28.9 ± 0.32 mM) showed a moderate Compound 20, a semicarbazone with an un-substituted phe-
5
0
enzyme inhibitory activity. However, shifting of fluoro group nyl ring, showed a moderate enzyme inhibitory activity
0
0
to C-3 in compound 13 (IC ¼ 73.0 ± 0.27 mM) resulted in a (IC ¼ 43.5 ± 0.44 mM). Compounds 21 and 26 with a –Cl at
5
0
50
00
0
decreased activity. Similar trend was observed in compounds C-3 were found to be inactive, irrespective of C-4 substitu-
1
4 and 15 where a decrease in DPP-IV inhibitory activity was tion. Compounds 22, bearing a trifuoromethyl groups at C-
00
00
observed when bromo group is shifted from C-2
3 , was found to be inactive. However, when trifluoromethyl
(IC50 ¼ 49.6 ± 0.07 mM), was present at C-4 in compounds 23 and 27 a moderate
00
00
(
IC50 ¼ 34.0 ± 2.06 mM)
to
C-3
respectively. Further decrease in enzyme inhibitory activity enzyme inhibition was observed (IC50 values 37.46 ± 0.97 and
was observed in compound 16 where bromo group is pre- 87.5 ± 0.7 mM, respectively). The only structural difference was
0
0
0
sent at C-4 (IC ¼ 161.7 ± 6.47 mM). However, compounds 6 the presence of –OH group at C-4 in compound 27 which
5
0

8
ARSHIA ET AL.
Figure 4d. Structure–activity relationship of semicarbazone analogs with chloro and trifluoromethyl substituted aryl part.
resulted in a decreased inhibitory potential of this com- 3.3. Molecular docking studies
pound. Comparable inhibition was observed in compounds
The compounds that were found to be active against DPP-IV
in in vitro biochemical assay were subjected to docking stud-
ies to decipher their molecular interactions. DPP-IV is a
homodimer consisting of subunits A and B. These subunits
are further divided into a/b-hydrolase and b-propeller
domains. The enzyme is a type-II transmembrane protein
with the membrane-spanning region from 7 to 28 amino
acid residues. The a/b-hydrolase domain starts with amino
acids 39–51 and 506–766 and is closest to the membrane.
While the b-propeller domain consists of residues 55–497.
As sitagliptin is used as reference drug in the in vitro assay,
docking pose of identified inhibitors were also compared to the
reference drug. The crystal structure used for docking analysis is
25 and 29 (IC50 ¼ 66.04 ± 0.77, and 59.37 ± 0.17 mM, respect-
ively). Strong electron donating effect of chloro group when
present at ortho position of benzene, might be responsible
of the observed inhibitory activities of these analogues.
Compound 28 showed only a weak inhibition of DPP-IV
enzyme (IC50 ¼ 136.4 ± 2.89 mM). This compound is substi-
tuted with a trifluoromethyl at ortho and a chloro group is
present at para position. However, the effect of these substi-
tutions was only visible only when a hydroxyl group was at
0
C-4 of benzophenone ring. Though compound 24 bearing
same substitution at aryl part but lacks a hydroxyl group at
0
C-4 of benzophenone part was found to be inactive
(Figure 4d).
1
x70, co-crystallized with the drug sitagliptin (Figure 5(a) and
Hence, preliminary SAR studies revealed that trifluoro-
(b)). It is a recombinant sequence deprived of membrane-span-
0
methyl, halogen substitutions, C-4 hydroxyl and CONH/CSNH
are particularly important for the inhibition of enzyme DPP-
IV. Combined effect of these groups may influence the bind-
ing ability of these molecules with amino acid residues pre-
sent in the active site or hydrophobic pocket of enzyme
ning region. The N-terminal region of 1x70 starts with Ser39,
which is the soluble form of the protein found in plasma.
The co-crystallized sitagliptin in the DPP-IV binding site showed
hydrogen bondings with the carboxyl and hydroxyl groups of
Glu205 and Tyr662, respectively. The trifluorophenyl and triazole
DPP-IV. Synthesis of more derivatives with structural varia- moieties of sitagliptin made p–p interactions with the phenol ring
tions and validation of identified inhibitors using in vivo of Tyr666 and benzene moiety of Phe357 Figures 6, and S2.
models and molecular mechanistic studies will be required
to demonstrate the efficacy of these leads for the inhibition DPP-IV with IC50 values 15.0 ± 0.6 and 34.06 ± 2.06 mM, respect-
of DPP-IV enzyme. ively, and hence chosen for docking studies.
Compounds 9 and 14 were found to be most active against

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Figure 5. (a) 3D Molecular surface representation of DPP-IV (PDB ID: 1X70) prepared by protein preapartion wizard tool illustrating the overall structure of enzyme.
b) The co-crystalized sitagliptin depicting the binding pose in the active site.
(
Figure 6. Modelled posed of drug sitagliptin in the binding site of DPP-IV (PDB ID: 1X70).
Docking of compound 9 in the binding site of DPP-IV hydrogen bonds with Glu205 Figures 8. and S4. The molecular
showed p–p interactions with Tyr666 and Phe357 through its interactions of these compounds along with sitagliptin in the
benzene moieties. While the hydroxyl benzyl moiety form binding site of DPP-IV are summarized in Table S1.
hydrogen bonding interaction with His126 figures 7, and S3.
Compound 14 showed a flip in the orientation of the docked
pose, as compared to compound 9. The bromobenzene moiety
formed p–p interactions with the indole moiety of Trp629. A
3
.4. Cytotoxicity studies
halogen bond was also observed with guanidinum group of Mouse fibroblast 3T3 cells were used for initial cytotoxic
Arg125. The hydroxyl benzyl moiety interacted through evaluation. Most active compounds 8–15, 17, 20, 23, 26, 27

1
0
ARSHIA ET AL.
Figure 7. Docked pose of compound 9 in the binding site of enzyme DPP-IV. Hydrogen bonds are represented as yellow dotted lines. p–p interaction is repre-
sented as cyan dotted lines.
Figure 8. Docked pose of compound 14 in the binding site of enzyme DPP-IV. Hydrogen bonds are represented as yellow dotted lines. p–p interaction is repre-
sented as cyan dotted lines.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
and 29 were evaluated for their cytotoxicity. Compounds 8, the development of novel therapeutic agents for type 2 dia-
4, 17, and 27 showed a weak cytotoxicity with IC50 values betes with minimal side effects.
9.0 ± 0.2, 27.8 ± 1.9, 25.7 ± 0.3, and 24.6 ± 0.3 mM, respectively.
Whereas compound 29 was found to be highly cytotoxic
IC50 ¼ 8.2 ± 0.5 mM) against this cell line. All other com-
pounds were found non-cytotoxic.
1
1
Disclosure statement
(
No potential conflict of interest was reported by the authors.
Funding
3.5. Inhibition of DPP-IV in in situ Caco-2 cellular assay
Authors are grateful to the Higher Education Commission (HEC),
Pakistan, for providing financial support under the Indigenous Ph.D.
Fellowship for 5000 Scholars Phase-II program and Pakistan Academy of
Sciences for financial support to Project No. 5-9/PAS/440.
Inhibitors identified through in vitro studies sometimes do
not correlate in in vivo models, because standard enzyme
inhibition assays with purified enzymes do not stimulate cer-
tain factors that might affect inhibitor’s bioactivity. We, there-
fore, further evaluated the efficacy of our identified inhibitors
using Caco-2 cellular assay. It has been reported that Caco-2
cell line expresses many membrane bound enzymes, and
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In conclusion, we report here benzophenone-based thiosemi-
carbazone and semicarbazone derived DPP-IV inhibitors.
Insights into the structure–activity relationship reveals that
halogen substituents, ortho to thioamide (compounds 9, 12,
and 14) are contributing significantly in the inhibitory activity
of DPP-IV. As for the semicarbazone derivatives, we observed
that trifluoromethyl is mainly responsible for the enzyme
inhibitory activity. Varied enzyme inhibitory activities, showed
by thio- and semicarbazone derivatives can be correlated
with different aryl substitutions, as well as hydroxyl group at
0
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potential inhibitors in in situ Caco-2 assay demonstrated their
inhibitory potential under intestinal cell conditions. These
compounds offer excellent starting point for medicinal chem-
ists to explore new chemical scaffolds and optimize existing
chemical entities with improved therapeutic profile. Further
optimization of these compounds for better efficacy lead to

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