molecules

Article
Synthesis of Novel Triazinoindole-Based Thiourea
Hybrid: A Study on α-Glucosidase Inhibitors and
Their Molecular Docking
Muhammad Taha 1, * , Foziah J. Alshamrani 2,3 , Fazal Rahim 4, *, Shawkat Hayat 4 , Hayat Ullah 4 ,
Khalid Zaman 4 , Syahrul Imran 5,6 , Khalid Mohammed Khan 7 and Farzana Naz 8
1 Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
2 Neurology Department, King Fahad hospital of University, Imam Abdulrahman Bin Faisal University,
P.O. Box 1982, Dammam 34211, Saudi Arabia; Fshamrani@uod.edu.sa
3 Department of Neuroscience Research, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
4 Department of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan;
shawkathayat866@yahoo.com (S.H.); ayaanwazir366@gmail.com (H.U.); khalidchemist69@yahoo.com (K.Z.)
5 Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor D.E., Malaysia; imran@isiswa.uitm.edu.my
6 Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor D.E., Malaysia
7 H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University
of Karachi, Karachi 75270, Pakistan; khalid.khan@iccs.edu
8 Department of Chemistry, Jinnah University for Women, 5-C, Nazimabad, Karachi 74600, Pakistan;
farzanahej@yahoo.com
* Correspondence: mtaha@iau.edu.sa (M.T.); fazalstar@gmail.com (F.R.); Tel.: +966502057350 (F.R.)




Received: 12 September 2019; Accepted: 21 October 2019; Published: 23 October 2019


Abstract: A new class of triazinoindole-bearing thiosemicarbazides (1–25) was synthesized and

evaluated for α-glucosidase inhibitory potential. All synthesized analogs exhibited excellent inhibitory
potential, with IC50 values ranging from 1.30 ± 0.01 to 35.80 ± 0.80 µM when compared to standard
acarbose (an IC50 value of 38.60 ± 0.20 µM). Among the series, analogs 1 and 23 were found to be the
most potent, with IC50 values of 1.30 ± 0.05 and 1.30 ± 0.01 µM, respectively. The structure–activity
relationship (SAR) was mainly based upon bringing about different substituents on the phenyl rings.
To confirm the binding interactions, a molecular docking study was performed.

Keywords: synthesis; triazinoindole; thiosemicarbazide; alpha-glucosidase; molecular docking

study; SAR

1. Introduction
Diabetes mellitus is a chronic health-threatening metabolic disease that is caused by insufficient
insulin secretion and is categorized as hypoglycemia/hyperglycemia [1]. In type II diabetes mellitus,
enhanced postprandial glucose levels can increase the risk of developing stroke, atherosclerosis, and
other coronary diseases [2]. In order to treat type II diabetes and its complications, the inhibition of
digestive enzyme-like α-glucosidase is an effective approach that can reduce postprandial glucose
and risk factors [3]: α-glucosidase is located in the epithelium cell lining of the small intestine and is
responsible for the conversion of polysaccharides and disaccharides into glucose. The inhibition of
α-glucosidase is directly associated with the blood glucose level, and its inhibition is vital due to the
potential effect of a decrease in postprandial blood glucose levels [4]. In order to delay rapid blood
glucose production, certain α-glucosidase inhibitors such as acarbose and voglibose are used clinically,

Molecules 2019, 24, 3819; doi:10.3390/molecules24213819 www.mdpi.com/journal/molecules

Molecules 2019, 24, x FOR PEER REVIEW 2 of 16
Molecules 2019, 24, 3819 2 of 16
Molecules 2019,
potential 24, of
effect x FOR PEER REVIEW
a decrease in postprandial blood glucose levels [4]. In order to delay2rapid of 16 blood

glucose production, certain α-glucosidase inhibitors such as acarbose and voglibose are used
potential effect of a decrease in postprandial blood glucose levels [4]. In order to delay rapid blood
though there
clinically, are certain
though there side effect
certainthat
are α-glucosidasesideinclude
effect abdominal
thatsuch
includepain, diarrhea, pain,
abdominal and other gastrointestinal
diarrhea, and other
glucose production, certain inhibitors as acarbose and voglibose are used
disorders in
gastrointestinal chronic
clinically, though
therapy
disorders
there are in
[5]. Therefore,
chronic
certain
in
therapy
side effect
order to
that [5].
treat postprandial
Therefore,
include abdominalinpain,
hyperglycemia,
order to treat
diarrhea,
a search for
and postprandial
other
efficient and safeaα-glucosidase
hyperglycemia,
gastrointestinal search forinefficient
disorders inhibitors
chronic and is needed.
safe
therapy α-glucosidase
[5]. Therefore,inhibitors
in orderisto needed.
treat postprandial
Triazinoindole scaffolds
scaffolds possess
possess excellent
excellent biological
biological
hyperglycemia, a search for efficient and safe α-glucosidase inhibitors is needed. potential against malarial and viral
diseases. Substituted
Triazinoindole
diseases. Substitutedscaffolds triazinoindole scaffolds
possess scaffolds
triazinoindole are
excellent biological of considerable interest
potential against
are of considerable malarial to
interest due their
and excellent
viral
their excellent
diseases. Substituted
antihypertensive
antihypertensive triazinoindole
antidepressantscaffolds
[6], antidepressant are of considerable[8],
[7], anti-inflammatory
anti-inflammatory interest due to their
antihypoxic
antihypoxic [9], excellent
[9], antifungal,
antifungal, and
and
antihypertensive
antibacterial
antibacterial [6],
activities antidepressant
[10].
[10]. Selected [7], anti-inflammatory
triazinoindole compounds[8], antihypoxic
act as [9],
potential antifungal,
drugs in and
treating the
antibacterial
common activities [10]. Selected triazinoindole compounds act as potential drugs in treating the
common cold cold [11–14].
[11–14].
common cold [11–14].
Our
Our research
researchgroup grouphas hasbeen
beenworking
workingon onthethedesign
design andandsynthesis
synthesis of of
heterocyclic
heterocyclic compounds
compounds in
Our research group has been working on the design and synthesis of heterocyclic compounds
search
in in of
search potential lead compounds for many years, and we have found promising results [15–29].
searchofofpotential leadcompounds
potential lead compounds forfor many
many years,
years, andand we have
we have foundfound promising
promising results [15–29].
We
We have
have already reported
reported on
on triazinoindole
triazinoindole analogs
analogs asas potent
potent
We have already reported on triazinoindole analogs as potent α-glucosidase inhibitors α-glucosidase
α-glucosidase inhibitors
inhibitors [30].
[30].
[30]. Thus, we Thus,
Thus, we
we decided
decided to to screen
screen a a library
library ofof triazinoindole-bearing
triazinoindole-bearing thiosemicarbazide
thiosemicarbazide
decided to screen a library of triazinoindole-bearing thiosemicarbazide analogs for α-glucosidase analogs
analogs for α-glucosidase
α-glucosidase
activity
activity (Figure
activity (Figure1).
(Figure 1).
1).

R R
N
NN N S OO
S H H
N N HN S N SH
N R N N S N N S
N R N
NH N HN
H N N HN R
N 1
R1
Previously reported New synthetic analogues 1-25
Previously reported
-Glucosidase inhibitors New-glucosidase
Potent synthetic analogues
inhibitors1-25
-Glucosidase inhibitors Potent -glucosidase inhibitors
Figure 1. Rationale of the current work.
Figure 1. Rationale of the current
Figure 1. current work.
work.
2. Results and Discussion
2. Results and Discussion
2. 2.1.
Results and Discussion
Chemistry
2.1. Chemistry
A new class of triazinoindole-based thiosemicarbazide analogs (1–25) was carried out in three
2.1. Chemistry
steps.
A new class of triazinoindole-based thiosemicarbazide analogs (1–25) was carried out in three steps.
AInnew classstep,
the first of triazinoindole-based
thiosemicarbazide wasthiosemicarbazide
reacted and refluxedanalogs (1–25)
with isatin in H2was
O in carried out in three
the presence
In the first step, thiosemicarbazide was reacted and refluxed with isatin in H2 O in the presence of
of potassium carbonate to yield 5H-triazinoindole-3-thiol as an intermediate product (I). The
steps.
potassium carbonate to yield 5H-triazinoindole-3-thiol as an intermediate product (I). The intermediate
intermediate
In the first(I)step,
was then mixed and refluxed
thiosemicarbazide waswith different
reacted andsubstituted phenacyl
refluxed with bromides
isatin in H2O ininEtOH
the presence
(I) in
was
thethen mixedofand
presence Et refluxed with different substituted phenacyl bromides in EtOH in the presence
3N to give triazinoindole derivatives as a second intermediate product (II).
of potassium carbonate to yield 5H-triazinoindole-3-thiol as an intermediate product (I). The
of (Scheme
Et3 N to 1)
give triazinoindole derivatives as a second intermediate product (II). (Scheme 1)
intermediate (I) was then mixed and refluxed with different substituted phenacyl bromides in EtOH
in the presence of Et3N to give triazinoindole derivatives as a second intermediate product (II).
(Scheme 1)

Scheme 1. Synthesis of triazinoindole derivatives.

Scheme 1. Synthesis of triazinoindole derivatives.
In the second step, hydrazine hydrate was reacted and refluxed with different isothiocyanates
in methanol to yield
In the second a thiosemicarbazide
step, derivative
hydrazine hydrate as an intermediate
was reacted and refluxed(III).
with (Scheme 2) isothiocyanates in
different
methanol to yield a thiosemicarbazide derivative
Scheme 1. Synthesis as an intermediate
of triazinoindole (III). (Scheme 2)
derivatives.

In the second step, hydrazine hydrate was reacted and refluxed with different isothiocyanates
in methanol to yield a thiosemicarbazide derivative as an intermediate (III). (Scheme 2)
Molecules 2019, 24, x FOR PEER REVIEW 3 of 16
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Molecules 2019, 24, x FOR PEER REVIEW 3 of 16
Molecules 2019, 24, x FOR PEER REVIEW 3 of 16
Molecules 2019, 24, 3819 3 of 16
Molecules 2019, 24, x FOR PEER REVIEW 3 of 16
Molecules 2019, 24, x FOR PEER REVIEW 3 of 16

Molecules 2019, 24, x FOR PEER REVIEW 3 of 16

Scheme 2. Synthesis of thiosemicarbazide derivatives.

Scheme 2. Synthesis of thiosemicarbazide derivatives.
Scheme 2. Synthesis of thiosemicarbazide derivatives.
Scheme 2.
In the third step, intermediate product Synthesis
(II) of thiosemicarbazide
was reacted derivatives.
and refluxed with intermediate product
(III) In
in the
glacial In theacid
acetic thirdtostep,
give intermediate
Scheme
Schemethe Synthesis
final
2.
2. product
of
product,
Synthesis of (II) wasderivatives.
reacted
thiosemicarbazide
triazinoindole-bearing
thiosemicarbazide and thiosemicarbazide
refluxed with intermediate
derivatives.
derivatives. (1–25). product
third step, intermediate
Scheme product
2. Synthesis (II) was reacted and
of thiosemicarbazide refluxed with intermediate product
(III) In the
in 1). third
glacial step,
2019, 24, acetic intermediate
to give the final product, triazinoindole-bearing thiosemicarbazideproduct
acid REVIEW product (II) was reacted and refluxed with intermediate (1–25).
(Scheme
(III) 3,Molecules
in glacialTable acetic acid xtoFOR PEER
give the final product, triazinoindole-bearing thiosemicarbazide (1–25). 3 of 16
In
In In(III)
the
the
the in
third
(Scheme glacial
third
third step,
3, acetic
Table
step,
step, 1). acid
intermediate
intermediate
intermediate to give the
product
product
product (II) final
(II)
was
(II) wasproduct,
reacted
reacted
was and
reactedtriazinoindole-bearing
and refluxed
refluxed
refluxed
and with thiosemicarbazide
with intermediate
intermediate
intermediate
with product product (1–25).
product
(Scheme 3, Table 1).
(III)
(III)in
(III) (Scheme
ininglacial
glacial 3, Table
glacial acetic
acetic
acetic acid1).
acid
acid to
to give
togive
givethethe
the final
final product,
product,
final product, triazinoindole-bearing
triazinoindole-bearing thiosemicarbazide
thiosemicarbazide
triazinoindole-bearing (1–25).
(1–25). (1–25).
thiosemicarbazide
(Scheme
(Scheme 3,
3,Table
Table 1).
1).
(Scheme 3, Table 1). Scheme 2. Synthesis of thiosemicarbazide derivatives.

In the third step, intermediate product (II) was reacted and refluxed with intermediate product
(III) in glacial acetic acid to give the final product, triazinoindole-bearing thiosemicarbazide (1–25).
(Scheme 3, Table 1).

Scheme 2. Synthesis of thiosemicarbazide derivatives.

In the third step, intermediate product (II) was reacted and refluxed with intermediate product
(III) in glacial acetic acid to give the final product, triazinoindole-bearing thiosemicarbazide (1–25).
(Scheme
Scheme3, Table 1).
3. Synthesis of triazinoindole-based thiosemicarbazide analogs (1–25).
Scheme 3. Synthesis of triazinoindole-based thiosemicarbazide analogs (1–25).
Scheme 3. Synthesis
Scheme 3. Synthesisofoftriazinoindole-based thiosemicarbazide
triazinoindole-based thiosemicarbazide analogs
analogs (1–25).
(1–25).
Scheme
Table 1. Structure Scheme 3. Synthesis
3. Synthesis
of triazinoindole-based of triazinoindole-based
of triazinoindole-based
thiosemicarbazide thiosemicarbazide
thiosemicarbazide
analogs and their analogs analogs (1–25).
(1–25).activity.
α-glucosidase
Table 1.
Scheme Structure of triazinoindole-based thiosemicarbazide analogs and their α-glucosidase activity.
Table
Table 1.1.Table
Structure
1. Structure of3.
of Synthesis of triazinoindole-based
triazinoindole-based
of triazinoindole-based
Structure thiosemicarbazide
R thiosemicarbazide
thiosemicarbazide
analogs
R1 analogs
and their
ICand
analogs
α-glucosidase
their (1–25).
activity.
α-glucosidase activity.
Table
S.triazinoindole-based
1. Structure thiosemicarbazide
No.of triazinoindole-based analogs
thiosemicarbazide and
50 (μM) their
analogs α-glucosidase
and activity. activity.
their α-glucosidase
R R R1 R1
ICIC
50 (μM) IC50 (μM)
Table 1. S. Structure S. No. S. No.
ofS.triazinoindole-based
No. R R1
thiosemicarbazide R150 (μM)
analogs and their
No.
S. No.
R R R1 IC50α-glucosidase
(μM)IC50 (µM) activity.
Cl
R
S. No.of triazinoindole-based
Scheme 3. Synthesis
R1
Cl 1.30IC
±
Cl
thiosemicarbazide
50 (μM) (1–25).
0.05
1 Cl 1.30 ± 0.05 analogs
1.30 ±Cl0.05
1.30 ± 0.05
1 1 Cl
1 1 1.30 ± 0.05
1.30 ± 0.05
Table 1. Structure of triazinoindole-based Cl
1 O thiosemicarbazide analogs
Cl andCl
Cltheir α-glucosidase activity.
O
O Cl ± 0.05
1.30
O
S. No.1 R O R1 IC50 (μM)
Cl
O
2.60
Cl 2.60 ± 0.05
± 0.05
22 1.30 ± 2.60 ± 0.05
2 Scheme
1 3. Synthesis of triazinoindole-based2.60 ± 0.05
0.05
thiosemicarbazide analogs (1–25).
2.60 ± 0.05
2 2.60 ± 0.05
2 NO
NO Cl
2 OO 2 2
Table 1. Structure of triazinoindole-based
O O NO2.60
thiosemicarbazide ± 0.05
2 analogs and their α-glucosidase activity.
NO
2 O
O
2
NO2
S. No. R R1 IC50 (μM)
Cl
NO2
O Cl
2.60 ±Cl0.05
3 ClCl
7.5 Cl
Cl
± 0.20
2
3 3 1
Cl 7.5 ± 0.20 1.30 ± 0.057.5 ± 0.20
3 Cl Cl 7.5 ± 0.20
3 NO2 Cl 7.5 ±Cl
Cl0.20
3O 7.5 ± 0.20
O
3 Cl 7.5 ± 0.20
Cl
2.60 ± 0.05
3 2 Cl 7.5 ± 0.20

O NO2

Cl
 1
Cl
O

Molecules
Molecules 24, x24,
2019,
2019, FOR3819
PEER REVIEW 4 of 416of 16
Molecules 2019, 24, x FOR PEER REVIEW 2.60 ± 0.05 4 of 16
Molecules 2019, 3.
Scheme 24,Synthesis
2 PEER
x FOR of REVIEW
triazinoindole-based thiosemicarbazide analogs (1–25). 4 of 16
Table 1. Cont.
Table 1. Structure of triazinoindole-based NO2 analogs and their α-glucosidase activity.
O thiosemicarbazide
Molecules 2019, 24, x FOR PEER REVIEW
S. No. R R1 IC
50 (µM) 4 of 16
Molecules 2019, 24, x FOR PEER REVIEW
R R1 IC50 (μM) 4 of 16
S. No.
4 12.80 ± 0.30
4 12.80 ± 0.30
4 Cl 12.80 ± 0.30
Cl
Scheme 3. Synthesis 1.30 ± 0.05
of triazinoindole-based thiosemicarbazide analogs (1–25).
4 1 Cl 12.80 ± 0.30
3 7.5 ± 0.20
44 Cl 12.80 ± 0.30
Table
Molecules 1. 24,
2019, Structure of triazinoindole-based
x FOR PEER REVIEW O thiosemicarbazide analogs and their12.80 ± 0.30 activity.3 of 16
α-glucosidase
Molecules 2019, 24, x FOR PEER REVIEW 4 of 16
Molecules 2019, 24, x FOR PEER REVIEW 4 of 16
S. No. R R1 IC50 ±
5.20 (μM)
0.30
5 5.20 ± 0.30
5 5.20 ± 0.30
5 Cl 2.60 ± 0.05
O
2 O 1.30 ± 0.05
5 1 O 5.20 ± 0.30
44 5.20
5.20 ±
12.80 ±±0.30
0.30
55 O NO2Cl 12.80 ±0.30
0.30
O
Br
O
O Br
Br
6 Cl 25.3 ± 0.50
Scheme
6 25.3 ± 0.50
2. Synthesis of thiosemicarbazide derivatives.
6 2.60 ± 0.05 25.3 ± 0.50
2 Br
Cland refluxed
6
In the third step, intermediate
3 product (II) was reacted 7.5 ±Br0.20with intermediate
25.3 ± 0.50product
5.20
5.20 ±
± 0.30
0.30
(III) in glacial acetic acid to give the final
55 O product, triazinoindole-bearing
NO2 thiosemicarbazide
25.3 ± 0.50 (1–25).
66 25.3 ± 0.50
Molecules 2019, 24,(Scheme
x FOR PEER3, REVIEW
Table 1). 4 of 16
O
O
33.30 ± 0.60
7 Cl 33.30 ± 0.60
7 PEER REVIEW Br 33.30 ± 0.60
Molecules 2019, 24, x FOR 4 of 16
7 Br Br
7
3 Cl Br
7.5 ± 0.20
33.30 ± 0.60
4 12.80 ± 0.30
Molecules 2019, 24, x FOR PEERMolecules
REVIEW 2019, 24, x FOR PEER REVIEW
66 25.3
25.3 ±
33.30
33.30 ±0.50
±±
40.60
of 16
0.60
0.50
4 of 16
77
Br
Br
17.5 ± 0.30
4
8 PEER REVIEW
Molecules 2019, 24, x FOR 17.5 ± 0.3012.80 ± 0.30 4 of 16
8 17.5 ± 0.30
8 8 17.5 ± 0.30
4 4 12.80 ± 0.30 12.80 ± 0.30
5.20 ± 0.30
5 17.5
17.5 ±
33.30 ±±0.30
0.60
88 33.30 ±0.30
0.60
77
Scheme 3. Synthesis of triazinoindole-based Br
thiosemicarbazide analogs
O 4 6.80 ±Br0.1012.80 (1–25).
± 0.30
9 6.80 ± 0.10 5.20 ± 0.30
9 9 59 thiosemicarbazide analogs and their6.80
Table 1. Structure of triazinoindole-based
± 0.106.80 ± 0.10
α-glucosidase activity.
NO2
NO
R 2 Br 5.20
O R1 ± 0.30 IC50 (μM) 5.20 ± 0.30
5 S. No. 5 NO 2 6.80
6.80 ±
± 0.10
0.10
99 17.5
17.5 ±
± 0.30
0.30
6 8 25.3 ± 0.50
O 8 O Br
Cl 35.80 ± 0.80
10 NO Br 35.80
1.30 ± ±0.05
Br 0.805.20 ± 0.30
10 1 5 NO22 Br 35.80 ± 0.80
10 35.80 ± 0.80
10 Cl
6OO
O Br O Br
25.3 ± 0.50
O Br 35.80 ± 0.80
10 26.80Br
± 0.7035.80
6.80 ± 0.80
6 10
69 25.3 ± 0.50 6.80 ±
26.80 ± 0.7025.3 ± 0.10
0.50
0.10
9 Br
33.30 ± 0.60 26.80 ± 0.70
11 11 O Br Br0.05
2.60 ± 26.80 ± 0.70
7 11 O Br
2 Br NO
NO22
11 NO 25.3 ± 0.50
6 2 26.80
NO2 NO2 26.80 ±
± 0.70
0.70
O NO 2 33.30 ± 0.60
7 Br
Br
11 Br 35.80
11
10
Br 35.80 ±
± 0.80
0.80
10 33.30 ± 0.60 33.30 ± 0.60
7 7 17.5NO
NO
± 0.30
2 Cl
2
8 Br O Br
O
3 Cl 7.5 ± 0.20 33.30 ± 0.60
26.80 ± 0.70
 6.80
Table 1. Structure of triazinoindole-based thiosemicarbazide analogs ± 0.10
and their α-glucosidase activity.
9 33.30 ± 0.60
Molecules 2019, 24, x FOR PEER REVIEW 3 of 16
7 R R1 Br IC50 (μM)
S. No.
NO2
Molecules 2019, 24, x FOR PEER REVIEW 4 of 16
Cl
Molecules 2019, 24, 3819 1.30 ± 0.05 5 of 16
1
Scheme
Molecules 2019, 24, x FOR PEER REVIEW 2. Synthesis of thiosemicarbazide
Br derivatives. 3 of 16
Molecules 2019, 24, x FOR
Molecules PEER
2019, 24, xREVIEW
FOR
35.80 ± 0.80 5 of 16
10 PEER
Molecules 2019, 24, x FOR
REVIEW
PEER REVIEW
Cl 5 of 16
5 of 16
In the third step, intermediate O (II)Table
product 1. Cont. and17.5
was reacted
± 0.30
refluxed with intermediate product
8
Molecules 2019, 24, x FOR PEER REVIEW
O 4 of 16
(III) in glacial acetic acid to give the final4 R product, triazinoindole-bearing thiosemicarbazide
12.80 ± 0.30 (1–25).
S. No. R1 IC50 (µM)
Molecules
(Scheme 3, Table 1). 2019, 24, x FOR PEER REVIEW 8.30 ± 0.20 8.30 ± 0.20 5 of 16
12 12 26.80 ± 0.708.30 ± 0.20
12
Scheme 2. Synthesis 2.60 ± 0.05
of thiosemicarbazide derivatives.
2 NO2 NO2 Br
12 11 NO2 8.30 ± 0.20
In theMolecules
third step,
2019,intermediate
24, x FOR PEERproduct
REVIEW (II) was reacted
NO and 6.80
refluxed 8.30 intermediate
± 0.10with ± 0.20 product 5 of 16
9 4O
12 2 12.80 ± 0.30
(III) in glacial acetic acid to give the final NO product,
2 triazinoindole-bearing thiosemicarbazide (1–25).
(Scheme 3, Table 1). NO2
Molecules 2019, 24, xScheme NO2 of thiosemicarbazide derivatives.5.20 ± 0.30
2. Synthesis
FOR PEER 5
REVIEW 5 of 16
Cl 8.30 ± 0.20
13
In the third step, intermediate 12
13
product (II) wasO reacted and23.30 ± 0.50
refluxed 23.30intermediate
with ± 0.50 product
13 23.30 ± 0.50
(III) in glacial acetic acid to give the final product, triazinoindole-bearing
NO2 Br Cl 35.80 thiosemicarbazide (1–25).
13 3 7.5 ±±0.20
0.808.30 ± 0.2023.30 ± 0.50
(Scheme 3, Table 1). 10 12 5.20 ± 0.30
5
13 23.30 ± 0.50
Molecules 2019, 24, x FOR PEER REVIEW
O NO2 Br 5 of 16
O
6 25.3 ± 0.50
Molecules 2019, 24,Scheme 3. Synthesis
x FOR PEER
26.80 ± 0.70
REVIEW of triazinoindole-based thiosemicarbazide analogs (1–25). 5 of 16
14 14
13 7.5 ± 0.20 23.30
7.5 ±±0.20
0.50
14 Br 8.30
7.5 ±±0.20
0.20
11 12 NO
14 of triazinoindole-based
Table 1. Structure thiosemicarbazide analogs and Brtheir α-glucosidase7.5 ±activity.
0.20
2 NO2
NO
NO 2
S. No. 13R NO2 R12 ± 0.2023.30
IC50 (μM)
8.30 ± 0.50
14
6 25.3
7.5 ±±0.20
0.50
12
NO2 NOthiosemicarbazide
Scheme 3. Synthesis of triazinoindole-based 2 analogs
33.30 (1–25).
± 0.60
15 Cl 5.80 ± 0.20
15
7 1.30 ± 0.05 5.80 ± 0.20
15 1 15 Br 5.80 ± 0.20
5.80 ± 0.20
Table 1. Structure of triazinoindole-based
14O thiosemicarbazide analogs and their 7.5 α-glucosidase
± 0.20 activity.
O Cl
13O O
S. No. R NO R12 IC50 (μM)23.30 ± 0.50
15 5.80 ± 0.20
Scheme 3. Synthesis of triazinoindole-based
14 thiosemicarbazide analogs7.5 ±±
33.30 (1–25).
0.20
0.60
13 7 O 23.30 ± 0.50
Table 1. Structure of triazinoindole-based
16 NO2 Clanalogs
thiosemicarbazide 1.8and
Brtheir α-glucosidase activity.
±± 0.20
16 1
16
8
16
1.30
2.60 ± 0.05 17.5
0.05 1.8 ±±0.20
0.30
1.8 ± 0.201.8 ± 0.20
15R IC50 (μM) 5.80 ± 0.20
S. 2No. O O F
R1 Cl
O O F
O NO2 F
16
14 O 1.8
7.5 ±
±±0.20
0.20
15 Cl 5.80 0.20
1.30 ± 0.05 17.5 ± 0.30
1 8 O NO2 F
17 O 8.80 ±0.20
0.20 8.80
17 14 17 Cl
Cl 7.5
2.60 ±± 0.05 6.80 ± 0.20
0.10
8.80 ± 0.20
17
9 8.80 ± 0.20
2 O
16 NO2 1.8 ± 0.20
NO2 NO2
NO2
3 NO2 NO2 Cl 7.5 ± 0.20 8.80 ± 0.20
17
15
O O F ± 0.05 5.80 ± 0.20
2.30 2.30
16 1.8 ±±
2.30
0.05
±0.20
0.05
NO 2
2.60 ± 0.05 6.80 ± 0.10
9 O
18 15
182 O F Br
5.80 ± 0.2035.80 ± 0.80 2.30 ± 0.05
18 Cl
18
10 Cl 2.30 ± 0.05
17 NOCl NO2 Cl 8.80 ± 0.20
O
O 2 Cl
O O Cl
3 O NO2 Cl Cl7.5 ± 0.20
18
16 1.8 ±±0.20
17 8.80 0.20
Cl
Cl Cl 26.80
Br 35.802.30 ±±±0.05
0.70
0.80
10 O NO2 F
16 1.8 ±Br0.20
19 11 Cl 2.30 ± 0.05
193 18 O Cl 7.5 ±±0.20
2.30 2.30 ±
0.05 2.30 ± 0.05
0.05
19O Cl
19 ClF2
NO Cl
Cl 2.30 ± 0.05
17 O Cl 8.80 ±±0.20
26.80 0.70
18
19 NO2 Br 2.30 ± 0.05
Cl
11 Cl
17 O 8.80 ± 0.20
NO2 2.30 ± 0.05
NO2
19 Cl 2.30 ± 0.05
18 Cl2.30 ± 0.05
Cl
Cl
 (III) in glacial acetic acid to14 give the final
15 product, triazinoindole-bearing thiosemicarbazide
7.5 ± 0.20 5.80 ± 0.20 (1–25).
(Scheme2019,
Molecules 3, Table 1). PEER REVIEW
24, x FOR 5 of 16
NO2 O
Molecules 2019, 24, x FOR PEER REVIEW
4 12.80 ± 0.30 5 of 16

MoleculesMolecules 2019, 24, x FOR PEER REVIEW

2019, 24, 3819 33.30
8.30 ±±0.20
0.60 6 of 16 4 of 16
12
7
15 16 Br 5.80 ± 0.20 8.30
1.8 ±±0.20
0.20
12 NO2 Table 1. Cont.
Molecules 2019, 24, x FOR PEERMolecules O
REVIEW2019, 24, x FOR PEER REVIEW O NO2 4 of 16 6 of 16
F
S. No. R R1 IC50 (µM)
5.20 ± 0.30
5 4 12.80 ± 0.30
Scheme 3. Synthesis of triazinoindole-based thiosemicarbazide analogs (1–25).
17.5 ± 0.30
O
8 of triazinoindole-based
Table 16
20 1. Structure 17
20 1.8 ± analogs
thiosemicarbazide 0.20 7.50
8.80 ±± 0.20
and 0.20
their α-glucosidase
7.50 ± 0.20 activity.
13 23.30 ± 0.50
4
Molecules 2019, 24, x FOR PEER REVIEW
12.80
R NO ± 0.30 R1 IC50 (μM)
S.13O
No. F2 23.30 ±40.50
of 16

Br analogs (1–25).
Scheme 3. Synthesis of triazinoindole-based thiosemicarbazide
2.30 ± 0.05
Cl 5.20 ± 0.30
56 thiosemicarbazide analogs
Table 1. Structure of triazinoindole-based 6.80
and± 0.10
their25.3
1.30 0.50
± 0.05
α-glucosidase activity.
9
17 1 8.80 ± 0.20
21 18 4.80 ± 0.20
21
4 S. No. R O R1
12.80 ± 0.30 IC50Cl(μM) 4.80 ± 0.20
Cl
14 NO O
NO2 2 5.20 ± 0.30 7.5 Cl
± 0.20
O Cl
5 Cl
14 NO 7.5 ± 0.20
2 Cl 2.30 ± 0.05
O 1
1.30 ±
Br 0.05
NO2
Br 35.80 ± 0.802.60 ± 0.05
10 26 Cl 33.30
25.3 ±±0.50
0.60
22 18 5.90 ± 0.10
22 7 O 5.90 ± 0.10
Cl Br
15 5.20 5.80
NO2Cl± 0.20 2.30 ± 0.05
19O O Br O Cl± 0.30
5 Cl
15 O 5.80 ± 0.20
O
6 25.3 ± 0.50 26.80 ± 0.70
O 2.60 ± 0.05
O
2 Br Cl
23 11 Cl 1.30 ± 0.01
NO 17.5 ± 0.30
23 8O 2 33.30 ± 0.60
1.30 ± 0.01
16
19 37 NO Br2 Cl 1.8 ±Cl
2.30 0.05 7.5 ± 0.20
±0.20
Cl 2
Br
Cl
16 NO 1.8 ± 0.20
6 O 25.3 ±F0.50
33.30 Cl
O ± 0.60 F
7
Br
24 3 Cl 7.5 ± 0.20 6.80 ± 0.10 15.3 ± 0.30
17 9 8.80 ± 0.20 17.5 ± 0.30
24 8 15.3 ± 0.30
17 NO NO2 8.80 ± 0.20
2
33.30
NO± 20.60
7 17.5 ± 0.30 2.30 ± 0.05
8 Br
Br 2.30 ±±0.05
35.80 0.80
10 6.80 ± 0.10
25 18 9.30 ± 0.30
9 Cl
18 O Cl
25O NO2 Cl 9.30 ± 0.30
17.5
O ± Cl
8 Acarbose 6.80 ± 0.30
0.10 26.80
38.60 ± 0.70
± 0.20
9 O
Br
11 Br
2.2. Biological Activity NO2 Acarbose 38.60 ± 35.80
0.20 ± 0.80
10
NO2
19
A new class of triazinoindole-bearing thiosemicarbazideCl 2.30 ± 0.05
analogs (1–25) was synthesized and was
evaluated for inhibitory potential. O Cl
All of the synthesized
α-glucosidase
2.2. Biological Activity 19 Cl scaffolds exhibited outstanding
2.30 ± 0.05
Br 6.80 ± 0.10± 0.01
inhibitory 9potential, with IC50 values ranging 35.80
from 1.30
± 0.80 Cl to 35.80 ± 0.80 µM when compared to
10 A new class of triazinoindole-bearing thiosemicarbazide analogs (1–25) was synthesized and
26.80 ±relationship
0.70
standard acarbose with an IC50 value of 38.60 ± 0.20 µM. The structure–activity (SAR) was
was evaluated for α-glucosidase inhibitory potential. All of the synthesized scaffolds exhibited
mainly based upon different
O 2 substation
NO pattern on phenyl rings. Br
outstanding inhibitory potential, with IC50 values ranging from 1.30 ± 0.01 to 35.80 ± 0.80 µ M when
We compared compound 1 (IC11 50 = 1.30 ± 0.05 µM) with a methoxy moiety at position 4 on one
compared to standard acarbose with an IC50 value of 38.60 ± 0.20 µ M. The structure–activity
phenyl ring and two chloro groups at positions 26.80 2 ±and
NO 20.70
3 on the second phenyl ring to scaffold 18
relationship (SAR) was mainly based upon different substation pattern on phenyl rings.
(IC50 = 2.30 ± 0.05 µM), which also had aBrmethoxy moiety at position 4 on one phenyl ring and two
We compared compound 1 (IC 35.80 ± 0.80
Br 50 = 1.30 ± 0.05 µ M) with a methoxy moiety at position 4 on one
chloro groups
10 at positions 3 and 4 on the second phenyl ring. The inhibition difference in these two
11
phenyl ring and two chloro groups at positions 2 and 3 on the second phenyl ring to scaffold 18 (IC50
= 2.30 ± 0.05 µ M), which
O NO 2 also had a methoxy moiety at position 4 on one phenyl ring and two chloro
groups at positions 3 and 4 on the second phenyl ring. The inhibition difference in these two scaffolds
26.80 ± 0.70
Br
11
Molecules 2019, 24, 3819 7 of 16
Molecules 2019, 24, x FOR PEER REVIEW 7 of 16

may havemay
scaffolds beenhave
due to two due
been chloroto groups
two chlorothat were
groups present in different
that were presentpositions on the
in different second phenyl
positions on the
ring. phenyl ring.
second
If we compare scaffold 99 (IC (IC5050 value 6.80 ± ± 0.10
0.10 µµM)
M) to scaffold 1717 (IC
(IC50
50 value 8.80 ± ± 0.20
0.20 µµM),
M),
both scaffolds had a nitro moiety on one phenyl ring, and in both cases the other phenyl ring was
unsubstituted. In In scaffold
scaffold 9, the nitro moiety was present at position 4, while in scaffold 17, the nitro
moiety was present at position 3 on the phenyl ring. The potential potential difference in these two scaffolds
may have been due to the nitro nitro moiety
moiety being
being inin aa different
different position
positionononone
onephenyl
phenylring.
ring.
Similarly, we
Similarly, we compared
comparedcompound
compound15 (IC5050 =
15(IC = 5.80 ±± 0.20
0.20 µµM)
M) with
with aa methoxy
methoxy moiety at position
4 on one phenyl ring and two methyl groups at positions 2 and 6 on the second second phenyl ring to scaffold
50 ==5.90
22 (IC50 5.90± ±0.10 µ M),
0.10 µM),which
which also had
also hada methoxy
a methoxy moiety
moietyat position 4 on4 one
at position phenyl
on one ringring
phenyl and and
two
methyl
two groups
methyl at positions
groups 2 and
at positions 3 on3the
2 and onsecond
the second phenyl ring.ring.
phenyl The inhibition difference
The inhibition in these
difference two
in these
scaffolds
two maymay
scaffolds havehavebeen duedue
been to two
to twomethyl
methyl groups
groups that
thatwere
werepresent
presentinindifferent
different positions
positions on the
second phenyl ring.
It was observed over the whole study that the phenyl ring substituents’ nature as well as their
positions greatly affected the the inhibitory
inhibitory potential
potential of of the
the compound.
compound. A docking study was done to
understand the binding interactions of the most active scaffolds scaffolds with
with the
the enzyme
enzyme active
active site.
site.

2.3. Docking Study

2.3. Docking Study
Docking studies were carried out on scaffolds 1, 16, and 23, which which displayed the most potent
inhibitory potential
potential among
amongthe thewhole
wholeseries.
series.Prior
Priortoto docking
docking and and analysis
analysis of the
of the binding
binding mode mode of
of the
the
mostmost active
active compound,
compound, the docking
1, the1,docking methodmethod was validated
was validated throughthrough the control
the control docking docking of a
of a native
native inhibitor.
inhibitor. AcarboseAcarbose was docked
was docked into into α-glucosidase
α-glucosidase from
from sugar
sugar beet
beet (PDBcode:
(PDB code:3W37)
3W37) andand was
compared
compared by superimposing
superimposing the the native
native ligand
ligand in
in the protein,
protein, as
as mentioned
mentioned in in Imran
Imran et. (2016) [24].
et. al. (2016) [24].
Prior to docking and analysis of the binding mode of the active compounds, the docking method was
validated through the control
control docking of a native inhibitor.
inhibitor. Acarbose was docked into α-glucosidase
α-glucosidase
from sugar beet (PDB code: 3W37) and was compared by superimposing the native ligand in the
protein
protein (Figure
(Figure 2a).
2a). The rmsd value between the docked and actual pose of acarbose was found to be
0.65 Å. Even though α-glucosidase
α-glucosidase from sugar beet shared aa relatively
relatively low
low homology
homology withwith Baker’s
Baker’s
yeast α-glucosidase
α-glucosidase (16% identity), the active site was highly conserved, and the main interactions of
the ligand remained the same. Another Another control docking
docking was done on the target protein, isomaltase
from Baker’s yeast, using α- α-d-glucopyranose,
D-glucopyranose, whichwhich was the native ligand located in the active site
(Figure 2b). The rmsd value for the docked pose and the native ligand was found to to be
be 0.93
0.93 Å.
Å.

Figure 2. Validation
Validationthrough
throughsuperimposition
superimpositionofof(a)
(a)acarbose
acarboseinin3W37
3W37and (b)(b)
and α-D -glucopyranose in
α-d-glucopyranose
3A4A.
in 3A4A.

The
The Chemscore
Chemscorevalues
valuesof of
thethe
active compounds
active are reported
compounds in Tablein2,Table
are reported and these values
2, and correlated
these values
well with the
correlated ICwith
well 50 values.
the ICThe resultsThe
50 values. obtained
resultsshowed that
obtained scaffold
showed 1 recorded
that scaffold the highest Chemscore
1 recorded the highest
Molecules 2019, 24, 3819 8 of 16

Molecules
value, −89.32019, 24, x FOR
kJ/mol, PEER REVIEW
followed by compound 23, which recorded a Chemscore value of −87.7 kJ/mol, 8 of 16

and finally compound 16, which displayed the lowest Chemscore value, −74.5.
Chemscore value, −89.3 kJ/mol, followed by compound 23, which recorded a Chemscore value of
−87.7 kJ/mol, and finally Tablecompound 16,value
2. Chemscore whichfordisplayed the lowest
active compounds Chemscore
1, 16, and 23. value, −74.5.

Compounds Chemscore
Table 2. Chemscore value for (kJ/mol) IC50
active compounds 1, (µM ± SD)
16, and 23.
1 −89.3 1.30 ± 0.05
Compounds Chemscore (kJ/mol) IC50 (μM ±SD)
16 1 −74.5
−89.3 1.301.80 ± 0.20
± 0.05
23 16 −87.7
−74.5 1.801.30 ± 0.01
± 0.20
23 −87.7 1.30 ± 0.01
TheTheresults
results from
from the docking
the docking ofofthese
theseactive
activecompounds
compoundsshowed showedthat thatthey
theywere
were able
abletoto
form
form
several hydrogen bonds within the cavity. The hydrogen on the nitrogen of
several hydrogen bonds within the cavity. The hydrogen on the nitrogen of triazinoindole formed a triazinoindole formed
a hydrogen
hydrogen bond bond with
with the
the backbone
backbone (Oε2)
(Oε2)of ofGlu276,
Glu276,andandthethecatalytic
catalyticresidue
residuewaswasinvolved
involvedininthe the
hydrolysis
hydrolysis reaction
reactionat at
a distance
a distance ofof
2.16 Å.Å .
2.16
InInthe case
the caseofof
compound
compound one
1, 1, ofof
one thethe
nitrogens
nitrogens ononthethe
triazine
triazine moiety
moietyestablished
established a hydrogen
a hydrogen
bonding interaction with the residue of hydrophobic patch Phe300 at a distance
bonding interaction with the residue of hydrophobic patch Phe300 at a distance of 3.42 Å . The of 3.42 Å. The sulfur
sulfur
linkage
linkage displayed hydrogen bonding with the side chain (O) of Glu304 at a distance of 2.23 Å .Å.
displayed hydrogen bonding with the side chain (O) of Glu304 at a distance of 2.23 An
An interaction
interaction involving
involving a halide
a halide bondbond
waswas observed
observed between
between a chlorine
a chlorine substituent
substituent at the at
meta meta
theposition
position
and the and thechain
side side chain
(O) of (O)Thr307
of Thr307
at aatdistance
a distance
of of 1.89Å Å.
1.89 An aromatic
. An aromatic ring
ring containing
containinga amethoxy
methoxy
substituent
substituent formed an electrostatic π-hydrogen interaction with His239 (Hε1) ata adistance
formed an electrostatic π-hydrogen interaction with His239 (Hε1) at distanceofof2.64
2.64Å.Å.
AsAsforfor
the triazine moiety, an electrostatic π-hydrogen involving the residue
the triazine moiety, an electrostatic π-hydrogen involving the residue of hydrophobic patchof hydrophobic patch
Phe177
Phe177was wasexpected
expectedtotostabilize
stabilizethetheligand–enzyme
ligand–enzymecomplexcomplexalongside
alongsideTyr71,
Tyr71,which
whichformed
formedanan
electrostatic π-hydrogen interaction with one of the hydrogens from the
electrostatic π-hydrogen interaction with one of the hydrogens from the triazinoindole moiety triazinoindole moiety atat
aa
distance
distance ofof3.93 Å ÅFigure
3.93 Figure 3. 3.

Figure
Figure 3. 3. Docking
Docking position
position ofof compound
compound 1 in
1 in thethe active
active site
site ofof
thethe α-glucosidase
α-glucosidase enzyme.
enzyme.

The
The analog
analog 2323 docking
docking study
study revealed
revealed that
that this
this scaffold
scaffold was
was capable
capable ofof forming
forming several
several hydrogen
hydrogen
bonds
bonds within
within thethe cavity.
cavity. The
The residue
residue Glu276,
Glu276, through
through itsits backbone
backbone (Oε2),
(Oε2), participated
participated inin hydrogen
hydrogen
bondinginteractions
bonding interactionswithwiththe
theamino
aminogroup
group of
of triazinoindole compound
compound 23 23at
ataadistance
distanceofof2.43
2.43Å Å.
. As
for the triazine moiety, electrostatic
As for the triazine moiety, electrostatic π-hydrogen was involved in the side chain of residue
involved in the side chain of residue Phe157, Phe157,
which
which waswas expected
expected to stabilize
to stabilize the ligand–enzyme
the ligand–enzyme complex.hydrogen
complex. Another Anotherformed
hydrogen formed an
an electrostatic
electrostaticinteraction
π-hydrogen π-hydrogen interaction
with Tyr71. Itwithwas Tyr71. It was also
also observed that observed
His239 was thatcapable
His239ofwas capable
forming anof
forming an electrostatic interaction with the oxygen of the nitro substituent at
electrostatic interaction with the oxygen of the nitro substituent at the meta position. An aromaticthe meta position. An
aromatic
ring ringofconsisting
consisting of dichloro were
dichloro substituents substituents
stabilizedwere stabilized
through throughinteractions
electrostatic electrostatic interactions
with Pro309
with Pro309
through through
electrostatic electrostatic
π-hydrogen π-hydrogen
interactions interactions.
(Figure 4). (Figure 4)
Molecules 2019, 24, 3819 9 of 16
Molecules 2019, 24, x FOR PEER REVIEW 9 of 16
Molecules 2019, 24, x FOR PEER REVIEW 9 of 16

Figure
Figure 4.
4. Docking
Docking position
Docking position of
position of compound
of compound 23
compound 23 in
23 in the
the active
active site
site of
of the
the α-glucosidase
α-glucosidase enzyme.
α-glucosidase enzyme.

The
The docking
docking results
results for compound
for compound
compound 16 16 displayed
displayed fewer interactions
fewer interactions compared
interactions compared
compared to compounds
to compounds
compounds 111
and
and 23. Some of
23. Some
Some of the
of the interactions
the interactions that
interactions that remained
that remained the
remained the same
the same were
same were the
were the interactions
the interactions
interactions of hydrogen
of hydrogen
hydrogen on on the
the
nitrogen of triazinoindole,
triazinoindole, which
which formed
formed a
a hydrogen
hydrogen bond
bond with
with the
the backbone
nitrogen of triazinoindole, which formed a hydrogen bond with the backbone (Oε2) of Glu276 at
backbone (Oε2)
(Oε2) of Glu276
Glu276 at aa
distance of
distance of1.84
of 1.84Å,ÅÅand
1.84 ,, and
andhydrogen
hydrogen
hydrogenformed electrostatic
formed
formed electrostatic interactions
π-hydrogen
π-hydrogen
electrostatic π-hydrogen with Tyr71
interactions
interactions withatTyr71
with a distance
Tyr71 at
at aa
of 3.98
distanceÅ.ofOn3.98theÅ other
. On hand,
the an
other aromatic
hand, an ring consisting
aromatic ring of a fluoro
consisting of substituent
a fluoro at the
substituent
distance of 3.98 Å . On the other hand, an aromatic ring consisting of a fluoro substituent at the para para
at position
the para
displayed
position an electrostatic
position displayed
displayed an interaction
an electrostatic
electrostatic with Glu304.
interaction
interaction withItGlu304.
with was observed
Glu304. It wasthat
It was the CH
observed
observed 3 ofthe
that
that methoxy
the CH
CH33 of the para
ofatmethoxy
methoxy
position
at
at the
the paraof position
para the otherof
position ofaromatic
the
the other
otherring displayed
aromatic
aromatic ring displayed interactionsinteractions
displayed π-hydrogen
ringπ-hydrogen π-hydrogen with Phe157
interactions at aPhe157
with
with distance
Phe157 atof
at aa
4.12 Å.
distance (Figure
of 4.12 5)
Å . (Figure
distance of 4.12 Å . (Figure 5) 5)

Figure
Figure 5.
5. Docking position of compound 16 in the
the active site
site of the
the α-glucosidase
α-glucosidase enzyme.
enzyme.
Figure 5. Docking position of compound 16 in the active site of the α-glucosidase enzyme.

3.
3. Conclusions
Conclusions
Conclusions
In
In conclusion,
conclusion, we we synthesized
synthesized 25
synthesized 25 analogs
analogs of triazinoindole-bearing
of triazinoindole-bearing thiosemicarbazide
thiosemicarbazide and
triazinoindole-bearing thiosemicarbazide and
evaluated
evaluated them against the α-glucosidase enzyme.
α-glucosidase enzyme.
evaluated them against the α-glucosidase enzyme. All All of
All of the
of the synthesized
synthesized scaffolds
the synthesized scaffolds exhibited
exhibited
outstanding
outstanding inhibitory
outstanding inhibitorypotential,
inhibitory potential,with
potential, withIC
with 50 values
ICIC ranging
values
values
50 50 ranging
ranging from
from 1.30
from ±± 0.01
1.301.30 0.01 to
to 35.80
± 0.01 ±± 0.80
to 35.80
35.80 0.80±µµMM when
0.80 µM
when
compared
when to
compared standard
to acarbose
standard with
acarbose an
withIC
compared to standard acarbose with an IC50 value 50 value
an IC 50 of 38.60 ± 0.20 µ M. It was confirmed through the
of
value 38.60
of ± 0.20
38.60 ± µ M.
0.20 It
µM.wasIt confirmed
was confirmedthrough
through
the
SAR
the that
SARSAR polar-
thatthat and
polar-
polar- electron-withdrawing
andand electron-withdrawing
electron-withdrawing groups
groups
groups on
onon the
the
the phenyl
phenylrings
phenyl ringshad
rings hadaaalot
had lotof
lot ofinfluence
of influence on
influence on the
on the
potency of the compounds. AA docking study was done
done to understand the
the binding
potency of the compounds. A docking study was done to understand the binding interactions of the interactions of the
most
most active
active scaffolds.
scaffolds.

4.
4. Experiment
Experiment
Molecules 2019, 24, 3819 10 of 16

4. Experiment

4.1. General Method for the Synthesis of Triazinoindole-Bearing Thiosemicarbazide Analogs (1–25)
A new class of triazinoindole-bearing thiosemicarbazide analogs (1–25) was carried out in
three steps.
In the first step, thiosemicarbazide (10 mmol) was reacted and refluxed with isatin (10 mmol) in
H2 O in the presence of potassium carbonate (5 mmol) to yield 5H-triazinoindole-3-thiol as intermediate
(I). Intermediate product (I) (5 mmol) was then mixed and refluxed with different substituted phenacyl
bromides (5 mmol) in EtOH in the presence of Et3 N to give a triazinoindole derivative as the second
intermediate product (II).
In the second step, hydrazine hydrate (2 mL) was reacted and refluxed with different
isothiocyanates (1 mmol) in methanol to yield a thiosemicarbazide derivative as intermediate
product (III).
In the third step, the intermediate (II) was reacted and refluxed with an equimolar intermediate
(III) in glacial acetic acid to give the final product, triazinoindole-bearing thiosemicarbazide.
2-(2-((5H-[1,2,4]triazino [5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-(2,3-dichlorophenyl)
hydrazine-1-carbothioamide (1) Yield: 62%; 1 H-NMR: (500 MHz, DMSO-d6 ), δ 13.30 (s, 1H, NH), 11.25 (s,
2H, NH), 7.84 (d, J = 7.3 Hz, 2H, Ar), 7.54 (d, J = 6.2 Hz, 1H, Ar), 7.45 (s, 1H, Ar), 7.35 (t, J = 5 Hz, 2H,
Ar), 7.26 (dd, J = 1, 6.65 Hz, 1H, Ar), 7.10 (m, 4H, Ar), 5.2 (s, 2H, CH2 ), 3.81 (s, 3H, OCH3 ). 13 C-NMR
(125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 159, 150.9, 141.2, 131.8, 131.8, 130.3, 128.2, 127.0, 126.9,
126.7, 123, 122.3, 121.2, 119.7, 119.7, 118.3 114.1, 114.1, 111, 104, 55.2, 41.2. HREI-MS: m/z, calcd for
C25 H19 Cl2 N7 OS2 [M]+ 567.0470; found: 567.0458.
2-(2-((5H-[1,2,4]triazino [5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-(4-nitrophenyl)hydrazine
-1-carbothioamide (2) Yield: 68%; 1 H-NMR: (500 MHz, DMSO-d6 ), δ 11.25 (s, 1H, NH), 10.86 (s, 2H, NH),
8.24 (d, J = 7.3 Hz, 2H, Ar), 7.83 (d, J = 7.2 Hz, 2H, Ar), 7.79 (d, J = 7.65 Hz, 2H, Ar), 7.53 (d, J = 6.2 Hz,
1H, Ar), 7.34 (d, J = 1H, Ar), 7.10 (t, J = 6.3, 1H, Ar), 6.98 (t, J = 7.5, 3H, Ar), 4.91 (s, 2H, CH2 ), 3.81 (s,
3H, OCH3 ). 13 C-NMR (125 MHz, DMSO-d6 ), δ 171.9, 165.8, 163.1, 159, 150.9, 141.2, 131.8, 131.8, 130.3,
128.2, 127, 126.9, 126.7, 123, 122.3, 121.2, 119.7, 119.7, 118.3 114.1, 114.1, 111, 104, 55.2, 38.2. HREI-MS:
m/z, calcd for C25 H20 N8 O3 S2 [M]+ 544.1100; found: 544.1088.
2-(2-((5H-[1,2,4]triazino [5,6-b]indol-3-yl)thio)-1-([1,10 -biphenyl]-4-yl)ethylidene)-N-(2,3-dichlorophenyl)
hydrazine-1-carbothioamide (3) Yield: 68%; 1H-NMR: (500 MHz, DMSO-d6 ), δ 11.25 (s, 1H, NH),
10.86 (s, 2H, NH), 8.24 (d, J = 7.3 Hz, 2H, Ar), 7.83 (d, J = 7.2 Hz, 2H, Ar), 7.79 (d, J = 7.65 Hz, 2H, Ar),
7.53 (d, J = 6.2 Hz, 1H, Ar), 7.34 (d, J = 6.5 Hz, 1H, Ar), 7.10 (t, J = 6.3 Hz, 1H, Ar), 6.98–6.93 (m, 3H, Ar),
4.91 (s, 2H, CH2), 3.81 (s, 3H, OCH3 ). 13 C-NMR (125 MHz, DMSO-d6 ), δ 171.9, 165.8, 163.1, 159, 150.9,
141.2, 131.8, 131.8, 130.3, 128.2, 127, 126.9, 126.7, 123, 122.3, 121.2, 119.7, 119.7, 118.3 114.1, 114.1, 111,
104, 55.2, 38.2. HREI-MS: m/z, calcd for C25 H20 N8 O3 S2 [M]+ 544.1100; found: 544.1088.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-([1,10 -biphenyl]-4-yl)ethylidene)-N-(p-tolyl)hydrazine-1-
carbothioamide (4) Yield: 72%; 1 H-NMR: (500 MHz, DMSO-d6 ), δ 11.25 (s, 2H, NH), 10.86 (s, 1H, NH),
8.2 (d, J = 7 Hz, 1H, Ar), 8.01 (m, 2H, Ar), 7.9 (d, J = 7 Hz, 1H, Ar), 7.80 (d, J = 6.2 Hz, 1H, Ar), 7.7 (m,
5H, Ar), 7.6 (d, J = 7.1 Hz, 2H, Ar), 7.4 (m, 3H, Ar), 7.1 (t, J = 6.25 Hz, 1H, Ar), 6.9 (d, J = 6.5 Hz, 1H,
Ar), 4.9 (s, 2H, CH2 ), 3.0 (s, 3H, CH3 ). 13 C-NMR (125 MHz, DMSO-d6 ), δ 192.8, 166.2, 163.1, 146.4,
144.8, 141, 139.5, 139.4, 138.8, 134.7, 133, 129.0, 129.0, 128.9, 128.4, 127.5, 127.0, 126.8, 126.8, 126.4,
126.2,122.4, 122.3, 121.4, 119.8, 117.5, 112.6, 111.0, 106.9, 38.2, 21.3. HREI-MS: m/z, calcd for C31 H25 N7 S2
[M]+ 559.1613; found: 559.1601.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-(p-tolyl)hydrazine-1-
carbothioamide (5) Yield: 73%; 1 H-NMR: (500 MHz, DMSO-d6 ), δ 13.33 (s, 1H, NH), 11.25 (s,2H, NH),
7.8 (d, J = 7.25 Hz, 2H, Ar), 7.79 (d, J = 7.2 Hz, 2H, Ar), 7.54 (d, J = 6.2 Hz, 2H, Ar), 7.35 (m, 2H, Ar),
7.11 (t, J = 6.25 Hz, 2H, Ar), 7.02 (m, 2H, Ar), 5.2 (s, 2H, CH2 ), 3.80 (s, 3H, -OCH3 ), 2.42 (s, 3H, -CH3 ).
Molecules 2019, 24, 3819 11 of 16

13 C-NMR (125 MHz, DMSO-d6 ), δ 165.8, 163.1, 159.0, 150.9, 141.2, 131.8, 130.3, 127.0, 127.0, 127.0, 126.9,
126.7, 122.3, 122.3, 121.0, 119.7, 119.7,114.1, 114.1, 114.1, 114.0, 111.0,104.6, 55.2, 38.2, 22.0. HREI-MS:
m/z, calcd for C26 H23 N7 OS2 [M]+ 513.1405; found: 513.1390.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-([1,10 -biphenyl]-4-yl)ethylidene)-N-(2-bromophenyl)hydrazine-
1-carbothioamide (6) Yield: 77%; 1 H-NMR: (500 MHz, DMSO-d6 ), δ 11.25 (s, 2H, NH), 10.86 (s, 1H, NH),
8.0 (d, J = 7 Hz, 1H, Ar), 7.9 (d, J = 7 Hz, 1H, Ar), 7.7 (d, J = 6.2 Hz, 1H, Ar), 7.5 (d, J = 7 Hz, 2H, Ar), 7.4
(d, J = 7.1 Hz, 2H, Ar), 7.36 (m, 3H, Ar), 7.34 (t, J = 6.25 Hz, 2H, Ar), 7.24 (d, J = 6.6 Hz, 1H, Ar), 7.21 (t,
J = 7.3 Hz, 1H, Ar), 7.17 (d, J = 7 Hz, 1H, Ar), 7 (t, J = 6.9 Hz, 1H, Ar), 6.9 (d, J = 6.5 Hz, 1H, Ar), 4.9 (s,
2H, CH2 ). 13 C-NMR (125 MHz, DMSO-d6 ), δ 192.8, 166.2, 163.1, 146.4, 144.8, 141, 139.5, 139.4, 138.8,
134.7, 133, 129.0, 129.0, 128.9, 128.4, 127.5, 127.0, 126.8, 126.8, 126.4, 126.2,122.4, 122.3, 121.4, 119.8, 117.5,
112.6, 111.0, 106.9, 38.2. HREI-MS: m/z, calcd for C30 H22 BrN7 S2 [M]+ 623.0561; found: 623.0550.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(p-tolyl)ethylidene)-N-(2-bromophenyl)hydrazine-1-
carbothioamide (7) Yield: 68%; 1 H-NMR: (500 MHz, DMSO-d6 ), δ 12.5 (s, 2H, NH), 11.25 (s, 1H, NH),
8.28 (d, J = 6.45 Hz, 1H, Ar), 8.01 (d, J = 6.75 Hz, 3H, Ar), 7.69 (t, J = 6.65 Hz, 2H, Ar), 7.5 (d, J = 6.97 Hz,
2H, Ar), 7.43 (m, 4H, Ar), 4.8 (s, 2H, CH2 ), 2.42 (s, 3H, CH3 ). 13 C-NMR (125 MHz, DMSO-d6 ), δ 171.9,
165.8, 163.1, 151.1, 141.2, 137.2, 131.9, 131.2, 130.4, 130.4, 130.4, 129.3, 129.2, 125.6, 125.6, 125.4, 122.3,
121.2, 119.7, 119.7, 111.0, 109.7, 105.8, 38.2, 20.7. HREI-MS: m/z, calcd for C25 H20 BrN7 S2 [M]+ 561.0405;
found: 561.0392.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(p-tolyl)ethylidene)-N-(p-tolyl)hydrazine-1- carbothioamide
(8) Yield: 65%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 12.5 (s, 2H, NH), 11.4 (s, 1H, NH), 8.25 (d, J = 6.45 Hz,
1H, Ar), 7.98 (d, J = 6.75 Hz, 3H, Ar), 7.69 (m, 2H, Ar), 7.48 (d, J = 6.97 Hz, 2H, Ar), 7.39 (m, 4H, Ar), 4.9
(s, 2H, CH2 ), 2.42 (s, 6H, CH3 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 151.1, 141.2, 137.2,
131.9, 131.2, 130.4, 129.3, 129.3, 129.3, 129.2, 125.6, 125.6, 125.4, 122.3, 121.2, 119.7, 119.7, 111.0, 109.7,
105.8, 38.2, 20.7, 20.7. HREI-MS: m/z, calcd for C26 H23 N7 S2 [M]+ 497.1456; found: 497.1440.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-nitrophenyl)ethylidene)-N-phenylhydrazine-1-
carbothioamide (9) Yield: 71%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 13.34 (s, 1H, NH), 11.25 (s,
2H, NH), 7.79 (d, J = 6.75 Hz, 3H, Ar), 7.54 (d, J = 5.8 Hz, 3H, Ar), 7.36 (ddd, J = 0.9, 6.4 Hz, 1H, Ar),
7.28 (dd, J = 6.55 Hz, 3H, Ar), 7.11 (m, 1H, Ar), 6.97 (d, J = 6.5 Hz, 2H, Ar), 5.1 (s, 2H, CH2 ). 13 C-NMR
(125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 151.1, 141.2, 137.2, 131.9, 131.2, 130.4, 130.4, 130.4, 129.3,
129.3, 129.3, 129.2, 125.6, 125.4, 122.3, 121.2, 119.7, 119.7, 111.0 105.8, 38.2. HREI-MS: m/z, calcd for
C24 H18 N8 O2 S2 [M]+ 514.0994; found: 514.0980.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-(2-bromophenyl)hydrazine-1
-carbothioamide (10) Yield: 71%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 13.30 (s, 1H, NH), 11.25 (s, 1H, NH),
10.86 (s, 1H, NH), 7.84 (t, J = 7.2 Hz, 2H, Ar), 7.54 (d, J = 6.2 Hz, 2H, Ar), 7.35 (t, J = 6.35, 2H, Ar), 7.10 (t,
J = 6.55, 2H, Ar), 7.03 (d, J = 7.1 Hz, 2H, Ar), 6.99 (m, 2H, Ar), 4.80 (s, 2H, CH2 ), 3.81 (s, 3H, OCH3 ).
13 C-NMR (125 MHz, DMSO-d ): δ 171.9, 165.8, 163.1, 151.1, 141.2, 137.2, 131.9, 131.2, 130.4, 130.4, 130.4,
6
129.3, 129.2, 125.6, 125.6, 125.4, 122.3, 121.2, 119.7, 119.7, 111.0, 109.7, 105.8, 38.2, 55.2. HREI-MS: m/z,
calcd for C25 H20 BrN7 OS2 [M]+ 577.0354; found: 577.0342.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(3-nitrophenyl)ethylidene)-N-(2-bromophenyl)hydrazine-1-
carbothioamide (11) Yield: 70%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 13.30 (s, 1H, NH), 11.25 (s, 2H, NH),
8.71 (m, 2H, Ar), 8.37 (d, J = 6.5 Hz, 1H, Ar), 8.33 (d, J = 6.35, 1H, Ar), 8.21 (m, 2H, Ar), 7.95 (s, 1H, Ar),
7.75 (t, J = 6.6 Hz, 1H, Ar), 7.55 (d, J = 6.25 Hz, 1H, Ar), 7.36 (t, J = 6.15 Hz, 1H, Ar), 7.11 (t, J = 6.2, 1H,
Ar), 6.9 (d, J = 6.5, 1H, Ar), 5.5 (s, 2H, CH2 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 151.1,
141.2, 137.2, 131.9, 131.2, 130.4, 130.4, 130.4, 129.3, 129.3, 129.3, 129.2, 125.6, 125.4, 123.3, 121.5, 119.7,
119.7, 111.0 105.8, 38.2. HREI-MS: m/z, calcd for C24 H17 BrN8 O2 S2 [M]+ 592.0099; found: 592.0084.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(3-nitrophenyl)ethylidene)-N-(p-tolyl)hydrazine-1-
carbothioamide (12) Yield: 70%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 11.25 (s, 2H, NH), 10.86 (s, 1H,
Molecules 2019, 24, 3819 12 of 16

NH), 7.80 (m, 3H, Ar), 7.68 (s, 1H, Ar), 7.54 (d, J = 4.8 Hz, 2H, Ar), 7.35 (t, J = 6.4, 1H, Ar), 7.25
(m, 3H, Ar), 7.11 (t, J = 6.3, 1H, Ar), 6.98 (m, 1H, Ar), 4.8 (s, 2H, CH2 ), 1.9 (s, 3H, CH3 ). 13 C-NMR
(125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 151.1, 141.2, 137.2, 131.9, 131.2, 130.4, 130.4, 130.4, 129.3,
129.3, 129.3, 129.2, 125.6, 125.4, 122.3, 121, 119.7, 119.7, 111.0 105.8, 38.2, 21.0. HREI-MS: m/z, calcd for
C25 H20 N8 O2 S2 [M]+ 528.1151; found: 528.1136.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-([1,10 -biphenyl]-4-yl)ethylidene)-N-(2,6-dimethylphenyl)
hydrazine-1-carbothioamide (13) Yield: 64%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 11.25 (s, 2H, NH), 10.86
(s, 1H, NH), 8.0 (d, J = 7 Hz, 1H, Ar), 7.9 (d, J = 7 Hz, 1H, Ar), 7.7 (d, J = 6.2 Hz, 1H, Ar), 7.5 (d, J = 7,
2H, Ar), 7.4 (d, J = 7.1 Hz, 2H, Ar), 7.36 (m, 3H, Ar), 7.34 (d, J = 6.25 Hz, 2H, Ar), 7.21 (d, J = 7.3 Hz, 1H,
Ar), 7.17 (d, J = 7 Hz, 1H, Ar), 7 (t, J = 6.9 Hz, 1H, Ar), 6.9 (d, J = 6.5 Hz, 1H, Ar), 4.9 (s, 2H, CH2 ), 1.88
(s, 6H, CH3 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 192.8, 166.2, 163.1, 146.4, 144.8, 141, 139.5, 139.4, 138.8,
134.7, 133.5, 129.0, 129.0, 128.9, 128.4, 127.5, 127.0, 126.9, 126.8, 126.4, 126.2,122.4, 122.3, 121.4, 119.8,
117.5, 112.6, 111.0, 106.9, 38.2, 21.0, 21.0. HREI-MS: m/z, calcd for C32 H27 N7 S2 [M]+ 573.1769; found:
573.1755.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(3-nitrophenyl)ethylidene)-N-(2,6-dimethylphenyl)hydrazine-
1-carbothioamide (14) Yield: 70%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 12.57 (s, 2H, NH), 11.28 (s, 1H, NH),
8.36 (t, J = 6.5 Hz, 1H, Ar), 8.6(s, 1H, Ar), 8.28 (d, J = 6.4, 2H, Ar), 7.96 (m, 3H, Ar), 7.56 (d, J = 6.75, 2H,
Ar), 7.43 (m, 2H, Ar), 5(s, 2H, CH2 ), 1.91 (s, 6H, CH3 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8,
163.1, 151.1, 141.2, 137.2, 131.9, 131.2, 130.4, 130.4, 130.4, 129.3, 129.3, 129.3, 129.2, 125.6, 125.4, 122.3,
121, 119.7, 119.7, 111.0, 105.8, 38.2, 22.1 22.1. HREI-MS: m/z, calcd for C26 H22 N8 O2 S2 [M]+ 542.1307;
found: 542.1293.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-(2,6-dimethylphenyl)
hydrazine-1-carbothioamide (15) Yield: 75%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 11.25 (s, 2H, NH), 10.86
(s, 1H, NH), 7.84 (d, J = 7.25 Hz, 3H, Ar), 7.54 (d, J = 6.2 Hz, 1H, Ar), 7.34 (d, J = 6.5 Hz, 1H, Ar), 7.10
(t, J = 6.3, 1H, Ar), 6.99 (t, J = 7.2 Hz, 5H, Ar), 4.4 (s, 2H, CH2 ), 3.81 (s, 3H, OCH3 ), 2.3 (s, 6H, CH3 ).
13 C-NMR (125 MHz, DMSO-d ): δ 165.8, 163.1, 159.0, 150.9, 141.2, 131.8, 130.3, 127.0, 127.0, 127.0,
6
126.9, 126.7, 122.3, 122.3, 121.0, 119.7, 119.7,114.1, 114.1, 114.1, 114.0, 111.0,104.6, 55.2, 38.2, 22.2, 22.2.
HREI-MS: m/z, calcd for C27 H25 N7 OS2 [M]+ 527.1562; found: 527.1551.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-(4-fluorophenyl)hydrazine
-1-carbothioamide (16) Yield: 74%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 12.5 (s, 2H, NH), 10.86 (s, 1H, NH),
8.28 (d, J = 6.5 Hz, 1H, Ar), 7.90 (d, J = 7.3 Hz, 2H, Ar), 7.66 (m, 1H, Ar), 7.55 (d, J = 6.75 Hz, 1H, Ar),
7.43 (d, J = 6.4 Hz, 2H, Ar), 7.39 (m, 1H, Ar), 7.08 (d, J = 7.3 Hz, 2H, Ar), 7.02 (d, J = 6.75 Hz, 2H, Ar),
4.94 (s, 2H, CH2 ), 3.46 (s, 3H, OCH3 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 191.5, 166.3, 163.4, 163.3 146.7,
146.3, 140.9, 140.2, 131.5, 131.1, 130.8, 130.6, 129.2, 128.7, 128.6, 122.4, 122.2, 121.7, 121.3, 117.5, 114.0,
113.9, 112.6, 55.5, 38.0. HREI-MS: m/z, calcd for C25 H20 FN7 OS2 [M]+ 517.1155; found: 517.1143.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(3-nitrophenyl)ethylidene)-N-phenylhydrazine-1-
carbothioamide (17) Yield: 69%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 11.27 (s, 2H, NH), 10.86 (s,
1H, NH), 8.6 (s, 1H, Ar), 8.36 (d, J = 6.5, 3H, Ar), 8.19 (m, 1H, Ar), 7.9(d, J = 5.2, 1H, Ar), 7.75 (m, 2H,
Ar), 7.55 (d, J = 6.15 Hz, 2H, Ar), 7.36 (t, J = 6.35, 1H, Ar), 7.18 (t, J = 6.3, 1H, Ar), 6.98 (d, J = 6.4, 1H,
Ar), 4.9 (s, 2H, CH2 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 151.1, 141.2, 137.2, 131.9,
131.2, 130.4, 130.4, 130.4, 129.3, 129.3, 129.3, 129.2, 125.6, 125.4, 122.3, 121.2, 119.7, 119.7, 111.0, 105.8,
38.2. HREI-MS: m/z, calcd for C24 H18 N8 O2 S2 [M]+ 514.0994; found: 514.0980.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl) ethylidene)-N-(3,4-dichlorophenyl)
hydrazine-1-carbothioamide (18) Yield: 71%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 12.6 (s, 2H, NH), 11.25 (s,
1H, NH), 8.28 (d, J = 6.45 Hz, 1H, Ar), 8.10 (d, J = 7.3 Hz, 2H, Ar), 7.69 (m, 2H, Ar), 7.61 (s, 1H, Ar), 7.55
(d, J = 6.7 Hz, 1H, Ar), 7.43 (t, J = 6.3 Hz, 2H, Ar), 7.12 (d, J = 7.35 Hz, 2H, Ar), 4.9 (s, 2H, CH2 ), 3.81(s,
3H, OCH3 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 159, 150.9, 141.2, 131.8, 131.8, 130.3,
Molecules 2019, 24, 3819 13 of 16

128.2, 127, 126.9, 126.7, 123, 121.3, 121.2, 119.7, 119.7, 118.3 114.1, 114, 111, 104, 55.2, 38.2. HREI-MS: m/z,
calcd for C25 H19 Cl2 N7 OS2 [M]+ 567.0470; found: 567.0458.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-([1,10 -biphenyl]-4-yl)ethylidene)-N-(3,4-dichlorophenyl)
hydrazine-1-carbothioamide (19) Yield: 77%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 12.5 (s, 2H, NH), 11.3 (s,
1H, NH), 8.27 (d, J = 6.5 Hz, 1H, Ar), 8.21 (d, J = 6.95 Hz, 2H, Ar), 7.91 (d, J = 6.9 Hz, 2H, Ar), 7.80
(d, J = 6.2 Hz, 2H, Ar), 7.69 (d, J = 7Hz, 2H, Ar), 7.55 (m, 4H, Ar), 7.47(m, 3H, Ar), 5.03 (s, 2H, CH2 ).
13 C-NMR (125 MHz, DMSO-d ): δ 192.8, 166.2, 163.1, 146.4, 144.8, 141, 139.5, 139.4, 138.8, 134.7, 133,
6
129.0, 129.0, 128.9, 128.4, 127.5, 127.0, 126.9, 126.8, 126.4, 126.2,122.6, 122.3, 121.4, 119.8, 117.5, 112.6,
111.0, 106.9, 38.2. HREI-MS: m/z, calcd for C30 H21 Cl2 N7 S2 [M]+ 613.0677; found: 613.0664.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(p-tolyl)ethylidene)-N-(2,6-dimethylphenyl)hydrazine-1-
carbothioamide (20) Yield: 68%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 11.25 (s, 2H, NH), 10.86 (s, 1H, NH),
7.79 (d, J = 6.6 Hz, 2H, Ar), 7.53 (d, J = 4.1 Hz, 3H, Ar), 7.35 (t, J = 6.45 Hz, 1H, Ar), 7.23 (d, J = 6.5 Hz,
3H, Ar), 7.10 (t, J = 6.3 Hz, 1H, Ar), 6.97 (d, J = 6.5, 1H, Ar), 2.3(s, 6H, CH3 ), 4.8 (s, 2H, CH2 ), 1.91 (s, 3H,
CH3 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 151.1, 141.2, 137.2, 131.9, 131.2, 130.4,
129.3, 129.3, 129.2, 129.2, 125.6, 125.6, 125.4, 122.3, 121.2, 119.7, 119.7, 111.0, 109.7, 105.8, 38.2, 21.0, 21.0,
20.7. HREI-MS: m/z, calcd for C27 H25 N7 S2 [M]+ 511.1613; found: 511.1600.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(p-tolyl)ethylidene)-N-(3,4-dichlorophenyl)hydrazine-1-
carbothioamide (21) Yield: 68%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 11.25 (s, 2H, NH), 10.86 (s, 1H, NH),
7.77 (d, J = 6.4 Hz, 2H, Ar), 7.54 (d, J = 4.8 Hz, 2H, Ar), 7.32 (t, J = 6.2 Hz, 1H, Ar), 7.23 (d, J = 6.5 Hz,
3H, Ar), 7.10 (t, J = 6.25 Hz, 2H, Ar), 6.97 (s, 1H, Ar), 4.9 (s, 2H, CH2 ), 1.91 (s, 3H, CH3 ). 13 C-NMR (125
MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 159, 150.9, 141.2,131.8, 131.8, 130.3, 128.2, 127, 126.9, 126.7, 123,
121.3, 121.2, 119.7, 119.7, 118.3 114.1, 114, 111, 104, 38.2, 20.1. HREI-MS: m/z, calcd for C25 H19 Cl2 N7 S2
[M]+ 551.0520; found: 551.0510.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-(2,3-dimethylphenyl)
hydrazine-1-carbothioamide (22) Yield: 62%; 1 H-NMR: (500 MHz, DMSO-d6 ): δ 11.25 (s, 2H, NH), 10.86
(s, 1H, NH), 7.79 (d, J = 6.6 Hz, 2H, Ar), 7.51 (d, J = 4.5 Hz, 3H, Ar), 7.30 (t, J = 6.45 Hz, 1H, Ar), 7.20 (d,
J = 6.7 Hz, 3H, Ar), 7.10 (t, J = 6.3 Hz, 1H, Ar), 6.93 (d, J = 6.5, 1H, Ar), 4.8 (s, 2H, CH2 ), 3.8 (s, 3H,
OCH3 ), 2.3 (s, 6H, CH3 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 151.1, 141.2, 137.2, 131.9,
131.2, 130.4, 129.3, 129.3, 129.3, 129.2, 125.6, 125.6, 125.4, 122.3, 121.2, 119.7, 119.7, 111.0, 109.7, 105.8,
55.2, 38.2, 21.0, 21.0. HREI-MS: m/z, calcd for C27 H25 N7 OS2 [M]+ 527.1562; found: 527.1551.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(3-nitrophenyl)ethylidene)-N-(2,3-dichlorophenyl)hydrazine-
1-carbothioamide (23) Yield: 65%; 1 H-NMR (500 MHz, DMSO-d6 ): δ 11.29 (s, 2H, NH), 10.86 (s, 1H, NH),
8.72 (d, J = 6.4 Hz, 1H, Ar), 8.37 (m, 1H, Ar), 8.23 (d, J = 6.2, 1H, Ar), 7.96 (s, 1H, Ar), 7.75 (m, 3H, Ar),
7.56 (d, J = 6.25, 1H, Ar), 7.37 (t, J = 6.25, 1H, Ar), 7.12 (t, J = 6.25, 1H, Ar), 6.98 (d, J = 6.5, 1H, Ar), 4.8 (s,
2H, CH2 ). 13 C-NMR (125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 159, 150.9, 141.2, 131.8, 131.8, 130.3,
129.2, 127, 126.9, 126.7, 123, 122.3, 121.2, 119.7, 119.7, 118.3 114.1, 114, 111, 104, 38.2. HREI-MS: m/z,
calcd for C24 H16 Cl2 N8 O2 S2 [M]+ 582.0215; found: 582.0202.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-([1,10 -biphenyl]-4-yl)ethylidene)-N-phenylhydrazine-1-
carbothioamide (24) Yield: 74%; 1 H-NMR (500 MHz, DMSO-d6 ): δ 11.27 (s, 2H, NH), 10.86 (s, 1H, NH),
8.2 (d, J = 6.8 Hz, 2H, Ar), 7.75 (m, 7H, Ar), 7.55 (d, J = 6.2 Hz, 1H, Ar), 7.49 (t, J = 6.3 Hz, 3H, Ar), 7.39
(m, 3H, Ar), 7.19 (t, J = 6.25 Hz, 1H, Ar), 6.98 (d, J = 6.45 Hz, 1H, Ar), 5.03 (s, 2H, CH2 ). 13 C-NMR
(125 MHz, DMSO-d6 ): δ 192.8, 166.2, 163.1, 146.4, 144.8, 141, 139.5, 139.4, 138.8, 134.7, 133, 129.0, 129.0,
128.9, 128.4, 127.5, 127.0, 126.8, 126.8, 126.4, 126.2, 122.4, 122.3, 121.4, 119.8, 117.5, 112.6, 111.0, 106.9,
38.2. HREI-MS: m/z, calcd for C30 H23 N7 S2 [M]+ 545.1456; found: 545.1439.
2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4-methoxyphenyl)ethylidene)-N-phenylhydrazine-1-
carbothioamide (25) Yield: 73%; 1 H-NMR (500 MHz, DMSO-d6 ): δ 11.25 (s, 2H, NH), 10.6 (s, 1H, NH),
7.84 (d, J = 7.3 Hz, 3H, Ar), 7.54 (d, J = 6.25 Hz, 1H, Ar) 7.4 (m, 1H, Ar), 7.36 (t, J = 6.4 Hz, 1H, Ar), 7.11
Molecules 2019, 24, 3819 14 of 16

(t, J = 6.75 Hz, 2H, Ar), 6.99 (t, J = 7.1 Hz, 5H, Ar), 4.9 (s, 2H, CH2 ), 3.84 (s, 3H, OCH3 ). 13 C-NMR
(125 MHz, DMSO-d6 ): δ 171.9, 165.8, 163.1, 159.0,150.9, 141.2, 131.8, 130.4, 127.0, 126.9, 126.9, 126.9,
126.7, 122.3, 121.2, 119.7, 119.7,114.2, 114.0, 114.0, 114.0, 111.0, 104.6, 55.2, 38.2. HREI-MS: m/z, calcd for
C25 H21 N7 OS2 [M]+ 499.1249; found: 499.1233.

4.2. α-Glucosidase Assay Protocol

The α-glucosidase activity was executed according to Fazal et al. [31]. The following chemicals
were used (concentrations):

1. 70 µL of 50 mM phosphate buffer (pH 6.8);

2. 10 µL (0.5 mM in methanol) test compounds; and
3. 10 µL (0.057 units, Sigma Inc.) of enzyme solution in buffer.

For details on the experiment, kindly see Reference [31].

4.3. Molecular Docking

Molecular docking was performed on the active compounds to identify possible binding modes
that explained the reason for their potency. The method used for molecular docking was as mentioned
in our previous paper with slight modifications [32]. The molecular docking study was conducted using
a homology model for α-glucosidase. The structures of all compounds were prepared using Chem3D by
CambridgeSoft. The geometry and energy of the structures were optimized using MMFF94. GOLD was
used to identify the binding modes of the active compounds responsible for the activity. The Chemscore
fitness function with default settings was employed in this study. The protein sequence for Baker’s
yeast α-glucosidase (MAL12) was obtained from uniprot (http://www.uniprot.org). A homology model
for Saccharomyces. cerevisiae glucosidase was built using the crystal structure of α-d-glucose-bound
isomaltase from S. cerevisiae (PDB ID: 3A4A), which shares a 72% identical and 85% similar sequence to
α-glucosidase. The sequence alignment and homology modeling were performed using Swiss-Model,
which is a fully automated homology modeling pipeline (SWISS-MODEL) managed by the Swiss
Institute of Bioinformatics. The docking results were visualized using Discovery Studio visualizer 3.5
and PyMol. The homology model was evaluated using PROCHECK.

Author Contributions: Conceptualization, M.T. and F.J.A.; methodology, F.R.; software, S.I.; validation, S.I., and
F.N.; formal analysis, H.U.; investigation, S.H.; resources, K.Z.; data curation, M.T. and F.R.; writing—original
draft preparation, X.X.; writing—review and editing, X.X.; visualization, X.X.; supervision, K.M.K. project
administration, F.J.A., F.R. and; funding acquisition, F.J.A.
Funding: The authors would like to acknowledge the Higher Education Commission of Pakistan for providing a
research grant under the National Research Program for Universities (Project Nos. 5721 & 5092).
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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