Synthesis of novel triazinoindole-based thiourea hybrid: A study on α-glucosidase inhibitors and their molecular docking

Article abstract of DOI:10.3390/molecules24213819

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.

Full text of DOI:10.3390/molecules24213819

Article
Synthesis of Novel Triazinoindole‐Based Thiourea
Hybrid: A Study on α‐Glucosidase Inhibitors and
Their Molecular Docking
1,
2,3
4,
4
4
Muhammad Taha *, Foziah J. Alshamrani , Fazal Rahim *, Shawkat Hayat , Hayat Ullah ,
4
5,6
7
8
Khalid Zaman , Syahrul Imran , Khalid Mohammed Khan and Farzana Naz
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
Neurology Department, King Fahad hospital of University, Imam Abdulrahman Bin Faisal University,
P.O. Box 1982, Dammam 34211, Saudi Arabia; Fshamrani@uod.edu.sa
Department of Neuroscience Research, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
Department of Chemistry, Hazara University, Mansehra‐21300, Khyber Pakhtunkhwa, Pakistan;
shawkathayat866@yahoo.com (S.H.); ayaanwazir366@gmail.com (H.U.);
2
3
4
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
Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam 40450, Selangor D.E., Malaysia
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Pakistan, khalid.khan@iccs.edu
6
7
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
Molecules 2019, 24, 3819; doi:10.3390/molecules24213819
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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, though there are certain side effect that include abdominal pain, diarrhea, and other
gastrointestinal disorders in chronic therapy [5]. Therefore, in order to treat postprandial
hyperglycemia, a search for efficient and safe α‐glucosidase inhibitors is needed.
Triazinoindole scaffolds possess excellent biological potential against malarial and viral
diseases. Substituted triazinoindole scaffolds are of considerable interest due to their excellent
antihypertensive [6], antidepressant [7], anti‐inflammatory [8], antihypoxic [9], antifungal, and
antibacterial activities [10]. Selected triazinoindole compounds act as potential drugs in treating the
common cold [11–14].
Our research group has been working on the design and synthesis of heterocyclic compounds
in search of potential lead compounds for many years, and we have found promising results [15–29].
We have already reported on triazinoindole analogs as potent α‐glucosidase inhibitors [30]. Thus, we
decided to screen a library of triazinoindole‐bearing thiosemicarbazide analogs for α‐glucosidase
activity (Figure 1).
R
N
N
O
S
H
H
N
N
S
N
S
N
N
N
R
N
N
HN
H
R1
Previously reported
New synthetic analogues 1-25
-Glucosidase inhibitors
Potent -glucosidase inhibitors
Figure 1. Rationale of the current work.
2. Results and Discussion
2.1. Chemistry
A new class of triazinoindole‐based thiosemicarbazide analogs (1–25) was carried out in three
steps.
In the first step, thiosemicarbazide was reacted and refluxed with isatin in H2O in the presence
of potassium carbonate to yield 5H‐triazinoindole‐3‐thiol as an intermediate product (I). The
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.
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)

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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 3. Synthesis of triazinoindole‐based thiosemicarbazide analogs (1–25).
Table 1. Structure of triazinoindole‐based thiosemicarbazide analogs and their α‐glucosidase activity.
R
R1
IC50 (μM)
S. No.
Cl
Cl
1.30 ± 0.05
1
O
2.60 ± 0.05
2
3
NO2
O
Cl
Cl
7.5 ± 0.20

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4
5
6
12.80 ± 0.30
5.20 ± 0.30
O
Br
25.3 ± 0.50
33.30 ± 0.60
7
Br
17.5 ± 0.30
6.80 ± 0.10
8
9
NO2
Br
Br
35.80 ± 0.80
26.80 ± 0.70
10
11
O
NO2

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8.30 ± 0.20
12
13
NO2
23.30 ± 0.50
14
15
7.5 ± 0.20
NO2
5.80 ± 0.20
O
O
16
17
1.8 ± 0.20
F
8.80 ± 0.20
2.30 ± 0.05
NO2
18
19
Cl
Cl
Cl
O
2.30 ± 0.05
Cl

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20
21
7.50 ± 0.20
4.80 ± 0.20
Cl
Cl
22
23
5.90 ± 0.10
1.30 ± 0.01
O
Cl
Cl
NO2
24
25
15.3 ± 0.30
9.30 ± 0.30
O
Acarbose
38.60 ± 0.20
2.2. Biological Activity
A new class of triazinoindole‐bearing thiosemicarbazide analogs (1–25) was synthesized and
was evaluated for α‐glucosidase inhibitory potential. All of the synthesized scaffolds exhibited
outstanding inhibitory potential, with IC50 values ranging from 1.30 ± 0.01 to 35.80 ± 0.80 μM when
compared to standard acarbose with an IC50 value of 38.60 ± 0.20 μM. The structure–activity
relationship (SAR) was mainly based upon different substation pattern on phenyl rings.
We compared compound 1 (IC50 = 1.30 ± 0.05 μM) with a methoxy moiety at position 4 on one
phenyl ring and two chloro groups at positions 2 and 3 on the second phenyl ring to scaffold 18 (IC50
= 2.30 ± 0.05 μM), which 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

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may have been due to two chloro groups that were present in different positions on the second phenyl
ring.
If we compare scaffold 9 (IC50 value 6.80 ± 0.10 μM) to scaffold 17 (IC50 value 8.80 ± 0.20 μM),
both scaffolds had a nitro moiety on one phenyl ring, and in both cases the other phenyl ring was
unsubstituted. In 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 difference in these two scaffolds
may have been due to the nitro moiety being in a different position on one phenyl ring.
Similarly, we compared compound 15 (IC50 = 5.80 ± 0.20 μM) with a methoxy moiety at position
4 on one phenyl ring and two methyl groups at positions 2 and 6 on the second phenyl ring to scaffold
22 (IC50 = 5.90 ± 0.10 μM), which also had a methoxy moiety at position 4 on one phenyl ring and two
methyl groups at positions 2 and 3 on the second phenyl ring. The inhibition difference in these two
scaffolds may have been due to two methyl groups that were present in different 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 inhibitory potential of the compound. A docking study was done to
understand the binding interactions of the most active scaffolds with the enzyme active site.
2.3. Docking Study
Docking studies were carried out on scaffolds 1, 16, and 23, which displayed the most potent
inhibitory potential among the whole series. Prior to docking and analysis of the binding mode of the
most active compound, 1, the docking method was validated through the control docking of a native
inhibitor. Acarbose was docked into α‐glucosidase from sugar beet (PDB code: 3W37) and was
compared by superimposing the native ligand in the protein, as mentioned in Imran 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 docking of a native inhibitor. Acarbose was docked into α‐glucosidase
from sugar beet (PDB code: 3W37) and was compared by superimposing the native ligand in the
protein (Figure 2a). The rmsd value between the docked and actual pose of acarbose was found to be
0.65 Å. Even though α‐glucosidase from sugar beet shared a relatively low homology with Baker’s
yeast α‐glucosidase (16% identity), the active site was highly conserved, and the main interactions of
the ligand remained the same. Another control docking was done on the target protein, isomaltase
from Baker’s yeast, using α‐D‐glucopyranose, which 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 be 0.93 Å.
Figure 2. Validation through superimposition of (a) acarbose in 3W37 and (b) α‐D‐glucopyranose in
3A4A.
The Chemscore values of the active compounds are reported in Table 2, and these values
correlated well with the IC50 values. The results obtained showed that scaffold 1 recorded the highest

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Chemscore value, −89.3 kJ/mol, followed by compound 23, which recorded a Chemscore value of
−87.7 kJ/mol, and finally compound 16, which displayed the lowest Chemscore value, −74.5.
Table 2. Chemscore value for active compounds 1, 16, and 23.
Compounds Chemscore (kJ/mol) IC50 (μM ± SD)
1
16
23
−89.3
−74.5
−87.7
1.30 ± 0.05
1.80 ± 0.20
1.30 ± 0.01
The results from the docking of these active compounds showed that they were able to form
several hydrogen bonds within the cavity. The hydrogen on the nitrogen of triazinoindole formed a
hydrogen bond with the backbone (Oε2) of Glu276, and the catalytic residue was involved in the
hydrolysis reaction at a distance of 2.16 Å.
In the case of compound 1, one of the nitrogens on the triazine moiety established a hydrogen
bonding interaction with the residue of hydrophobic patch Phe300 at a distance of 3.42 Å. The sulfur
linkage displayed hydrogen bonding with the side chain (O) of Glu304 at a distance of 2.23 Å. An
interaction involving a halide bond was observed between a chlorine substituent at the meta position
and the side chain (O) of Thr307 at a distance of 1.89 Å. An aromatic ring containing a methoxy
substituent formed an electrostatic π‐hydrogen interaction with His239 (Hε1) at a distance of 2.64 Å.
As for the triazine moiety, an electrostatic π‐hydrogen involving the residue of hydrophobic patch
Phe177 was expected to stabilize the ligand–enzyme complex alongside Tyr71, which formed an
electrostatic π‐hydrogen interaction with one of the hydrogens from the triazinoindole moiety at a
distance of 3.93 Å Figure 3.
Figure 3. Docking position of compound 1 in the active site of the α‐glucosidase enzyme.
The analog 23 docking study revealed that this scaffold was capable of forming several hydrogen
bonds within the cavity. The residue Glu276, through its backbone (Oε2), participated in hydrogen
bonding interactions with the amino group of triazinoindole compound 23 at a distance of 2.43 Å. As
for the triazine moiety, electrostatic π‐hydrogen was involved in the side chain of residue Phe157,
which was expected to stabilize the ligand–enzyme complex. Another hydrogen formed an
electrostatic π‐hydrogen interaction with Tyr71. It was also observed that His239 was capable of
forming an electrostatic interaction with the oxygen of the nitro substituent at the meta position. An
aromatic ring consisting of dichloro substituents were stabilized through electrostatic interactions
with Pro309 through electrostatic π‐hydrogen interactions. (Figure 4)

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Figure 4. Docking position of compound 23 in the active site of the α‐glucosidase enzyme.
The docking results for compound 16 displayed fewer interactions compared to compounds 1
and 23. Some of the interactions that remained the same were the interactions of hydrogen on the
nitrogen of triazinoindole, which formed a hydrogen bond with the backbone (Oε2) of Glu276 at a
distance of 1.84 Å, and hydrogen formed electrostatic π‐hydrogen interactions with Tyr71 at a
distance of 3.98 Å. On the other hand, an aromatic ring consisting of a fluoro substituent at the para
position displayed an electrostatic interaction with Glu304. It was observed that the CH3 of methoxy
at the para position of the other aromatic ring displayed π‐hydrogen interactions with Phe157 at a
distance of 4.12 Å. (Figure 5)
Figure 5. Docking position of compound 16 in the active site of the α‐glucosidase enzyme.
3. Conclusions
In conclusion, we synthesized 25 analogs of triazinoindole‐bearing thiosemicarbazide and
evaluated them against the α‐glucosidase enzyme. All of the synthesized scaffolds exhibited
outstanding inhibitory potential, with IC50 values ranging from 1.30 ± 0.01 to 35.80 ± 0.80 μM when
compared to standard acarbose with an IC50 value of 38.60 ± 0.20 μM. It was confirmed through the
SAR that polar‐ and electron‐withdrawing groups on the phenyl rings had a lot of influence on the
potency of the compounds. A docking study was done to understand the binding interactions of the
most active scaffolds.
4. Experiment

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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
H2O 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 Et3N 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‐
1
dichlorophenyl)hydrazine‐1‐carbothioamide (1) Yield: 62%; 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 C25H19Cl2N7OS2 [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‐
1
nitrophenyl)hydrazine‐1‐carbothioamide (2) Yield: 68%; 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
13
(s, 2H, CH2), 3.81 (s, 3H, OCH3). 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 C25H20N8O3S2 [M] 544.1100; found: 544.1088.
2‐(2‐((5H‐[1,2,4]triazino
[5,6‐b]indol‐3‐yl)thio)‐1‐([1,1′‐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
13
(m, 3H, Ar), 4.91 (s, 2H, CH2), 3.81 (s, 3H, OCH3). 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 C25H20N8O3S2 [M] 544.1100; found: 544.1088.
2‐(2‐((5H‐[1,2,4]triazino[5,6‐b]indol‐3‐yl)thio)‐1‐([1,1′‐biphenyl]‐4‐yl)ethylidene)‐N‐(p‐tolyl)hydrazine‐1‐
1
carbothioamide (4) Yield: 72%; 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,
13
Ar), 4.9 (s, 2H, CH2), 3.0 (s, 3H, CH3). 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 C31H25N7S2 [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‐
1
carbothioamide (5) Yield: 73%; 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).
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,

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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 C26H23N7OS2 [M] 513.1405; found: 513.1390.
2‐(2‐((5H‐[1,2,4]triazino[5,6‐b]indol‐3‐yl)thio)‐1‐([1,1′‐biphenyl]‐4‐yl)ethylidene)‐N‐(2‐
1
bromophenyl)hydrazine‐1‐carbothioamide (6) Yield: 77%; 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,
13
J = 6.5 Hz, 1H, Ar), 4.9 (s, 2H, CH2). 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 C30H22BrN7S2 [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‐
1
carbothioamide (7) Yield: 68%; 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
13
Hz, 2H, Ar), 7.43 (m, 4H, Ar), 4.8 (s, 2H, CH2), 2.42 (s, 3H, CH3). 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 C25H20BrN7S2 [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
1
(8) Yield: 65%; 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,
13
Ar), 4.9 (s, 2H, CH2), 2.42 (s, 6H, CH3). 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 C26H23N7S2 [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‐
1
carbothioamide (9) Yield: 71%; 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 =
13
6.55 Hz, 3H, Ar), 7.11 (m, 1H, Ar), 6.97 (d, J = 6.5 Hz, 2H, Ar), 5.1 (s, 2H, CH2). 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 C24H18N8O2S2
+
[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‐
1
bromophenyl)hydrazine‐1‐carbothioamide (10) Yield: 71%; 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,
13
CH2), 3.81 (s, 3H, OCH3). 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, 55.2. HREI‐MS: m/z, calcd for C25H20BrN7OS2 [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
1‐carbothioamide (11) Yield: 70%; 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 =
13
6.2, 1H, Ar), 6.9 (d, J = 6.5, 1H, Ar), 5.5 (s, 2H, CH2). 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 C24H17BrN8O2S2 [M] 592.0099; found:
592.0084.

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2‐(2‐((5H‐[1,2,4]triazino[5,6‐b]indol‐3‐yl)thio)‐1‐(3‐nitrophenyl)ethylidene)‐N‐(p‐tolyl)hydrazine‐1‐
1
carbothioamide (12) Yield: 70%; H‐NMR: (500 MHz, DMSO‐d6): δ 11.25 (s, 2H, NH), 10.86 (s, 1H, 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),
13
7.11 (t, J = 6.3, 1H, Ar), 6.98 (m, 1H, Ar), 4.8 (s, 2H, CH2), 1.9 (s, 3H, CH3). 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 C25H20N8O2S2 [M]
528.1151; found: 528.1136.
2‐(2‐((5H‐[1,2,4]triazino[5,6‐b]indol‐3‐yl)thio)‐1‐([1,1′‐biphenyl]‐4‐yl)ethylidene)‐N‐(2,6‐
1
dimethylphenyl)hydrazine‐1‐carbothioamide (13) Yield: 64%; 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
13
(s, 2H, CH2), 1.88 (s, 6H, CH3). 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 C32H27N7S2 [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‐
1
dimethylphenyl)hydrazine‐1‐carbothioamide (14) Yield: 70%; 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,
13
3H, Ar), 7.56 (d, J = 6.75, 2H, Ar), 7.43 (m, 2H, Ar), 5(s, 2H, CH2), 1.91 (s, 6H, CH3). 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
+
C26H22N8O2S2 [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‐
1
dimethylphenyl)hydrazine‐1‐carbothioamide (15) Yield: 75%; 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,
13
6H, CH3). 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, 22.2,
+
22.2. HREI‐MS: m/z, calcd for C27H25N7OS2 [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‐
1
fluorophenyl)hydrazine‐1‐carbothioamide (16) Yield: 74%; 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
13
(d, J = 6.75 Hz, 2H, Ar), 4.94 (s, 2H, CH2), 3.46 (s, 3H, OCH3). 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 C25H20FN7OS2 [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‐
1
carbothioamide (17) Yield: 69%; 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,
13
2H, CH2). 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 C24H18N8O2S2 [M] 514.0994; found: 514.0980.

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2‐(2‐((5H‐[1,2,4]triazino[5,6‐b]indol‐3‐yl)thio)‐1‐(4‐methoxyphenyl)
ethylidene)‐N‐(3,4‐
1
dichlorophenyl)hydrazine‐1‐carbothioamide (18) Yield: 71%; 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,
13
2H, CH2), 3.81(s, 3H, OCH3). 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, 55.2,
+
38.2. HREI‐MS: m/z, calcd for C25H19Cl2N7OS2 [M] 567.0470; found: 567.0458.
2‐(2‐((5H‐[1,2,4]triazino[5,6‐b]indol‐3‐yl)thio)‐1‐([1,1′‐biphenyl]‐4‐yl)ethylidene)‐N‐(3,4‐
1
dichlorophenyl)hydrazine‐1‐carbothioamide (19) Yield: 77%; 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,
13
CH2). 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, 126.9, 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 C30H21Cl2N7S2 [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‐
1
carbothioamide (20) Yield: 68%; 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,
13
3H, CH3). 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 C27H25N7S2 [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‐
1
carbothioamide (21) Yield: 68%; 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,
13
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). 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 C25H19Cl2N7S2
+
[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‐
1
dimethylphenyl)hydrazine‐1‐carbothioamide (22) Yield: 62%; 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),
13
3.8 (s, 3H, OCH3), 2.3 (s, 6H, CH3). 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 C27H25N7OS2 [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‐
1
dichlorophenyl)hydrazine‐1‐carbothioamide (23) Yield: 65%; 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),
13
6.98 (d, J = 6.5, 1H, Ar), 4.8 (s, 2H, CH2). 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 C24H16Cl2N8O2S2 [M] 582.0215; found: 582.0202.
2‐(2‐((5H‐[1,2,4]triazino[5,6‐b]indol‐3‐yl)thio)‐1‐([1,1′‐biphenyl]‐4‐yl)ethylidene)‐N‐phenylhydrazine‐1‐
1
carbothioamide (24) Yield: 74%; 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
13
(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). C‐NMR (125

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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 C30H23N7S2 [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‐
1
carbothioamide (25) Yield: 73%; 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
13
(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). 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
+
C25H21N7OS2 [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 Farzana Naz; 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 conflicts of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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