Isatin based thiosemicarbazide derivatives as potential inhibitor of α-glucosidase, synthesis and their molecular docking study

Article abstract of DOI:10.1016/j.molstruc.2020.128922

A new series of isatin based thiosemicarbazide derivatives 1–15 were synthesized and characterized by ¹H NMR, ¹³C NMR and HR-EIMS. The synthetic derivatives were evaluated for α-glucosidase inhibitory potential. All compounds showed

Full text of DOI:10.1016/j.molstruc.2020.128922

Journal Pre-proof
Isatin based thiosemicarbazide derivatives as potential inhibitor of
α-glucosidase, synthesis and their molecular docking study
Fazal Rahim , Muhammad Taha , Naveed Iqbal , Shawkat Hayat ,
Faiza Qureshi , Imad Uddin , Khalid Zaman , Abdur Rab ,
Abdul Wadood , Nizam Uddin , Muhammad Nawaz ,
Syed Adnan Ali Shah , Khalid Mohammed Khan
PII:
S0022-2860(20)31247-3
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128922
MOLSTR 128922
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
2 April 2020
15 July 2020
15 July 2020
Please cite this article as: Fazal Rahim , Muhammad Taha , Naveed Iqbal , Shawkat Hayat ,
Faiza Qureshi , Imad Uddin , Khalid Zaman , Abdur Rab , Abdul Wadood , Nizam Uddin ,
Muhammad Nawaz , Syed Adnan Ali Shah , Khalid Mohammed Khan , Isatin based thiosemi-
carbazide derivatives as potential inhibitor of α-glucosidase, synthesis and their molecular docking
study, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128922
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Highlights:

Synthesis of Isatin bearing thiosemicarbazide analogs
 In vitro alpha glucosydase activity



Identification of a new class of alpha glucosydase activity
Structure Activity Relationship established
Molecular docking

Isatin based thiosemicarbazide derivatives as potential inhibitor of α-glucosidase, synthesis
and their molecular docking study
Fazal Rahima, Muhammad Taha*b, Naveed Iqbalc, Shawkat Hayata, Faiza Qureshid,e, Imad
Uddina, Khalid Zamana, Abdur Raba, Abdul Wadoodf, Nizam Udding, Muhammad Nawazd ,
Syed Adnan Ali Shahh,i, Khalid Mohammed Khanj,
aDepatment of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan
bDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia.
cDepartment of Chemistry, University of Poonch, Rawalakot, AJK, Pakistan
dDepartment of Nano-Medicine Research, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
eDeanship of Scientific Research, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam
31441, Saudi Arabia
fDepartment of Biochemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan
gDepartment of Chemistry, University of Karachi, Karachi-75270 Pakistan.
hAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia.
iFaculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
PuncakAlam, Selangor Darul Ehsan, Malaysia
jH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Pakistan
Abstract
A new series of isatin based thiosemicarbazide derivatives 1-15 were synthesized and
1
characterized by H-NMR, 13C-NMR and HR-EIMS. The synthetic derivatives were evaluated
for α-glucosidase inhibitory potential. All compounds showed excellent α-glucosidase inhibitory
potential having IC50 values ranging between 1.20 ± 0.10 to 35.60 ± 0.80 µM when compared
with the standard acarbose having IC50 value 38.60 ± 0.20 µM. Compound 3, 4, 5, 9, 10, 12 and
15 having IC50 values 2.20 ± 0.10, 3.5 ± 0.10, 1.20 ± 0.10, 5.20 ± 0.20, 3.60 ± 0.10, 4.60 ± 0.20
and 3.90 ± 0.10 µM, respectively, was found many fold better than the standard acarbose having
IC50 value 38.60 ± 0.20 µM. Structure activity relationship has been also established for all
newly synthesized compounds, mainly based on substitution pattern on phenyl ring. Through
molecular docking study the binding mode of active derivatives with α-glucosidase enzyme
active site was confirmed.
Keywords: Synthesis, isatin, thiosemicarbazide, SAR, α-glucosidase, molecular docking
*Corresponding Authors : fazalstar@gmail.com, Tel. : 0092-335-9528343 ; mtaha@iau.edu.sa; Tel 00966502057370

1.0. Introduction
α-Glucosidase (EC 3.2.1.20) is a membrane bound enzyme present in the brush-border surface of
small intestine which is responsible for digestion of carbohydrate. It catalyzes the cleavage of 1-
4 linked glycosidic bond of polysaccharide chain to monosaccharaide as a result releasing α-D-
glucose which help in the normal body function [1]. In human, the disorder of α-glucosidase
increase the blood glucose level associated with various diseases including azoospermia [2],
diabetes [3], viral infection [4], and pomp disease [5]. Therefore, in drug discovery α-glucosidase
is recognized an important target. α-Glucosidase enzyme is the restorative target to treat diabetes
disease [6,7]. Specially acarbose, voglibose, and miglitol are clinical approved drugs that are
used as α-glucosidase inhibitors and furthermore they are also act as anti-hepatitis [8], anti-
cancer [9] and anti-HIV agents [10].
Isatin is an important class of nitrogen containing aromatic heterocyclic system present in
numerous plants, human tissues, and blood [11]. Since last few decades isatin and its derivative
gain considerable attention in drug designing in medicinal chemistry [12] and proved their broad
spectrum potentials as anticancer [13], antibacterial [14], antiviral [15] anticonvulsant [16], anti-
inflammatory [17], and antifungal agent [18]. Moreover, some isatin derivatives like sunitinib,
toceranib, nintedanib have been approved for clinical use.
On the other hand thiosemicarbazide and its derivatives occupy a prime place in medicinal
chemistry. Thiosemicarbazide analogs display a diverse range of pharmacological activities like
antifungal [19], anti-prolifrative [20], anti-amoebal [21, 22], antiviral [23], and antimalarial [24]
activities. From more than 50 years p-acetamidobenzaldehyde thiosemicarbazone, trade name
(tibon) has been used for inhibit the growth of Tubercular Mycobacterium [25].
Previously our group has reported isatin based Schiff base as potent α-glucosidase inhibitors
[26].

Fig. 1. Rational of the current study.
Based on our previous experience our continued research, we have synthesized a series of isatin
based thiosemicarbazide analogs and screened for their α-glucosidase inhibitory potential to seek
more potent α-glucosidase inhibitor (Fig. 1). Furthermore, structure-activity relationship has
been established and to explore the interaction of inhibitors with enzyme active site and
molecular docking studies were performed.
2.0. Material and method
For NMR analysis Avance Bruker AV-500 MHz spectrometer was used and DMSO-d6 was used
as solvent and tetramethylsilane was used as internal standard. The product formation was
confirmed via TLC plates which are aluminum plate coated with silica gel (kieselgel 60, 254, E.
Merck, Germany). UV lamp having wavelength ranging from 254 nm to 265 nm was used to
check spots on TLC.
2.1. General procedure for the synthesis of isatin based thiosemicarbazides
Varyingly substituted isothiocyanate reacted and refluxed with hydrazine hydrate in methanol for
3 h which afforded substituted N-phenyl hydrazine carbothioamide as intermediate product (I).

The progress of reaction was checked with the help of TLC. To get the pure product crystals the
solvent was evaporated on a rotary evaporator. Equimolar intermediate (I) was again reacted and
refluxed with different substituted isatin in methanol for 4-5 h to get final products 1-15. The
progress of reaction was checked with the help of TLC. After completion of reaction, solvent
was evaporated to obtained final products and recrystallized from methanol.
2.1.1. N-(2-Bromophenyl)-2- (5-chloro-2-oxoindolin-3-ylidene) hydrazine-1-carbothioamide
(1)
1
Yield: 75% HNMR: (500 MHz DMSO-d6) δ 12.62 (s, 1H, NH), 11.32 (s, 1H, NH), 10.93 (s,
1H, 7.8 (s, 1H, ArH), 7.77 (d, J = 6.8 Hz, 1H, ArH), 7.50 (m, 2H, ArH), 7.43 (m, 1H, ArH) 7.37
13
(m, 1H, ArH), 6.91 (d, J = 6.2 Hz, 1H, ArH). C- NMR (DMSO-d6, 125 MHz): δ 177.5, 162.3,
141.1, 137.4, 132.7, 131.2, 130.6, 130.6, 129.2, 128.2, 126.5, 122.3, 121.7, 120.8, 112.6. HREI-
MS: m/z Calcd for C15H10BrClN4OS, [M]+ 407.9447; Found; 407.9431.
2.1.2. 2-(2-Oxoindolin-3-ylidene)- N-(p-tolyl) hydrazine-1-carbothioamide (2)
1
Yield: 88% H-NMR: (500 MHz DMSO-d6) δ 12.75 (s, 1H, NH), 11.22 (s, 1H, NH), 10.70 (s,
1H, NH), 7.76 (d, J = 5.9 Hz, 1H, ArH), 7.43 (d, J = 6.4 Hz, 2H, ArH), 7.32 (t, J = 6.2 Hz, 1H,
ArH), 7.23 (d, J = 6.3 Hz, 1H, ArH), 7.17 (t, J = 6 Hz, 1H, ArH), 6.91 (d, J = 6.3 Hz, 1H, ArH),
13
2.33 (s, 3H, CH3). C-NMR (DMSO-d6, 125 MHz): δ 176.2, 162.6, 142.4, 135.8, 135.3, 132.0,
131.4, 128.9, 128.9, 125.9, 125.7, 125.6, 121.3, 119.9, 20.5, 11.0, HREI-MS: m/z Calcd for
C16H14N4OS, [M]+ 310.0888; Found; 310.0872.
2.1.3. N-(3,5-Dichlorophenyl) -2-(2-oxoindolin-3-ylidene) hydrazine-1-carbothioamide (3)
1
Yield: 76% HNMR: (500 MHz DMSO-d6) δ 12.91 (s, 1H, NH), 11.33 (s, 1H, NH), 10.92 (s,
1H, NH), 7.77 (s, 1H, ArH), 7.43 (d, J = 6.7 Hz, 2H, ArH), 7.32 (s, 1H, ArH), 7.20 (d, J = 6.7
Hz, 2H, ArH), 7.10 (s, 1H, ArH). 13C-NMR (DMSO-d6, 125 MHz): δ 176.2, 162.6, 152.4, 142.4,
141.6, 138.5, 131.5, 130.1, 130.1, 125.4, 125.4, 123.3, 121.3, 119.9, 111.1. HREI-MS: m/z Calcd
for C15H10Cl2N4OS, [M]+ 363.9952; Found; 363.9935.
2.1.4. N-(2,3-Dichlorophenyl) -2-(2-oxoindolin-3-ylidene) hydrazine-1-carbothioamide (4)
1
Yield: 76% HNMR: (500 MHz DMSO-d6) δ 12.90 (s, 1H, NH), 11.20 (s, 1H, NH), 10.90 (s,
1H, NH), 7.72 (d, J = 6.2 Hz, 1H, ArH), 7.63 (d, J =6.7 Hz, 1H, ArH), 7.53 (d, J = 6.3 Hz, 1H,
ArH), 7.47 (t, J = 6.7 Hz, 1H, ArH), 7.40 (t, J = 6.4 Hz, 1H, ArH), 7.12 (t, J =6 Hz, 1H, ArH),

6.92 (d, J =6.5 Hz, 1H, ArH). 13C-NMR (125 MHz. DMSO-d6,): δ 177.5, 162.6, 142.6, 138.1,
132.9, 131.9, 131.6, 130.2, 129.3, 129.2, 128.0, 122.4, 121.2, 119.7, 111.1. HREI-MS: m/z Calcd
for C15H10Cl2N4OS, [M]+ 363.9952; Found; 363.9935.
2.1.5. N-(2,3-dichlorophenyl) -2-(5-nitro-2-oxoindolin-3-ylidene) hydrazine-1-
carbothioamide (5)
1
Yield: 75% HNMR: (500 MHz DMSO-d6) δ 12.63 (s, 1H, NH), 11.83 (s, 1H, NH), 11.15 (s,
1H, NH), 8.63 (s, 1H, ArH), 8.23 (d, J = 6.9 Hz, 1H, ArH), 7.74 (d, J = 6.4 Hz, 1H, ArH), 7.54
(d, J = 6.7 Hz, 2H, ArH), 7.53 (t, J = 6.6 Hz, 1H, ArH), 7.12 (d, J = 6.2 Hz, 1H, ArH). 13C-NMR
(125 MHz, DMSO-d6,): δ 177.7, 162.8, 142.7, 138.0, 132.0, 130.8, 129.5, 128.1, 127.1, 123.0,
120.7, 116.5, 112.1, 111.2, 109.9. HREI-MS: m/z Calcd for C15H9Cl2N5O3S [M]+ 408.9803;
Found; 408.9787.
2.1.6. N-(2,6-Dimethylphenyl) -2-(2-oxoindolin-3-ylidene) hydrazine-1-carbothioamide (6)
1
Yield: 75% HNMR: (500 MHz DMSO-d6) δ 12.79 (s, 1H, NH), 11.24 (s, 1H, NH), 10.62 (s,
1H, 7.78 (d, J = 6.4 Hz, 1H, ArH), 7.36 (t, J = 6.7 Hz, 1H, ArH), 7.25 (m, 3H, ArH), 7.15 (t, J =
13
6.2 Hz 1H, ArH), 6.92 (d, J = 6.3 Hz, 1H, ArH), 2.22 (s, 6H, CH3). C-NMR (DMSO-d6, 125
MHz): δ 176.9, 162.6, 142.4, 136.1, 136.1, 132.0, 131.2, 127.7, 127.4, 127.4, 122.5, 122.3,
121.2, 119.9, 111.0, 18.0, 17.8. HREI-MS: m/z Calcd for C17H16N4OS [M]+ 324.1045; Found;
324.1028.
2.1.7. N-(2-Bromophenyl) -2-(2-oxoindolin-3-ylidene) hydrazine-1-carbothioamide (7)
Yield: 75% 1HNMR: (500 MHz DMSO-d6) δ 10.71 (s, 1H, NH), 10.55 (s, 1H, NH), 9.53 (s, 1H,
NH), 7.30 (d, J = 6.1 Hz, 2H, ArH), 7.16 (t, J = 6.2 Hz, 2H, ArH), 6.98 (t, J = 6.2 Hz, 2H, ArH),
6.82 (d, J = 6.6 Hz, 2H, ArH). 13C-NMR (DMSO-d6, 125 MHz): δ 177.6, 162.7, 138.6, 128.6,
126.9, 126.2, 124.4, 122.6, 122.2, 121.3, 120.9, 117.5, 117.4, 117.1, 109.9. HREI-MS: m/z Calcd
for C15H11BrN4OS, [M]+ 373.9837; Found; 373.9822.
2.1.8. 2-(5-Chloro-2-oxoindolin-3-ylidene) -N-(p-tolyl)hydrazine-1-carbothioamide (8)
1
Yield: 73% HNMR: (500 MHz DMSO-d6) δ 12.53 (s, 1H, NH), 11.34 (s, 1H, NH), 10.81 (s,
1H, 7.83 (s, 1H, ArH), 7.41 (d, J = 6.5 Hz, 2H, ArH), 7.40 (m, 1H, ArH), 7.22 (d, J = 6.2 Hz, 2H,
ArH) 6.91 (d, J = 6.2 Hz, 1H, ArH), 2.33 (s, 3H, CH3). 13C-NMR (DMSO-d6, 125 MHz): δ 176.2,

162.4, 141.0, 135.6, 135.5, 130.8, 130.5, 128.8, 128.6, 126.5, 125.2, 124.5, 121.8, 120.7, 112.5,
20.6. HREI-MS: m/z Calcd for C16H13ClN4OS, [M]+ 344.0499; Found; 344.0482.
2.1.9.
2-(5-Chloro-2-oxoindolin-3-ylidene)
-N-(3,4-dichlorophenyl)
hydrazine-1-
carbothioamide (9)
1
Yield: 80% HNMR: (500 MHz DMSO-d6) δ 12.73 (s, 1H, NH), 11.44 (s, 1H, NH), 10.95 (s,
1H, NH), 8.13 (s, 1H, ArH), 7.78 (d, J = 6.3 Hz 2H, ArH), 7.55 (s, 1H, ArH) 7.44 (d, J = 6.2 Hz
2H, ArH). 13C-NMR (DMSO-d6, 125 MHz): δ 176.2, 162.5, 141.2, 138.3, 137.1, 131.6, 130.8,
130.5, 128.0, 126.7, 126.2, 125.4, 124.9, 121.7, 111.3. HREI-MS: m/z Calcd for C15H9Cl3N4OS,
[M]+ 397.9563; Found; 397.9548.
2.1.10. 2-(5-Chloro-2-oxoindolin-3-ylidene) -N-(2,3-dichlorophenyl) hydrazine-1-
carbothioamide (10)
1
Yield: 75% HNMR: (500 MHz DMSO-d6) δ 12.63 (s, 1H, NH), 11.32 (s, 1H, NH), 10.91 (s,
1H, NH), 7.74 (s, 1H, ArH), 7.66 (m, 1H, ArH), 7.57 (m, 1H, ArH), 7.44 (s, 1H, ArH), 7.42 (m,
1H, ArH), 6.94 (d, J = 6.2 Hz, 1H, ArH). 13C-NMR (DMSO-d6, 125 MHz): δ 177.7, 162.3, 141.3,
138.0, 131.9, 131.6, 130.8, 130.7, 130.4, 129.4, 128.1, 126.5, 121.6, 120.8, 112.6. HREI-MS:
m/z Calcd for: C15H9Cl3N4OS, [M]+ 397.9563; Found; 397.9549.
2.1.11. 2-(5-Chloro-2-oxoindolin-3-ylidene) -N-(2,6-dimethyl phenyl) hydrazine-1-
carbothioamide (11)
1
Yield: 73% HNMR: (500 MHz DMSO-d6) δ 12.53 (s, 1H, NH), 11.33 (s, 1H, NH), 10.73 (s,
1H, NH), 7.81 (s, 1H, ArH), 7.39 (d, J = 6.3 Hz, 1H, ArH), 7.23 (m, 4H, ArH) 6.90 (d, J =6.2 Hz,
1H, ArH), 2.33 (s, 6H, CH3). 13C- NMR (DMSO-d6, 125 MHz): δ 176.9, 162.3, 141.0, 136.1,
135.9, 135.5, 130.8, 130.4, 127.8, 127.6, 127.4, 126.5, 121.9, 120.8, 112.4, 17.7, 17.6. HREI-
MS: m/z Calcd for C17H15ClN4OS, [M]+ 358.0638; Found; 358.0655.
2.1.12. N-(2-Bromophenyl) -2-(5-nitro-2-oxoindolin-3-ylidene) hydrazine-1-carbothioamide
(12)
1
Yield: 73% HNMR: (500 MHz DMSO-d6) δ 12.52 (s, 1H, NH), 11.81 (s, 1H, NH), 11.14 (s,
1H, NH), 8.65 (s, 1H, ArH), 8.23 (m, 1H, ArH), 7.72 (d, J = 6.3 Hz, 1H, ArH), 7.50 (t, J =6.2
Hz, 2H, ArH), 7.31 (m, 1H, ArH), 7.15 (d, J = 6.5 Hz, 1H, ArH). 13C-NMR (DMSO-d6, 125

MHz): δ 177.7, 162.9, 147.5, 142.7, 137.4, 132.7, 130.8, 130.5, 129.3, 128.2, 127.0, 122.4,
120.8, 116.6, 111.2. HREI-MS: m/z Calcd for C15H10BrN5O3S, [M]+ 418.9688; Found; 418.9672.
2.1.13. N-(3,4-Dichlorophenyl)-2- (5-nitro-2-oxoindolin-3-ylidene) hydrazine-1-
carbothioamide (13)
1
Yield: 80% HNMR: (500 MHz DMSO-d6) δ 12.61 (s, 1H, NH), 11.93 (s, 1H, NH), 11.19 (s,
1H, 8.68 (s, 1H, ArH), 8.27 (d, J = 6.5 Hz 1H, ArH), 7.78 (d, J = 6.3 Hz, 1H, ArH), 7.55 (t, J =
6.2 Hz, 2H, ArH), 7.33 (m, 1H, ArH) 7.15 (d, J =6.2 Hz 1H, ArH). 13C-NMR (DMSO-d6, 125
MHz): δ 176.8, 162.8, 147.4, 143.5, 136.2, 135.9, 130.1, 127.8, 127.4, 126.9, 124.0, 123.0,
122.9, 121.0, 112.1. HREI-MS: m/z Calcd for: C15H9Cl2N5O3S, [M]+ 408.9803; Found;
408.9787.
2.1.14. 2-(5-Nitro-2-oxoindolin-3-ylidene)-N-(p-tolyl) hydrazine-1-carbothioamide (14)
1
Yield: 80% HNMR: (500 MHz DMSO-d6) δ 12.53 (s, 1H, NH), 11.82 (s, 1H, NH), 11.12 (s,
1H, NH), 8.66 (s, 1H, ArH), 8.25 (m, 1H, ArH), 7.47 (d, J = 6.3 Hz, 2H, ArH), 7.22 (d, J =6.2
Hz, 2H, ArH), 7.13 (d, J = 6.2 Hz, 1H, ArH), 2.34 (s, 3H, CH3). 13C-NMR (DMSO-d6, 125
MHz): δ 176.3, 162.9, 147.4, 142.7, 135.6, 130.0, 129.8, 128.8, 127.0, 126.9, 125.6, 125.5,
120.8, 116.4, 111.1, 20.6. HREI-MS: m/z Calcd for C16H13N5O3S, [M]+ 355.0739; Found;
355.0722.
2.1.15.
N-(3,4-Dichlorophenyl)-2-(6-nitro-2-oxoindolin-3-ylidene)
hydrazine-1-
carbothioamide (15)
1
Yield: 75% HNMR: (500 MHz DMSO-d6) δ 12.61 (s, 1H, NH), 11.80 (s, 1H, NH), 11.12 (s,
1H, NH), 8.63 (s, 1H, ArH), 8.31 (m, 1H, ArH), 8.00 (d, J = 6.4 Hz, 1H, ArH), 7.74 (m, 2H,
ArH), 7.13 (d, J = 6.3 Hz, 1H, ArH). 13C-NMR (DMSO-d6, 125 MHz): δ 177.2, 162.8, 147.6,
142.7, 141.6, 138.0, 132.1, 130.1, 129.5, 128.1, 127.1, 123.0, 120.7, 116.5, 111.2. HREI-MS:
m/z Calcd for C15H9Cl2N5O3S, [M]+ 408.9803; Found; 408.9786.
3.0. Molecular docking protocol
Molecular docking study was carried out for all the synthetic derivative against α-glucosidase
enzyme using Molecular Operating Environment (MOE) software package [27]. Due to the
unavailability of the crystallographic structure of the corresponding α-glucosidase enzyme, we

used the homology modeling structural coordinates described by Carreiro et al. [28] for the α-
glucosidase enzyme. Next, the predicted homology model was subjected for protonation, and
then energy was minimized till 0.05 Gradient using Forcefield MMFF94s. The 3D coordinates
for all the derivatives were built in MOE. During the MD approach, all the derivatives were set
to flexible during docking, so that to obtain minimal energy protein-ligand complex. Later, the
docked complexes were ranked based on the docking scores (S). Finally, the predicted ligand-
protein complexes were analyzed for molecular interactions using PyMol v 1.7 Oliveira et al.
[29].
3.1. α-Glucosidase Assay
The α-glucosidase activity was performed according to Imran et al. [30]. The following
chemicals were used (concentrations)
1. 70 µL of 50 mM phosphate buffer (pH 6.8)
2. 10 µL of (0.5 mM in methanol) test compound, and
3. 10 µL of (0.05 units, Sigma Inc.) of α-glucosidase (CAS number 9001-42-7) enzyme
solution in buffer.
4.0. Results and Discussion
4.1. Chemistry
Here in this work fifteen (1-15) analogs were synthesized by reacting variously substituted
isothiocynate with hydrazine hydrate in methanol as solvent and refluxed the reaction mixture
for 3 h to afford substituted N-phenyl hydrazine carbothioamide as intermediate product (I)
(Scheme 1).
Scheme 1. Synthesis of substituted N-phenyl hydrazine carbothioamide (I).

The intermediate, substituted N-phenyl hydrazine carbothioamide (I) was the reacted with varied
substituted isatin in methanol and refluxed for 4-5 h to afford the target compounds (1-15)
(Scheme 2).
Scheme 2. Synthesis of isatin based thiosemicarbazide analog.
4.2. Bioactivities:
Isatin based thiosemicarbazides (1-15) have been synthesized and screened for α-glucosidase
inhibitory potential. All the synthetic derivatives displayed excellent α-glucosidase inhibitory
activity having IC50 value ranging between 1.20 ± 0.10-35.60 ± 0.80 µM as compared to
standard acarbose having IC50 value 38.60 µM. Compounds 3 (IC50 = 2.20 ± 0.10 µM), 4 (IC50 =
3.5± 0.10 µM), 5 (IC50 = 1.2 ± 0.10 µM), 9 (IC50 = 5.20 ± 0.20 µM), 10 (IC50 = 3.60 ± 0.10 µM),
12 (IC50 = 4.60 ± 0.20 µM), and 15, (IC50 = 3.90 ± 0.10 µM) exhibited excellent inhibitory
activity among the series (Table 1). Structure-activity relationships are mainly depending upon
substitution pattern on phenyl ring. All other analogs also exhibited good inhibitory activity.
Compound 5 (IC50 = 1.20 ± 0.10 µM) having 2,3-dichloro substitution on phenyl ring of
isothiocynate and 5-nitro substitution on phenyl ring of isatin was found to be most potent
among the series. The high potency of this analog may be due to the presence of electron
withdrawing groups on both of the phenyl rings. If we compare derivative 5 with residue 12
(IC50 = 4.60 ± 0.20 µM) having 3,4-dichlorosubstitution on phenyl ring of isothiocynate and 5-
nitro substitution on phenyl ring of isatin. The difference in the activity of these molecules may
be due to the different position of chloro group on the phenyl ring of isothiocynate.
Similarly, by comparing scaffolds 3 (IC50 = 2.20 ± 0.10 µM) with 4 (IC50 = 3.5 ± 0.10 µM) both
have di-chloro moieties present on the phenyl ring of isothiocynate, however, there is difference
in their position. Compound 3 have 3,5-dichloro moiety while 4 have 2,3-dicholo moieties
present on the phenyl ring of isothiocynate part. Molecules 3 was found to be more active than 4,

this higher activity indicates that the chloro groups at 3,5 position are more favorable for
inhibition.
On the same pattern, if we compare compound 8 (IC50 = 13.30 ± 0.30 µM) having 5-chloro group
present on phenyl ring of isatin and 4-methyl group on phenyl ring of isothiocynate part with
compound 11 (IC50 = 9.40 ± 0.30 µM) having 5-chloro group present on phenyl ring of isatin and
2-methyl group on phenyl ring of isothiocynate part. The little difference in their activity may be
due to the different position of methyl group on isothiocynate part. The decline in the potential of
these analogs may be due to the presence of electron donating methyl group on the phenyl ring
of isatin part.
On comparing compound 9 (IC50 = 5.20 ± 0.20 µM) with 10 (IC50 = 3.60 ± 0.10 µM) both
compounds have 5-chloro substitution on phenyl ring of isatin while di-chloro substitution on the
phenyl ring of isothiocynate part. Compound 9 having 3,4-dichlorosubstitution while compound
10 having 2,3-dichloro substitution and compound 10 was found to be more potent than
compound 9, the difference in their activity may be due the different positions of chloro group on
the phenyl ring.
Derivative 7 (IC50 = 35.60 ± 0.80µM) was find to be the least potent among the series is having
2-bromo substituent on phenyl ring of isothiocynate part. The decline in the potential of this
analog may be due to the presence of bromine group which might cause some steric hindrance
due to its large size. Generally, electron donating group and large size substituent greatly
decrease the inhibitory potential of these analogs. To understand the binding interaction of the
most active molecules with enzyme active site docking study was also carried out.
Table1
Structures and α-glucosidase inhibitory activity of isatin based thiosemicarbazide analogs.
S. No. Structure
IC50 (µM ±SEMa)

31.50 ± 0.70
1
8.30 ± 0.30
2.20 ± 0.10
2
3
3.5 ± 0.10
1.20 ± 0.10
4
5
11.20 ± 0.30
35.60 ± 0.80
6
7
13.30 ± 0.30
8

5.20 ± 0.20
3.60 ± 0.10
9.40 ± 0.30
4.60 ± 0.213
9
10
11
12
28.60 ± 0.60
8.30 ± 0.30
13
14
15
3.90 ± 0.10
Acarbose
38.60 ± 0.86
SEMa (Standard error mean)
4.3. Molecular Docking

The molecular docking (MD) study was carried out to know the binding mode of the synthetic
molecules with the active site of enzyme. Generally, from the post-docking analysis, we have
noticed that all the enlisted derivatives possess both electron donating group (EDG) and electron
withdrawing group (EWG) at various position over benzene ring. Additionally, it was observed
that the position (-para, -ortho or -meta) ultimately change the activity of enzyme. Consequently,
the MD results revealed that all the compounds participate in binding with the catalytic residue
that play a significant role in enhancing inhibition activity against the α-glucosidase enzyme.
The most potent compounds among the series include compound 5, 3, 4, 10, 15, and 12 have
particularly shown significant interactions with active sites of the α-glucosidase enzyme. In-
depth protein-ligand interaction analysis for the most active compound 5 (1.20 ± 0.10) exhibits
and supports the high potency against the enzyme which adopts most favorable interactions with
active site residues, such as His111, Phe157, Asp214, Arg212, Arg312 and Asp 408 (Fig. 2 A).
The high potency might be due to the presence of (di-Cl) EWG and has a more deactivating
effect on some positions, i.e., -para and -ortho at the benzene ring than others. Halides are -
ortho-para directing groups but unlike most -ortho-para directors, halides mildly deactivate the
arene. Two properties can explain this unusual behavior. First, the halogens are very
electronegative; they cause inductive withdrawal (withdrawal of electrons from the carbon atom
of benzene). Second, halogens possess lone pair of electron and donate electron density through
π-bonding. These -ortho-para directors with the electron from the benzene ring and hence trigger
the positive charge over benzene, so further this ring catches other and make them self-stable in
the reaction center (i.e., here Phe157 adopt π-H interaction with the benzene ring), hence
enzymatic activity enhances. As the variation occurs in the position from -ortho to -para or
reveres ultimately reduce enzymatic activity which was observed same for active compound 3, 4,
10, 15 in the series (Fig. 2B-F).
From molecular docking study it was concluded that all the compounds exhibited enough
favorable interactions with the catalytic residues. The observed gradual decline in docking score
as well as in the mode of interaction of each compound may be based on the attached electron
withdrawing position. Additionally, we have noticed that some compounds possess the same
group over benzene ring but showed less activity against the enzyme, so the following less
activity might be due to the attached another side ED on the compound and hence participating
in enhancing or decline the enzyme inhibition activity.

Fig. 2. The in-depth ligand-protein interaction profile for synthesized derivatives against the
alpha-glucosidase enzyme. The binding mode of the most potent analog (A) for compound 5, (B)
for derivative 3, (C) for residue 4, (D) for molecule 10, (E) for scafold 15, and (F) for compound
12. The double-sided arrows indicate the π-H interaction. The docking score (DS) of each
complex labeled for each.
5.0. Conclusion
In conclusion a new library of isatin based thiosemicarbazides 1-15 synthesized characterized
1
through HNMR, 13CNMR and HR/EIMS. The synthesized analogs were evaluated for α-
glucosidase inhibitory potential in search of lead candidate. All compounds showed excellent α-
glucosidase inhibitory potential having IC50 values ranging between 1.20 ± 0.10-35.60 ± 0.80
µM when compared with the standard acarbose having IC50 value 38.60 ± 0.20 µM. Through
molecular docking study the binding mode of active analogs with α-glucosidase enzyme was
confirmed
Acknowledgement:
Authors would like to acknowledge Higher Education Commission of Pakistan for providing a
research grant under National Research Program for Universities under project No. 5721 & 5092
and Authors would like to acknowledge the Ministry of Higher Education (MoHE) for financial
support under the Fundamental Research Grant Scheme (FRGS) with sponsorship reference

numbers FRGS/1/2019/STG05/UITM/02/9. The author would also like to acknowledge
Universiti Teknologi MARA for the financial support under the reference number 600-
IRMI/FRGS 5/3 (424/2019).
CONFLICTS OF INTEREST
We declare that we have no conflict of interest
AUTHOR STATEMENT
We declare that all authors equally contributed in manuscripts
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GRAPHICAL ABSTRACT