Synthesis, crystal structure and Hirshfeld Surface analysis of benzamide derivatives of thiourea as potent inhibitors of α-glucosidase in-vitro

Article abstract of DOI:10.1016/j.bioorg.2020.104531

Benzamide based structural analogues 1–15 were synthesized, and evaluated for α-glucosidase inhibition activity in vitro for the first time. Compounds 1–9 were found to be known, while compounds 10–15 were found to be new. However, to the best of our knowledge we are reporting α-glucosidase inhibitory activity of these bezamide derivatives of thiourea for the first time. Compounds 1, 3, 6–8, 10–14 were found to be potent inhibitors of α-glucosidase within IC₅₀ range of 20.44–333.41 μM, in comparison to the standard inhibitor, acarbose (IC₅₀ = 875.75 ± 2.08 μM). Mode of the enzyme inhibition was determined on the basis of kinetic studies which demonstrated that compounds 8, and 10 were non-competitive and competitive inhibitors of α-glucosidase enzyme, respectively. These compounds were also evaluated for their DPPH radical scavenging activity, and cytotoxicity against 3T3 mouse fibroblast cell lines. All synthesized compounds showed a significant to moderate DPPH radical scavenging activity and appeared to be non-cytotoxic except compound 9 which showed cytotoxicity against 3T3 normal mouse fibroblast cell lines. A single crystal X-ray and Hirshfeld Surface analysis of a representative compound is also presented.

Full text of DOI:10.1016/j.bioorg.2020.104531

Journal Pre-proofs
Synthesis, Crystal Structure and Hirshfeld Analysis of Benzamide Derivatives
of Thiourea as Potent Inhibitors of α-Glucosidase in-vitro
Sidra Akhter, Saeed Ullah, SammerYousuf, Atia-tul-Wahab, Hina Siddiqui,
M. Iqbal Choudhary
PII:
S0045-2068(20)31829-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.104531
YBIOO 104531
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 April 2020
1 December 2020
2 December 2020
Please cite this article as: S. Akhter, S. Ullah, SammerYousuf, Atia-tul-Wahab, H. Siddiqui, M. Iqbal Choudhary,
Synthesis, Crystal Structure and Hirshfeld Analysis of Benzamide Derivatives of Thiourea as Potent Inhibitors of
α-Glucosidase in-vitro, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104531
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Synthesis, Crystal Structure and Hirshfeld Analysis of Benzamide
Derivatives of Thiourea as Potent Inhibitors of α-Glucosidase in-vitro
Sidra Akhtera, Saeed Ullaha, SammerYousufa, Atia-tul-Wahabb, Hina Siddiqui*a, and M. Iqbal
Choudhary*a,b,c
aH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi-75270, Pakistan
bDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
cDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah-21452, Saudi Arabia
ABSTRACT
Benzamide based structural analogues 1-15 were synthesized, and evaluated for α-glucosidase
inhibition activity in vitro for the first time. Compounds 1-9 were found to be known, while
compounds 10-15 were found to be new. However, to the best of our knowledge we are reporting
α-glucosidase inhibitory activity of these bezamide derivatives of thiourea for the first time.
Compounds 1, 3, 6-8, 10-14 were found to be potent inhibitors of α-glucosidase within IC50 range
of 20.44-333.41 µM, in comparison to the standard inhibitor, acarbose (IC50=875.75±2.08 µM).
Mode of the enzyme inhibition was determined on the basis of kinetic studies which demonstrated
that compounds 8, and 10 were non-competitive and competitive inhibitors of α-glucosidase
enzyme, respectively. These compoundswere also evaluated for their DPPH radical scavenging
activity, and cytotoxicity against 3T3 mouse fibroblast cell lines. All synthesized compounds
showed a significant to moderate DPPH radical scavenging activity and appeared to be non-
cytotoxic except compound 9 which showed cytotoxicity against 3T3 normal mouse fibroblast cell
lines. A single crystal X-ray and Hirshfeld Surface analysis of a representative compound is also
presented.
O
S
O
O
R2 -NH2
60oC, 2-4 hrs
R2
Acetone
N
H
N
H
+
KSCN
Cl
NCS
R1
R1
R1
rt, 20min
HCl
1-15
Key words: 1-Benzamide derivatives, 2- Thiourea derivatives, 3- α-Glucosidaseinhibitors, 4- Diabetes, 5- Hirshfeld analysis

1.0: INTRODUCTION
α-glucosidase (EC 3.2.1.20) is an exo-type glycosidase enzyme, present on the brush boarders of
small intestine, and catalyzes the hydrolysis of carbohydrates from non-reducing end, yielding
glucose molecules. α-Glucosidase inhibitors (AGIs) slowdown the absorption of glucose into
blood, thus control the postprandial hyperglycemia.AGIs also provide cardiovascular benefits [1].
AGI’s have been termed as ‘untapped diamonds’ of diabeteology and also referred as starch
blocker [2].Therefore, α-glucosidase inhibition has become an important strategy for the
management of type 2 diabetes. Unlike most other types of diabetic drugs, they do not have a direct
effect on insulin secretion instead they work by slowing down the digestion of carbohydrates.
Although AGI’s are effective therapies against diabetes, they are also known to be associated with
various adverse effects, including abdominal bloating caused by fermentation of unabsorbed
carbohydrate, diarrhea, cramping, and colonic gas production [3]. Therefore, there is a need of
new, safe, and effective inhibitors of α-glucosidase enzyme for the treatment of type 2 diabetes
mellitus.
Thiourea forms an important class of organic compounds with diverse applications in the field of
coordination chemistry, therapeutic, metallurgy, analytical and agriculture chemistry [4-7].
Benzamide derivatives of thiourea are known to have a wide range of medicinal properties [8],
including anti-provocative [9], anti-obesity [10, 11], anti-bacterial [12, 13], anti-fungal [14, 15]
anti-glycation [16, 17], and carbonic anhydrase inhibition [18] activities. Metal complexes of
derivatives of thiourea are also well known for their biological activities [19-21] and also reported
to be used for cationic separation of metals [22, 23].
During the current study, phenyl substituted derivatives of thiourea (1-15) were synthesized. All
synthesized derivatives were found to be new except compounds 1-9. An in-vitro mechanism-
based biochemical screening has also been performed to evaluate their α-glucosidase inhibitory
activity. To the best of our knowledge, all the derivatives studied in this study are reported for the
first time as inhibitors of α-glucosidase enzyme. Compounds 1, 3, 6-8, 13, and 10-14 were found
to be potent inhibitors ofα-glucosidase enzyme as compared to the standard Acarbose
(IC50=875.75±2.08 µM). All synthesized derivatives 1-15 were also evaluated for their cytotoxic
effect on 3T3 normal fibroblast cell lines and DPPH radical scavenging activity. A single crystal
X-ray and Hirshfeld Surface analysis of a representative compound is also presented.

2.0: Experimental
2.1. General experimental conditions
The 1H-NMR spectra were acquired on Avance 300, 400, 500 and 600 MHz spectrometers (Bruker
Switzerland) using DMSO-d6. EI-MS analysis was carried out on JEOL JMS-600 mass
spectrometer with associated data system of MASPEC. Melting points of compounds were
recorded on Buchi 434 an electro-thermal melting point equipment. TLC analyses were carried on
pre-coated ALUGRAM, SIL, G/UV254 aluminum plates (Kieselgel 60, 20 x 20, 0.5mm thick, E,
Merck, Germany). Ultraviolet light of 254 nm was used to observe fluorescence quenching spots,
and 365 nm was used for visualization of thin layer chromatogram.
2.2. General synthetic procedure
O
S
O
O
R2 -NH2
60oC, 2-4 hrs
R2
Acetone
N
H
N
H
+
KSCN
Cl
NCS
R1
R1
R1
rt, 20 min
HCl
1-15
Scheme 1: General reaction scheme for the synthesis of benzamide derivatives of thiourea 1-15.
Compounds 1-15 were synthesized by the procedure described by Binzet and co-workers [24].
Substituted benzoyl chloride (1 mmol) was dissolved, followed by addition of 10 mL, i.e. 1 mmol
solution of potassium thiocynate in dry acetone and allowed to reflux for 20 minutes. After 20
minutes, mixture was cooled to room temperature, and 6 mL solution of 1 mmol primary amine,
prepared in dry acetone, was added into it with continuous stirring. After 2 hours, 5 mL of 0.2 N
HCl was added to obtained solid product which was washed with hexane and ethylacetate (9:1)
and recrystallized from 2:1 mixture of dichloromethane: methanol.
All the derivatives were obtained in amorphous state, except compounds 5, and 6, which were
obtained in crystalline state. In EI-MS all synthesized thiourea derivatives showed the molecular
ion peak (M+) except compound 3, that yielded molecular ion peak viasoft technique ESI-MS. FT-
IR spectra were taken within the range of 4000-400 cm-1by using KBr pellets. The 1H-NMR data
for all compounds was recorded in DMSO-d6 at 400 MHz.13C-NMR experiments were conducted

in DMSO-d6 for compounds 13, and 14 at100MHz instrument. UV absorptionstudies were carried
out between 250 to 400 nm.
2.2.1. N-(Phenylcarbamothioyl)benzamide (1):
White. Yield: 72%, m. p. 144-152 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3285; (C-H) 2925;
(C=S) 1261.λmax, nm (CH3OH) (ϵ, M−1cm−1): 212 (7271); 261 (10928);1H-NMR (400 MHz,
DMSO-d6): δ 12.59 (1H, s, N-H), 11.54 (1H, s, N-H), 7.97 (2H, d, J2′,6′ = 7.5Hz, H-2′, H-6′), 7.53
(2H, t, J3′(2′,4′) =J5′(4′,6′)= 7.7 Hz, H-3′,H-5′). 7.26 (1H, t, J4′(3′,5′)= 7.4 Hz, H-4′), 7.67 (3H, m, H-3″,
H-4″, H-5″), 7.42 (2H, t, J2″(3″,5″)=J6″(5″,3″)=9.3 Hz, H-2″, H-6″). EI-MS m/z (rel. int %): 256.2(67)
(M+), 135.2 (64), 1050(100), 77.0(70); HREI-MS: calculated for C14H12N2OS; 256.0670,
observed; m/z 256.0672.
2.2.2. 4-Fluoro-N-(phenylcarbamothioyl)benzamide (2):
White. Yield: 74%, m. p. 146-155°C. FT-IR (KBr pellet cm-1): νmax (N-H) 3272; (C-H) 2924;
(C=S) 1257; (C=O) (C-F) 746. λmax, nm (CH3OH) (ϵ, M−1cm−1):209 (3966); 261 (5322); H1-NMR
(400 MHz, DMSO-d6): δ 12.53 (1H, s, N-H), 11.59 (1H, s, N-H), 8.05 (2H, dd, J3″,5″=5.6 Hz, H-
3″, H-5″), 7.36 (2H, t, J2″ (3″,5″) =J6″(5″,3″)= 8.8 Hz, H-2″, H-6″), 7.26 (1H, t, J4′(3′,5′)= 8.0 Hz, H-4′),
7.67 (2H, d, J2′(3′)=J6′(5′)= 7.7 Hz, H-2′, H-6′), 7.42 (2H, t, J3′(2′,4′)=J5″(4′,6′)= 7.6 Hz, H-3′, H-5′). EI-
MS m/z (rel. int %): 274.2(49) (M+), 135.0(70), 123.0(100), 95(62); HREI-MS: calculated for
C14H11FN2OS; 274.0576, observed; m/z 274.0564.
2.2.3. N-(2-Chloro(phenylcarbamothioyl)-4-fluorobenzamide (3):
White. Yield: 68%, m. p. 200- 210 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3307; (C-H) 3024;
(C=S) 1234; (C=O) 1674; (C-Cl) 754;(C-F); 792; λmax, nm (CH3OH) (ϵ, M−1 cm−1): 212 (6843);
261 (7900); 1H-NMR (400 MHz, DMSO-d6): δ 12.66 (1H, s, N-H), 11.83 (1H, s, N-H), 8.08 (2H,
dd, J3″,5″=5.3 Hz, H-3″, H-5″), 8.01 (1H, d, J2′(3′)= 7.6 Hz, H-2′), 7.58 (1H, dd, J5′= 5.6 Hz, H-5′),
7.37 (4H, m, H-3′, H-4′, H-2″, H-6″). ESI-MS m/z (rel. int %): 309.0(62) (M+), 311.0(24) (M++2);
HRESI-MS: calculated for C14H10ClFN2OS; 308.7584, observed; m/z 309.7234.

2.2.4. N-(3,4-Dimethylphenylcarbamothioyl)-4-fluorobenzamide (4):
White. Yield: 88%, m. p. 157-166 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3350; (C-H) 2924;
(C=O); 1666, (C=S); 1260, (C-F); 793; λmax, nm (CH3OH) (ϵ, M−1cm−1): 220 (8839); 265
(14678);1H-NMR (400 MHz, DMSO-d6): δ 12.43 (1H, s, N-H), 11.51 (1H, s, N-H), 8.02 (2H, dd,
J3″,5″= 5.4 H, €; 7900 L mol-1 cm-1, H-3″, H-5″), 7.13 (1H , d, J2′(3′)= 7.6 Hz, H-2′), 7.37 (3H, m,
H-3′, H-2″, H-6″), 2.19 ( 3H, s, CH3), 2.18 (3H, s, CH3), 3.1 (1H, s, H-5′). EI-MS m/z (rel. int %):
302.2(19) (M+), 163.1(31), 123.1(100), 95(40); HREI-MS: calculated for C16H15FN2OS;
302.0889, observed; m/z 302.0886.
2.2.5. N-(4-Bromophenylcarbamothioyl)-4-fluorobenzamide (5):
White. Yield: 80%, m. p. 217-225 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3243; (C-H) 2927;
(C=S) 1262; (C=O) 1667; (C-F) 758; λmax, nm (CH3OH) (ϵ, M−1cm−1): 215 (10056); 261 (15113);
1H-NMR (400 MHz, DMSO-d6): δ 12.46 (1H, s, N-H), 11.62 (1H, s, N-H), 7.63 (2H, d, J3′,5′= 9.3
Hz, H-3, H-5′), 7.57 (2H, d , J2′,4′ = 9.2 Hz, H-2′, H-4′), 8.02 (2H, dd, J3″,2″=J5”,6″= 5.6 Hz, H-3″,
H-5″), 7.34 (2H, t, J2″(3″,6″)=J6″(5″,2″)= 8.8 Hz, H-2″, H-6″). EI-MS m/z (rel. int %): 352.0 (32)
M+,354.0(35) (M++2), 213.0(64), 171.0(43), 123.0(100); HREI-MS: calculated for
C14H10N2OSFBr; 351.9681, observed; m/z 351.9692 (M+).
2.2.6. 4-Fluoro-N-(4-methoxyphenylcarbamothioyl) benzamide (6):
White. Yield: 62%, m. p. 155-167 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3248; ν(C-H) 2926;
(C=S) 1243; (C=O) 1665; (C-F) 781; λmax, nm (CH3OH) (ϵ, M−1cm−1): 211 (5628); 271 (6706);1H-
NMR (400 MHz, DMSO-d6): δ 12.38 (1H, s, N-H), 11.53 (1H, s, N-H), 8.02 (2H, dd, J3″,5″= 5.6
Hz, H-3″, H-5″), 7.54 (2H, d, J3′(2′)=J5′(6′) = 8.8 Hz, H-3′,H-5′), 6.96 (2H, d, J2′(3′)=J6′(5′) = 2.4 Hz,
H-2′, H-6′), 7.36 (2H ,t , J2″(3″,6″)=J6″(2″,5″) =8.8 Hz, H-2″, H-6″), 3.76 (3H, s, OCH3). EI-MS m/z
(rel. int %): 304.2(8) (M+), 270.2(7), 165.7(18), 123.1(100), 95(25); HREI-MS: calculated for
C15H13FN2O2S; 304.0682, observed; m/z 304.0672.

2.2.7 4-Fluoro-N-(2-methoxyphenylcarbamothioyl) benzamide (7):
White. Yield: 74%, m. p. 171-178 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3390; (C-H) 2930;
(C=S) 1240; (C=O) 1677, (C-F); 747; λmax, nm (CH3OH) (ϵ, M−1cm−1) : 213 (6640); 321 (3760);
1H-NMR (400 MHz, DMSO-d6): δ 12.90 (1H, s, N-H), 11.79 (1H, s, N-H), 8.5 (1H,d, J2′(3′)= 9.3
Hz, H-2), 7.21 (1H , d , J2′(3′) = 2.4 Hz, H-2′), 8.04 (2H, dd, J3″,2″=J5″,6″= 5.2 Hz, H-3″, H-5″), 6.99
(1H, t, J5″(4″)=2.4 Hz, H-5″), 7.12 (1H, m, H-5″), 3.88 (3H, s, OCH3). EI-MS m/z (rel. int %):
304.2(12) (M+), 273.1(43), 165.1(14), 123.0(100), 95 (24); HREI-MS: calculated for
C15H13N2O2SF; 304.0682, observed; m/z 304.0683.
2.3.8. N-(4-Chlorobenzothiazol-2-yl)-4-fluorobenzamide (8):
White. Yield: 52%, m. p. 291-300 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3230; (C-H) 2923;
(C=S) 1232; (C=O) 1681; (C-F); 757; (C-Cl) 679; λmax, nm (CH3OH) (ϵ, M−1cm−1):222 (2840);
288 (1644); 1H-NMR (400 MHz, DMSO-d6): δ 13.03 (1H, s, N-H), 8.04 (2H, m, H-3′, H-5′), 8.20
(1H, dd, J2″=5.6 Hz, H-2″), 7.92 (1H, m, H-5″), 7.27(1H, t, J3′(2′) = 4.0 Hz, H-3′), 7.03 (1H, s, H-
5′), 7.37 (1H, m, H-2′). EI-MS m/z (rel. int %): 306.2(40) (M+), 307.2(18) (M++2) 263.2(14),
140.1(20), 123.1(100), 95(77); HREI-MS: calculated for C14H8ClFN2OS; 306.003, observed; m/z
306.0028.
2.2.9. 4-Fluoro-N-(2-methoxy-4-nitrophenylcarbamothioyl) benzamide (9):
Yellow. Yield: 81%, m. p. 202-210 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3242; (C-H) 2924;
(C=S) 1251; (C=O) 1677; (C-O) 1027; ( NO2) 1327; λmax, nm (CH3OH) (ϵ, M−1cm−1): 211 (4769);
284 (6447);.1H-NMR (400 MHz, DMSO-d6): 13.34 (1H, s, N-H), 11.94 (1H, s, N-H), 8.06 (2H, t,
J3″(2″,4″)=J5″(6″,4″) = 7.0 Hz, H-3″, H-5″), 7.37 (2H ,t , J2″(3″,6″)=J6″(2″,5″) =8.5 Hz, H-2″, H-6″), 9.10
(1H, dd, J2′(3′)=8.3 Hz, H-2′), 7.96 (1H, dd, J3′(2′,5′)= 8.8 Hz, H-3′), 7.90 (1H, s, H-5′), 4.4 (3H, s,
OCH3). EI-MS m/z (rel. int %): 349.1(3) (M+), 318.2(11), 210.0(9) 123.0(100), 95(50); HREI-MS:
calculated for C15H12N3O4SF; 349.0533, observed; m/z 349.0534.

2.2.10. N-(2, 5-Difluorophenylcarbamothioyl)-4-fluorobenzamide (10):
White. Yield: 92%, m. p. 164-170 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3418.6; (C-H) 2925;
(C=S) 1248; (C=O) 1676; (C-F) 711; λmax, nm (CH3OH) (ϵ, M−1cm−1): 212 (6310); 307 (4627);1H-
NMR (400 MHz, DMSO-d6): δ 12.69 (1H, s, N-H), δ 11.90 (1H, s, N-H), 8.06 (2H, dd, J3″,5″= 5.5
Hz, H-3″, H-5″), 8.13 (1H, m, H-5′), 7.36 (2H,t , J2″(3″,6″)= J6″(2″,5″) = 8.9 Hz, H-2″, H-6″), 7.41(1H,
m, H-4′), 7.2 (1H, m, H-3′) EI-MS m/z 310.2(52) (M+), 291(16), 171.2(28), 123.1(100), 95(82);
HREI-MS: calculated for C14H9F3N2OS; 310.0388, observed; m/z 310.0372.
2.2.11. N-(2,6-Diisopropylphenylcarbamothioyl)-4-fluorobenzamide (11):
White. Yield: 61%, m. p. 178-184 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3205; ν(C-H) 2964;
ν(C=S) 1246; ν(C=O) 1674; ν(C-F); 7581. λmax, nm (CH3OH) (ϵ, M−1cm−1):215 (10201); 244
(12633);1H-NMR (400 MHz, DMSO-d6): δ 11.88 (1H, s, N-H), 11.62 (1H, s, N-H)), 8.05 (2H, dd,
J3″,2″=J5″,6″= 5.4 Hz, H-3″, H-5″), 7.21 (2H,d, J3′,4′ =7.6 Hz, H-3′, H-5′), 8.62 (2H, t, J2″(3″,6″)
=
J6″(2″,5″) =8.9 Hz, H-2″, H-6″), 7.30 (1H, t, J3′(2′,4′)= 8.0 Hz, H-3′), 1.20 (6H, s, CH3), 1.10 (6H, s,
CH3). EI-MS m/z (rel. int %): 358.0(1) (M+), 325.0(33), 315.0(69), 186.1(22), 122.9(100), 63(82);
HREI-MS: calculated for C20H23FN2OS; 358.4728, observed; m/z 358.4732.
2.2.12. 4-Fluoro-N-(4-(2-methoxyethyl)-2-methylphenylcarbamothioyl) benzamide (12):
Yellow orange. Yield: 55%, m. p. 154-160 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3350; ν(C-H)
2925; ν(C=S) 1235; ν(C=O) 1666; ν(C-O) 1090; ν(C-F) 735; λmax, nm (CH3OH) (ϵ, M−1cm−1): 213
(6617); 261 (6617);.1H-NMR (400 MHz, DMSO-d6): δ 12.53 (1H, s, N-H), 11.58 (1H, s, N-H),
8.05 (2H, dd, J3″,2″=J5″,6″= 5.3 Hz, H-3″, H-5″), 7.65 (2H,d, J2″(3″,6″)=J6″(2″,5″)=9.0 Hz, H-2″, H-6″),
7.3 (2H, m, H-3′, H-5′), 7.3 (2H, d, J2′,3′= J6′,5′ = 9.0 Hz, H-2′, H-6′), 4.42 (2H, s, CH2), 3.29 (1H,
s, CH3). EI-MS m/z (rel. int %): 318.2(9) (M+), 259.2(13), 179.0(7), 122.9(100), 94.0 (35); HREI-
MS: calculated for C16H15FN2O2S; 318.0838, observed; m/z 318.0854.
2.2.13. N-(3, 4-Dimethylphenylcarbamothioyl)-4-(trifluoromethyl) benzamide (13):
White. Yield: 88%, m. p. 254-260 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3277; ν(C-H) 2923;
ν(C=S) 1259; ν(C=O) 1674; ν(C-F) 777; λmax, nm (CH3OH)(ϵ, M−1cm−1): 211 (6859); 268
(6161);1H-NMR (400 MHz, DMSO-d6): δ 12.56 (1H, s, N-H), 11.79 (1H, s, N-H), 7.40 (1H,s, H-
5′), 8.13 (2H, d, J3″,2″=J5″,6″= 8.5 Hz, H-3″, H-5″), 7.90 (2H ,d , J2″(3″,6″)= J6″(2″,5″)=8.3 Hz, H-2″, H-

6″), 7.17 (1H, d, J3′(2′)=8.1 Hz, H-3′), 7.43 (1H, d, J2′(3′)= 8.64 Hz, H-2′), 2.21 (3H, s, CH3), 2.22
13
(3H, s, CH3). C NMR (300 MHz, DMSO-d6): δ 167.2 (C-1), 178.3 (C-2), 125.5 (C-7″), 136.6
(C-4′′), 136.2 (C-1′′), 132.5 (C-1′), 135.5 (C-4′), 134.5 (C-5′), 129.5 (C-5″, 3″), 125.3 (C-2″, C-
6′′), 121.5 (C-2′), 125. 1 (C-3′, 6′), 19.3 (C-7′), 18.9 (C-8′); EI-MS m/z (rel. int %):352.2(13) (M+),
318.2(12), 173.0(100), 163.0 (27), 145.0 (59); HREI-MS: calculated for C17H15N2OSF3; 352.0857,
observed; m/z 352.0876.
2.2.14. N-(4-(Trifluoromethoxy) phenylcarbamothioyl)-4-(trifluoromethyl) benzamide (14):
White. Yield: 79%, m. p. 169-178 °C. FT-IR (KBr pellet cm-1): νmax (N-H) 3227; ν(C-H) 2923;
ν(C=S) 1275; ν(C=O) 1685; ν(C-O) 1012; ν (C-F) 717; λmax, nm (CH3OH) (ϵ, M−1cm−1): 208
(7918); 261 (9196);1H-NMR (400 MHz, DMSO-d6): δ 12.36 (1H, s, N-H), 11.89 (1H, s, N-H),
7.67 (1H,s, H-2′), 8.13 (2H, d, J3″,2″=J5″,6″= 8.2 Hz, H-3″, H-5″), 7.90 (2H ,d , J2″(3″,6″)= J6″(2″,5″)
=
7.6 Hz, H-2″, H-6″), 7.79 (2H, d, J3′,2′=J5′,6′= 9.0 Hz, H-3′, H-5′), 7.42 (2H, d, J2′,3′=J6′,5′= 8.4 Hz,
H-2′, H-6′), 7.40 (1H, d, J4′(5′)= 8.4 Hz, H-4′).13C-NMR (100 MHz, DMSO-d6). EI-MS m/z (rel. int
%): 408.2(17) (M+), 349.2(10), 219.2(42), 173.0(100), 145.0(71); HREI-MS: calculated for
C16H10N2O2SF6; 408.3182, observed; m/z 408.0355.
2.2.15. N-(3-(Methylthio) phenylcarbamothioyl)-4-(trifluromethyl)benzamide (15):
Pale yellow. Yield: 94%, m. p. 138-146°C. FT-IR (KBr pellet cm-1): νmax (N-H) 3322; ν(C-H)
2921; ν(C=S) 1261; ν(C=O) 1671; ν(C-F) 796; λmax, nm (CH3OH) (ϵ, M−1cm−1): 215 (10503); 26
(10274);1H-NMR (400 MHz, DMSO-d6): δ 12.35 (1H, s, N-H), 11.83 (1H, s, N-H), 7.67 (1H,s,
H-2′), 8.13 (2H, d, J3″,2″=J5″,6″= 8.0 Hz, H-3″, H-5″), 7.90 (2H ,d , J2″(3″,6″)= J6″(2″,5″) = 8.3 Hz, H-
2″, H-6″), 7.16 (1H, d, J6′(5′)= 7.4 Hz, H-6′), 7.35 (1H, t, J5′(4′,6′)= 7.6 Hz, H5′), 7.40 ( 1H, d, J4′(5′)
=
8.4 Hz, H-4′), 2.25 (3H, s, CH3). EI-MS m/z (rel. int %): 370.2(23) (M+), 336.2 (9), 173.1(100),
145.0(50); HREI-MS: calculated for C16H13N2OS2F3; 370.0421, observed; m/z 370.0427.
3.0:Results and Discussion:
3.1.0. Chemistry:
Structure elucidation of a representative compound (13) is presented here. It was obtained as an
amourphous white fluffy powderwith 88% yield.

High resolution mass, calculated for C17H15N2OSF3 (352.0857) was obtained by EI-MS technique,
atm/z 352.0876 as the (M+) molecular ion peak.The major fragments of the compounds appearded
at 718.2, 173.0, and 146.2m/z. FT-IR spectra showed sharp and strong stretching frequencies for
N-H, C-H, C=S, C=O and C-F at3277, 2923, 1259, 1674 and 777 cm-1, respectively.
1H-NMR spectroscopic study for compound 13 revealed that the protons of two secondary amines
(a and b, Figure-1) appeared as singlet at δ 12.56, and δ 11.79, respectively.C-2″ and C- 6″ protons
appeared at δ 8.13 as a doublet with J=8.3 Hz, because of same environment of aromatic ring.
Similarly, two other aromatic protons of C-3″ and C-5″ resonated at δ 7.90 ppm (J = 8.5 Hz) as a
doublet. One singlet of aromaticH-6′ appeared at δ 7.40. Proton C-2′ appeared at δ 7.43 (J = 8.6
Hz) as a doublet due to coupling with 3′ proton. WhileH-2′ resonated at the chemical shift δ 7.17as
a doublet (J = 8.1 Hz).
13C-NMR spectrum of compound 13 showed 16 carbon signals, including 7 quaternary carbons, 7
methine, and 2methyl carbons. Sulphur substituted quaternary carbon appeared as the most
downfield carbon at δ 178.6. Carbon of C=O appeared at δ 167.2. Methine carbons of aromatic
rings having similar environment appeared at δ 125.2 (C-3′/C-6′), δ 125.3 (C-2″/ C-6″), and 129.5
(C-3″/C-5″), whereas methine C-2′ appeared at δ 121.5. Two methyl carbons appeared at δ 135.5,
and δ 134.5. Other quaternary carbons appeared at δ 136.2 (C-1″), 139 (C-4′), 136.5 (C-4″), and
134.5 (C-5′).
125.1
7.17 (d)
J
3',2'= 8.15 Hz
7.43 (d)
J2',3'= 8.64 Hz
2.21 (s)
19.3
CH3
3'
4'
121.5
8.13 (d)
S
135.5
O
2'
1'
J2",3"= 8.46 Hz
125.3
7.90 (d)
5'
6'
J3",2"= 8.35 Hz
132.5
134.5
2"
3"
178.3
167.2
136.2
N
H
N
H
CH3
129.5
2.22 (s)
19.0
1"
6"
7.40 (s)
125.1
(a)
(b)
136.6
F3C
4"
5"
11.79 (s)
12.56 (s)
8.13(d)
J6",5"= 8.46 Hz
125.5
7.90 (d)
J5",6"= 8.35 Hz
125.3
129.5
Figure-1: 1H-NMR and 13C-NMR peak assignment of compound 13.
3.1.1. X-ray Diffraction Study:

3.1.1.1. Structure Features
The structure determination of compound 13 (Figure-2) represents that the structure is composed
of a planar trifluoro methyl substituted benzoyl ring (C1-C6) and a dimethyl substituted phenyl
rings (C9-C12) linked with each other via thiourea moiety along N1-C7 and N2-C9 bonds having
length of 1.3588 Å, and 1.7521 Å. The dihedral angle between these planar rings was found to be
22.72o with maximum deviation of 0.014(3) Å, for C9 from root mean square plane (r.m.s). The
bond length for trifluoro methyl attached to C3 was found to be 1.4361(15) Å. The bond length
for methyl substituent attached to C9 and C12 were found to be 15.081(3) Å, 15.042(3) Å,
respectively.
Figur-2: ORTEP view of X-ray structure of compound 13 at 50% ellipsoid probability.
3.1.1.2. Crystal Packing analysis
In the crystal lattice of compound 13 molecules were arranged in parallel repeating pattern running
along c-axis (Figure 3) via N2—H2N_ _ _S1, C10—H10_ _ _S1, C11—H11_ _ _S1, and C17—H17A_
_ _F3, hydrogen bonds with donor to acceptor distances 3.5772 (2) Å, 3.213 (2) Å, 3.668 Å and
3.368 (3) Å, respectively. The geometry of this compound along thioamide linkage is stabilized

by intra-molecular hydrogen bonds N1—H1N_ _ _O1 and C5—H5_ _ _S1 to form S6 graph set
motif with donor to acceptor distance 2.645(3) Å and 3.195 (2) Å, respectively.
Figure-3: Arrangement and hydrogen bond interactions of molecules of compound 13 in crystal
lattice.
3.1.1.3. Hirshfeld Surface
The Hirshfeld surface analysis is carried out (dnorm, curvedness and shape index) in order to analyze
the intermolecular interaction within the crystal, and 2D fingerprint plots were generated by using
Crystalexplorer3.1. The Hirshfeld surface analysis suggested that in compound 13 the major
contributions towards the crystal packing include S---H (11.6%) and H---H (35.9%) followed by
other interactions C-H (12.7%), F---H (11.7%), O---H (12.0%), and C---C (8. 7%).Two
dimensional finger plots showing the occurrence of all intermolecular contacts is represented in
Figure.4.

Figure 4: The dnorm mapped on Hirshfeld surface for visualizing the intermolecular contacts of the
compound 13.
Figure 5: Two-dimensional fingerprint plot of compound 13 showing the percentage contribution
of intermolecular interaction.

Table-1: Data Collection and refinement parameter of compound 13
Empirical formula
Formula weight
Temperature
C17 H15 F3 N2 O S
352.37
100(2) K
Wavelength
1.54178 Å
Crystal system
Space group
Triclinic
P -1
Unit cell dimensions
a = 4.6790(4) Å α= 77.582(5) °
b = 12.1130(11) Å β= 85.653(5)°
c = 14.5622(14) Å γ = 89.977(5)°
3
Volume
803.60(13) Å
Z
2
3
Density (calculated)
1.456 Mg/m
-1
Absorption coefficient
F(000)
2.153 mm
364
3
Crystal size
0.280 x 0.070 x 0.040 mm
Theta range for data collection
Index ranges
3.117 to 68.181°.
-5<=h<=5, -14<=k<=14, -17<=l<=17
21555
Reflections collected
Independent reflections
Completeness to theta = 67.679°
2952 [R(int) = 0.0628]
99.8 %

Absorption correction
Refinement method
None
2
Full-matrix least-squares on F
Data / restraints / parameters
2952 / 0 / 219
2
0.829
Goodness-of-fit on F
Final R indices [I>2sigma(I)]
R indices (all data)
R1 = 0.0457, wR2 = 0.1159
R1 = 0.0544, wR2 = 0.1239
n/a
Extinction coefficient
-3
Largest diff. peak and hole
0.500 and -0.298 e.Å
3.2.0
Bioactivity Evaluation:
Compounds 1-15 were evaluated for their α-glucosidase inhibitory activity in-vitro by employing
the reported procedure [25]. All tested compounds were found to be more active than the standard
drug acarbose (IC50=875.75±2.08 µM), except compounds 2, 4, 5, 9, and 14 which were found to
be insoluble under required buffer conditions.
The parent compound 1 having unsubstituted benzene rings attached to thiourea moiety appeared
as a potent inhibitor (IC50 = 171.21±0.23 µM) in comparison to the standard acarbose (IC50 =
875.75± 2.08 µM). In compounds 1 to 12 the R1substituent is Fluorine atom which is substituted
at para position of the phenyl ring, while the R2 substituent was varied. Substitution of alkyl (-
CH3, -CH(CH3)2) alkoxy(-OCH3), and halogens (Cl, Br, F) at different positions on benzamide
ring showed variation in the α-glucosidase inhibitory activities. Compound 11 in which the R2
isdi-ortho isopropyl group was found to be the most active member of the series with IC50 = 40.9
± 0.74 µM, followed by difluoro substituted phenyl ring in compound 10 (IC50=34.63 ± 0.16 µM),
further decrease in activity was observed in compound 3 (IC50 = 163.6± 0.61 µM) having chloro
substituent at ortho position of the phenyl ring. Compound 7 with an ortho-alkoxy group showed
more α-glucosidase inhibitory activity (IC50 = 105.2 ± 0.75 µM) than compound 6 having an

alkoxy substituent but at para position (IC50 = 333.41 ± 0.85 µM). A several fold decrease in the
inhibitory activity observed in compound 12 in which the methoxy methyl group is present at para
positions instead of alkoxygroup (IC50 =218.64 ± 1.14µM)
In compound 8 presence of heterocyclic ring with nitrogen and sulfur, fused with ortho chlorine
substituted phenyl ring, showed a potent activity (IC50 = 20.44 ± 1.73 µM), made it the most potent
compound in the series. SAR studies concluded that nitrogen and sulfur containing heterocyclic
rings apparently contribute towards in significant inhibitory activity against α-glucosidase. Kinetic
studies of compound 8 showed a non-competitive type of inhibition, it is revealed from inhibition
pattern that compound 8 binds with allosteric site residues of the α-glucosidase.
In compounds 13-15the R1 (F) was replaced with trifluoromethyl group. Compound 13 having
para and meta-methyl groups possessed potent inhibitory activity (IC50 = 60.41 ± 0.72 µM), which
is further decreased in para trifluoro methoxy substituted compound 14 (IC50 = 92.36 ± 0.11 µM).
In conclusion, SAR studies revealed that the number, position and nature of electron donating and
withdrawing groups significantly alter the inhibitory potential of synthesized thiourea derivatives.
Compounds 1-15 were also evaluated for DPPH radical scavenging activity [26, 27]. and
cytotoxicity against 3T3 mouse fibroblast cell line [28]. All compounds showed a weak to
moderate activity against DPPH radical scavenging activity and non-cytotoxic in nature, except
compound 12.
3
2
1
4
5
O
S
2
5
R2
1
3
4
N
H
N
H
6
R1
6
R1= F
Increase activitiy
Increase activitiy
halogens (Cl, Br, F)
R2=
R2= -O-CH3 (ortho)
R2= -O-CH3 (para)
Decrease activitiy
The summary of biological activity is given in Table 2.

Table 2: Summary of biological activities of compounds 1-15
α-G1ucosidase
Cytotoxicity
Assay
inhibition
DPPH Radical
Scavenging
Assay
(anti-
against 3T3
cell line
hyperglycemic)
Comp.#
-R1
-R2
IC50±SEM
IC50±SEM
IC50±SEM
[µM]
[µM]
[µM]
171.21±0.23
1
2
-
-
-
NA
NA
NA
206.7±3.6
4-Fluoro
ND
3
4
5
4-Fluoro
4-Fluoro
4-Fluoro
2-Chloro
163.6±0.61
ND
NA
NA
NA
NA
4,5-Dimethyl
4-Bromo
NA
ND
NA
117.2±1.87
6
7
4-Fluoro
4-Fluoro
4-Methoxy
333.41 ±0.69
105.2±0.82
NA
NA
209.3±2.12
2-Methoxy
4-chloro-2-
8
4-Fluoro
methylbenzo[d] 20.44±1.73
thiazole
NA
NA

29.1±1.9
2-Methoxy, 4-
Nitro,
9
4-Fluoro
ND
NA
10
11
4-Fluoro
4-Fluoro
2,5-Difluoro
34.63±0.16
NA
NA
NA
2,6-Diisopropyl 40.9±0.74
NA
218.64±1.14
4-
158.3±3.02
12
13
4-Fluoro
NA
NA
Methoxymethyl
60.41±0.72
226.5±0.34
4-Trifluoro 4,5-Dimethyl
4-
14
15
4-Trifluoro Trifluorometho 92.37±0.11
xy
NA
NA
NA
NA
3-
4-Trifluoro
ND
Methylsulfane
ACARBOSE
875.75±2.08
-
-
Standard
GALLIC ACID
-
-
26±3.15
-
-
CYCLOHEXAMIDE
0.8±0.20
*NA= Not active
*ND= Not dissolved
*SEM= Standard Error of Mean.
3.3.0
Kinetic Study:

Compounds 8 and 10 were selected for the evaluation of mechanism of inhibition. Lineweaver-
Burk plots were used to determine the type of inhibition of α-glucosidase enzyme. The reciprocal
of the rate of the reaction were plotted against the reciprocal of substrate concentrations to monitor
the effect of the inhibitor on both Km, and Vmax values.Compound 8 showed a non-competitive
inhibition with Ki value 26.2 ± 0.018 µM, in this type of inhibition Km remains constant and a
decrease in Vmax observed. Compound 10 appeared as a competitive inhibitor with the Ki 29.8 ±
0.0056 µM, with constant Vmax and increasing Km values.
Table 3: Kinetic study data of compounds 8, and 9 as α-glucosidase inhibitors.
Compounds
IC50 ± SEM
20.44±1.73
34.63±0.16
Ki ±SEM
Type of inhibition
Non-competitive
Competitive
8
26.2 ± 0.018
29.8 ± 0.0056
10

60
40
20
0
-20
-40
-60
-0.8 -0.6 -0.4 -0.2
0
0.2 0.4 0.6 0.8
-100 -80 -60 -40 -20
0
20 40 60 80 100
1/Substrate
Compound 8
(A)
(B)
80
60
40
20
0
-20
-40
-60
-80
-80 -60 -40 -20
0
20
40
60
80
Compound 8
(C)
Figure 4: The inhibition of α-glucosidase by compound 8 (A) Linweaver-Burk plot of reciprocal
of rate of reaction (velocities) vs reciprocal of substrate p-nitro phenyl-α-D-glucopyranoside in the
absence of (-), and in the presence of 80µM (e), 40µM (f), 20µM (g) and 10µM (h) of compound
8. (B) Secondary replot of Line weaver-Burk plot between the slopes of each line on Lineweaver-
Burk plot vs different concentrations of compound 8. (C) Dixon plot of reciprocal of rate of
reaction (velocities) vs different concentrations of compound 8.

600
400
200
0
-200
-400
-600
-800 -600 -400 -200
0
200 400 600 800 1000
1/Substrate
(A)
(B)
(C)
Figure 5: The inhibition of α-glucosidase by compound 10 (A) Lineweaver-Burk plot reciprocal
of rate of reaction (velocities) vs reciprocal of substrate p-nitro phenyl-α-D-glucopyranoside in the
absence of (-), and in the presence of 80µM (e), 40µM (f), and 10µM of compound 10. (B)
Secondary replot of Lineweaver-Burk plot between the slopes of each line on Lineweaver-Burk
plot vs different concentrations of compound 10. (C) Dixon plot of reciprocal of rate of reaction
(velocities) vs different concentrations of compound 10.

3.5.0.
Statistical Analysis:
EZ-Fit Enzyme Kinetics Program (Perrella Scientific Inc. Amherst, USA) was employed to
calculate the IC50 values. All the experiments were performed in triplicate, and variations in the
results are reported in Standard Error of Mean values (SEM).
3.6.0.
Conclusion:
In conclusion, we have synthesized a series of substituted benzamide based derivatives of thiourea
as novel α-glucosidase inhibitors. The promising results of study concluded that these derivatives
can be a potential candidate for further development and designing of new inhibitors of α-
glucosidase enzyme, however further studies are required towards their development as anti-
diabetic drug leads.
References:
[1]
S. Kalra, Alpha glucosidase inhibitors, J. Pak. Med. Assoc. 64 (2014) 474–476 PMID:
24864650.
[2]
C. Eleazu, A. Sampson, S. Saidu, K. Eleazu, C. Egedigwe-Ekeleme, Starch digestibility,
polyphenol contents and in vitro alpha amylase inhibitory properties of two varieties of cocoyam
(Colocassia esculenta and Xanthosoma mafafa) as affected by cooking, J. Food Meas. Charact.
(2018) 1047–1053. https://doi.org/10.1007/s11694-018-9720-9.
[3]
D. Halegoua-De Marzio, V.J. Navarro, Hepatotoxicity of cardiovascular and antidiabetic
drugs, in: Drug-Induced Liver Dis. (Third Ed.), Elsevier, 2013: pp. 519–540
https://doi.org/10.1016/B978-0-12-387817-5.00029-7.
[4]
analysis
bromophenylcarbamothioyl)-4-fluorobenzamide,
Communications 75(7) (2019) 1026-1029. https://doi.org/10.1107/S2056989019008569.
S. Akhter, M. I. Choudhary, H. Siddiqui, S. Yousuf, Crystal structure and Hirshfeld surface
of N-(2-chlorophenylcarbamothioyl)-4-fluorobenzamide and N-(4-
Acta Cryst. E: Crystallographic

[5]
Y. Zhang, X. Meng, H. Tang, M. Cheng, F. Yang, W. Xu, Design, synthesis, and biological
evaluation of novel substituted thiourea derivatives as potential anticancer agents for NSCLC by
blocking K-Ras protein-effectors interactions, J. Enzyme Inhib. Med. Chem. 35 (2020) 344–
353.https://doi.org/10.1080/14756366.2019.1702653.
[6]
D. Calla-Choque, F. Nava-Alonso, thiourea determination for the precious metals leaching
process by iodate titration, Rev. Mex. Ing. Química. 19 (2020) 275–284.
https://doi.org/10.24275/rmiq/IA539.
[7]
V.S. Nagrare, B.B. Fand, V.C.B. Naik, B. Naikwadi, V. Deshmukh, D. Sinh, Resistance
development in Cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)
to insecticides from Organophosphate, Thiadiazines and Thiourea derivatives, Int. J. Trop. Insect
Sci. 40 (2020) 181–188. https://doi.org/10.1007/s42690-019-00068-9.
[8]
A. Shakeel, A. A. Altaf, A.M. Qureshi, A. Badshah, Thiourea Derivatives in Drug Design
and Medicinal Chemistry: A Short Review, J. Drug Des. Med. Chem. 2 (2016) 10–20.
https://doi.org/10.11648/j.jddmc.20160201.12.
[9]
D. M. Vardhan, H. K. Kumara, M. B. Sridhara, D. Gowda, Urea/thiourea derivatives of
Gly/Pro conjugated 2, 3-dichlorophenyl piperazine as potent anti-inflammatory agents: SAR
studies, Indian J. Chem. 56 (2017) 1207-1211. http://nopr.niscair.res.in/handle/123456789/43081.
[10] A. Maalik, H. Rahim, M. Saleem, N. Fatima, A. Rauf, A. Wadood, M.I. Malik, A. Ahmed,
H. Rafique, M.N. Zafar, M. Riaz, L. Rasheed, A. Mumtaz, Synthesis, antimicrobial, antioxidant,
cytotoxic, antiurease and molecular docking studies of N-(3-trifluoromethyl)benzoyl-N′-aryl
thiourea
derivatives,
Bioorg. Chem
88
(2019)
102946.
https://doi.org/10.1016/j.bioorg.2019.102946.
[11] S. Qaiser, M.S. Mubarak, S. Ashraf, M. Saleem, Z. Ul-Haq, M. Safdar, A. Rauf, T. Abu-
Izneid, M.I. Qadri, A. Maalik, Benzilydene and thiourea derivatives as new classes of carbonic
anhydrase inhibitors: an in vitro and molecular docking study, Med. Chem. Res. (2020). pp.1-12.
https://doi.org/10.1007/s00044-020-02661-x.

[12] P. Cui, X. Li, M. Zhu, B. Wang, J. Liu, H. Chen, Design, synthesis and antibacterial
activities of thiouracil derivatives containing acyl thiourea as SecA inhibitors, Bioorg. Med. Chem.
Lett. 27 (2017) 2234–2237. https://doi.org/10.1016/j.bmcl.2016.11.060.
[13] S.S. Hassan, R.N. Shallan, M.M. Salama, Antibacterial and molecular study of thiourea
derivative ligand and its Dimethyltin (IV) complex with the superior of its Copper (II) complex as
a
hepatocellular antitumor drug, J. Organomet. Chem. 911 (2020) 12115.
https://doi.org/10.1016/j.jorganchem.2020.121115.
[14] C. Limban, A.V. Missir, M.T. Caproiu, A.M. Grumezescu, M.C. Chifiriuc, C. Bleotu, L.
Marutescu, M.T. Papacocea, D.C. Nuta, Novel Hybrid Formulations Based on Thiourea
Derivatives and Core@ Shell Fe3O4@ C18 Nanostructures for the Development of Antifungal
Strategies, Nanomaterials. 8 (2018) 47. https://doi.org/10.3390/nano8010047.
[15] A.A. Spasov, A.N. Geisman, V.A. Kosolapov, D.A. Babkov, A.I. Rashchenko, V.A.
Babkova, O.Y. Zakhar’yashcheva, A.A. Ozerov, Synthesis and Antiglycation Activity of Novel
S-Carboxyalkyl Derivatives of 2-Thiouracil, Pharm. Chem. J. 53 (2019) 610–615.
https://10.1007/s11094-019-02047-3.
[16] D.M.S. Vardhan, H.K. Kumara, H.P. Kumar, D.C. Gowda, Urea and Thiourea Derivatives
of Dipeptides Conjugated Piperazine Analogue as A New Class of AGE’s Inhibitors: Synthesis
and Molecular Docking Studies, Asian J. Res. Chem. 10 (2017) 459–469. https://10.5958/0974-
4150.2017.00075.
[17] M. Khan, N. Khan, K. Ghazal, S. Shoaib, Samiullah, I. Ali, M.K. Rauf, A. Badshah, M.N.
Tahir, A.-U. Rehman, Synthesis, characterization, crystal structure, in-vitro cytotoxicity,
antibacterial, and antifungal activities of nickel(II) and cobalt(III) complexes with acylthioureas,
J. Coord. Chem. 73(12) (2020) 1790-1805. https://doi.org/10.1080/00958972.2020.1793136.
[18] N. Korkmaz, O.A. Obaidi, M. Senturk, D. Astley, D. Ekinci, C.T. Supuran, Synthesis and
biological activity of novel thiourea derivatives as carbonic anhydrase inhibitors, J. Enzyme Inhib.
Med. Chem. 30 (2015) 75–80. https://doi.org/10.1007/s00044-020-02661-x.

[19] G. Binzet, H. Arslan, U. Flörke, N. Külcü, N. Duran, Synthesis, characterization and
antimicrobial activities of transition metal complexes of N, N-dialkyl-N′-(2-chlorobenzoyl)
thiourea derivatives, J. Coord. Chem. 59 (2006) 1395–1406.
https://doi.org/10.1080/00958970500512633.
[20] G. Binzet, N. Külcü, U. Flörke, H. Arslan, Synthesis and characterization of Cu (II) and Ni
(II) complexes of some 4-bromo-N-(di (alkyl/aryl) carbamothioyl) benzamide derivatives, J.
Coord. Chem. 62 (2009) 3454–3462. https://doi.org/10.1080/00958970903082200.
[21] B. Yu, Y. Liu, X. Peng, S. Hua, G. Zhou, K. Yan, Y. Liu, Synthesis, characterization, and
antitumor properties of Au (i)–thiourea complexes, Metallomics. (2020) 104-113.
https://doi.org/10.1039/C9MT00232D.
[22] W.I. Mortada, I.M.M. Kenawy, M.H. Abdel-Rhman, G.G. El-Gamal, S.M.N. Moalla, A
new thiourea derivative [2-(3-ethylthioureido) benzoic acid] for cloud point extraction of some
trace metals in water, biological and food samples, J. Trace Elem. Med. Biol. 44 (2017) 266–273.
https://doi.org/10.1016/j.jtemb.2017.09.003.
[23] K.G. Akpomie, O.M. Fayomi, C.C. Ezeofor, R. Sha’Ato, W.E. Van Zyl, Insights into the
use of metal complexes of thiourea derivatives as highly efficient adsorbents for ciprofloxacin
from contaminated water, Trans. R. Soc. South Africa. 74 (2019) 180–188.
https://doi.org/10.1080/0035919X.2019.1614695.
[24] G. Binzet, G. Kavak, N. Külcü, S. Özbey, U. Flörke, H. Arslan, Synthesis and
characterization of novel thiourea derivatives and their nickel and copper complexes, J. Chem.
2013 (2013) 1-9. https://doi.org/10.1155/2013/536562.
[25] M.A. Bashir, K. Javaid, M. Shaikh, M.I. Choudhary, H. Siddiqui, Tyramine Derivatives
as Potent Therapeutics for Type 2 Diabetes: Synthesis and In Vitro Inhibition of α-Glucosidase
Enzyme, Med. Chem. (2020) 1124 - 1135.
https://doi.org/10.2174/1573406416666200128114422.

[26] V.M. Thadhani, M.I. Choudhary, S. Ali, I. Omar, H. Siddique, V. Karunaratne, Antioxidant
activity of some lichen metabolites, Nat. Prod. Res. 25 (2011) 1827–1837.
https://doi.org/10.1080/14786419.2010.529546.
[27] J.M. Duarte-Almeida, A.V. Novoa, A.F. Linares, F.M. Lajolo, M.I. Genovese, Antioxidant
activity of phenolics compounds from sugar cane (Saccharum officinarum L.) juice, Plant Foods
Hum. Nutr. 61 (2006) 187. https://doi.org/10.1007/s11130-006-0032-6.
[28] M. Ayaz, M. Junaid, F. Ullah, A. Sadiq, F. Subhan, M.A. Khan, W. Ahmad, G. Ali, M.
Imran, S. Ahmad, Molecularly characterized solvent extracts and saponins from Polygonum
hydropiper L. show high anti-angiogenic, anti-tumor, brine shrimp, and fibroblast NIH/3T3 cell
line cytotoxicity, Front. Pharmacol. 7 (2016) 74. https://doi.org/10.1002/slct.201901870.

GRAPHICAL ABSTRACT
Synthesis, Crystal Structure and Hirshfeld Analysis of Benzamide Derivatives of Thiourea as
Potent Inhibitors of α-Glucosidase in-vitro
Sidra Akhtera, Saeed Ullaha, SammerYousufa, Atia-tul-Wahabb, Hina Siddiqui, *a and M. Iqbal
Choudhary*a,b,c
aH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi-75270, Pakistan
bDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
cDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah-21452, Saudi Arabia
O
S
O
O
R2 -NH2
60oC, 2-4 hrs
R2
Acetone
N
H
N
H
+
KSCN
Cl
NCS
R1
R1
R1
rt, 20min
HCl
1-15

Synthesis, Crystal Structure and Hirshfeld Analysis of Benzamide Derivatives of Thiourea as
Potent Inhibitors of α-Glucosidase in-vitro
Benzamide based structural analogues 1-15 were synthesized, and evaluated for α-glucosidase
inhibition activity invitro for the first time. Compounds 1-9 were found to be known, while
compounds 10-15 were found to be new. However, to the best of our knowledge we are reporting
α-glucosidase inhibitory activity of these bezamide derivative of thiourea for the first time.
Compounds 1, 3, 6-8, 10-14 were found to be potent α-glucosidase inhibitors within IC50 range of
20.44- 333.41 µM, in comparison to standard inhibitor, acarbose (IC50 = 875.75 ± 2.08 µM). Mode
of the enzyme inhibition was determined on the basis of kinetic studies which demonstrated that
compounds 8, and 10 were non-competitive and competitive inhibitors of α-glucosidase enzyme,
respectively.
Single crystal X-ray and Hirshfeld Surface analyses of a representative compound is also
presented.
O
S
O
O
R2 -NH2
60oC, 2-4 hrs
R2
Acetone
N
H
N
H
+
KSCN
Cl
NCS
R1
R1
R1
rt, 20min
HCl
1-15

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: