Investigation on the effect of alkyl chain linked mono-thioureas as Jack bean urease inhibitors, SAR, pharmacokinetics ADMET parameters and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2019.02.011

The increasing resistance of pathogens to common antibiotics, as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications, has stimulated the development of novel classes of molecules that target urease as an enzyme. In this context, the newly developed compounds on the basis of 1-heptanoyl-3-arylthiourea family were evaluated for Jack bean urease enzyme inhibition activity to validate their role as potent inhibitors of this enzyme. 1-Heptanoyl-3-arylthioureas were obtained in excellent yield and characterized through spectral and elemental analysis. All the compounds displayed remarkable potency against urease inhibition as compared to thiourea standard. It was found that novel compounds fulfill the criteria of drug-likeness by obeying Lipinski's rule of five. Particularly compound 4a and 4c can serve as lead molecules in 4D (drug designing discovery and development). Kinetic mechanism and molecular docking studies also carried out to delineate the mode of inhibition and binding affinity of the molecules.

Full text of DOI:10.1016/j.bioorg.2019.02.011

Accepted Manuscript
Investigation on the Effect of Alkyl Chain Linked Mono-thioureas as Jack Bean
Urease Inhibitors, SAR, Pharmacokinetics ADMET Parameters and Molecular
Docking Studies
Fayaz Ali Larik, Muhammad Faisal, Aamer Saeed, Pervaiz Ali Channar, Jan
Korabecny, Farukh Jabeen, Ihsan Ali Mahar, Mehar Ali Kazi, Qamar Abbas,
Ghulam Murtaza, Gul Shahzada Khan, Mubashir Hassan, Sung-Yum Seo
PII:
S0045-2068(18)31466-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.011
YBIOO 2786
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 December 2018
30 January 2019
3 February 2019
Please cite this article as: F. Ali Larik, M. Faisal, A. Saeed, P. Ali Channar, J. Korabecny, F. Jabeen, I. Ali Mahar,
M. Ali Kazi, Q. Abbas, G. Murtaza, G. Shahzada Khan, M. Hassan, S-Y. Seo, Investigation on the Effect of Alkyl
Chain Linked Mono-thioureas as Jack Bean Urease Inhibitors, SAR, Pharmacokinetics ADMET Parameters and
Molecular Docking Studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.011
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Investigation on the Effect of Alkyl Chain Linked Mono-thioureas as Jack Bean Urease
Inhibitors, SAR, Pharmacokinetics ADMET Parameters and Molecular Docking Studies
Fayaz Ali Larik*a, Muhammad Faisal*a, Aamer Saeed*a, Pervaiz Ali Channara, Jan
Korabecnyb, Farukh Jabeene, Ihsan Ali Mahara, Mehar Ali Kazid, Qamar Abbasc, Ghulam
Murtazaa, Gul Shahzada Khanf, Mubashir Hassanc, Sung-Yum Seoc
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
b
Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05
Hradec Kralove, Czech Republic.
c Department of Biological Sciences, College of Natural Sciences, Kongju National University,
56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of Korea.
d Department of Physiology, University of Sindh, Jamshoro-76080, Pakistan.
e Cardiovascular and Metabolic Research Unit, Laurentian University, 935 Ramsey Lake Road,
Sudbury, ON P3E 2C6, Canada.
f Department of Chemistry, Abdul Wali Khan University, Mardan, Khybder Pakhtunkhwa,
Pakistan.
Email: fayazali@chem.qau.edu.pk (Fayaz Ali Larik); mfaisal4646@gmail.com (Muhammad
Faisal); aamersaeed@yahoo.com (Aamer Saeed); Tel +92-51-9064-2128, Fax: +92-51-9064-
2241
1

Graphical abstract
2

Abstract
The increasing resistance of pathogens to common antibiotics, as well as the need to control
urease activity to improve the yield of soil nitrogen fertilization in agricultural applications, has
stimulated the development of novel classes of molecules that target urease as an enzyme. In this
context, the newly developed compounds on the basis of 1-heptanoyl-3-arylthiourea family were
evaluated for Jack bean urease enzyme inhibition activity to validate their role as potent
inhibitors of this enzyme. 1-Heptanoyl-3-arylthioureas were obtained in excellent yield and
characterized through spectral and elemental analysis. All the compounds displayed remarkable
potency against urease inhibition as compared to thiourea standard. It was found that novel
compounds fulfill the criteria of drug-likeness by obeying Lipinski’s rule of five. Particularly
compound 4a and 4c can serve as lead molecules in 4D (drug designing discovery and
development). Kinetic mechanism and molecular docking studies also carried out to delineate the
mode of inhibition and binding affinity of the molecules.
Keywords: Lipinski’s rules; acyl thioureas; kinetic mechanism; antioxidant; molecular
modeling; Jack bean urease; urease
1. Introduction
3

The enzyme urease (urea amidohydrolase; E.C.3.5.1.5) has a storied history and constantly been
exploited by medicinal chemist to develop potent and efficient inhibitors of this enzyme [1].
Urease helps in the hydrolysis of urea, an essential fertilizer for soil, by cleaving urea into
ammonia and carbon dioxide. Both the ammonia gas and carbon dioxide are not ecofriendly and
can cause catastrophic alterations in the crop production [2]. In addition to this, urease provide
an aid to Heliobacter pylori (H. Pylori) to survive in the stomach. Urease test is a rapid
diagnostic test for diagnosis of H. pylori infection in the human stomach. Settlement by H.pylori
can can stimulate pathogenesis of many human health conditions vis-à-vis pyelonephritis,
heptatic coma, peptic ulceration and urinary catheter encrustation [3].
Urease inhibitors can serve as key precursors to prevent stomach and urinary related disorders in
human beings and also to prevent soil from damage caused by the excess of ammonia release
from urea during hydrolysis [4]. Urease inhibitors have broadly classified mainly into two
categories (i) active site directed (substrate-like), and (ii) mechanism-based directed [5]. Several
classes of compounds are reported to show significant inhibition against urease enzyme, most
commonly hydroxamic acids (HXAs) [6], phosphorodiamidates (PPDs) [7], imidazoles [8],
phosphazene [9], thioureas [10] and related compounds [11].
Acyl thiourea family is versatile class of organic compounds representing basic scaffold in
medicinal and synthetic chemistry [12]. Acyl thioureas motif has been the valuable synthon in
the synthesis of wide variety of heterocyclic compounds [13–15]. In addition to being important
in synthetic chemistry, these show broad spectrum of biological activities such as antioxidant,
antifungal, antimicrobial, antiviral, anti-cancer, anti-tuberculosis, antibacterial, herbicidal and
insecticidal [16].
4

The structure-activity relationship [17] and literature data of thioureas-derived N-bridged alkyl
compounds [18–20] reveal that these compounds have wide bioactive spectrum. In view of these
particulars and by taking into account to extend alkyl chain up to six carbons, we designed and
synthesized acyl thioureas as Jack bean urease inhibitors. The inhibition ability for these new
low-molecular weight thioureas were in micromolar range. The results of our work would serve
as templates for the designing potent drugs against Jack bean urease enzyme.
2. Experimental
2.1 Methods and Materials
1H-NMR and 13C NMR spectra were recorder on a Brucker AM-300 spectrophotometer and
1
chemical shifts of H NMR and 13C NMR are reported in parts per million (ppm). Internal
1
standard TMS; the splitting of proton resonances in the reported H-NMR spectra are defined as
s = singlet, d = doublet, q = quartet, dd = doublet of doublets, se = septet and m = complex
pattern. The melting point was determined on Staurt SMP3 melting point apparatus and is
uncorrected. FTIR spectra were recorded using Shimadzu IR 460 spectrophotometer by
Attenuated Total Reflectance (ATR) method. The elemental analysis was performed on LECO-
932 CHNS analyzer.
For organic chemistry part, all experiments were performed under the specified conditions of
temperature. Solvents were distilled from the appropriate drying agents and degassed before use.
All solvents were purified by distillation, dried, saturated with nitrogen and stored over
molecular sieves 4 Å. Heptanoic acid, potassium thiocyanate, thionyl chloride, 2,4-dinitroaniline,
2,3-dichloroaniline, 4-bromoaniline, 4-chloroaniline, 3-nitroaniline, 2-chloroaniline, 2,4,6-
trimethylaniline, 4-nitroaniline, and 2-bromo-2-flouroaniline were purchased from Sigma-
Aldrich, Australia and used as received.
5

2.2 Experimental Data
2.2.1 1-Heptanoyl-3-(2, 4-nitrophenyl) thiourea (4a)
o
Yellow solid; Rf (n-hexane: ethylacetate 1:1); 0.49; m.p: 198 C; IR (neat, cm-1): 3234 (N-H),
1
1670 (C=O), 1549 (NH), 1261 (C=S). H-NMR (DMSO-d6), 300 MHz): 12.25 (s, 1H, NH),
11.63 (s, 1H, NH), 7.83 (d, 1H, J = 7.12 Hz, Ar-H), 7.53 (d, 1H, J = 7.52 Hz, Ar-H), 7.52 (d, 1H,
J = 7.52 Hz, Ar-H), 2.82 (t, 2H, J=3.3 Hz), 2.11 (quint, 2H, J=2.2 Hz), 1.93 (quint, 2H), 1.32
(quint, 2H), 0.92 (sex, 2H), 0.86 (t, 3H, J=1.1 Hz) ;13C-NMR (DMSO-d6); 75.5 MHz): 180.6,
175.4, 157.9, 152.1, 135.2, 129.7, 120.7, 118.2, 36.5, 31.5, 28.5, 25.7, 22.7, 15.1, Anal. Calcd.
For C14H18N4O5S, C, 47.44; H, 5.12; N, 15.81; S 9.04; Found: C, 47.33, H, 4.99; N, 15.73; S,
8.97.
2.2.2 1-Heptanoyl-3-(2, 3-dichlorophenyl) thiourea (4b)
Yellow solid; Rf (n-hexane: ethylacetate 1:1); 0.49; m.p: 198 oC; IR (neat, cm-1): 3234 (m, N-H),
1
1670 (C=O), 1549 (NH), 1261 (C=S). H-NMR (DMSO-d6), 300 MHz): 12.25 (s, 1H, NH),
11.63 (s, 1H, NH), 7.73 (d, 1H, J = 7.12 Hz, Ar-H), 7.46 (d, 1H, J = 7.52Hz, Ar-H), 7.36 (d, 1H,
J = 7.52, Ar-H), 2.91 (t, 2H, J=3Hz), 2.14 (quint, 2H, J=2Hz), 1.95 (quint, 2H), 1.34 (quint,
13
2H), 1.02 (sex, 2H), 0.91 (t, 3H, J=1Hz); C-NMR (DMSO-d6); 75.5 MHz): 179.6, 173.4,
154.19, 140.1, 135.2, 131.7, 125.7, 122.5, 37.3, 32.4, 27.4, 26.4, 23.5, 14.7, Anal. Calcd. For
C14H18Cl2N2OS, C, 50.44; H, 5.44; N, 8.41; S 9.64; Found: C, 50.34, H, 5.32; N, 8.23; S, 9.51.
2.2.3 1-Heptanoyl-3-(4-bromophenyl) thiourea (4c)
Yellow solid; Rf (n-hexane:ethylacetate 1:1); 0.51; m.p: 187 oC; IR (neat, cm-1): 3229 (m, N-H),
1
1685 (C=O), 1540 (N-H), 1279 (C=S). H-NMR (DMSO- d6), 300 MHz): 12.65 (s, 1H, NH),
12.03 (s, 1H, NH), 7.63 (dd, 1H, J= 6.3Hz, J=5.6Hz, Ar-H), 7.56 (dd, 1H, J = 7.1Hz, J=5.3 Hz
Ar-H), 2.42 (t, 2H, J=3Hz), 2.85 (t, 2H, J=3Hz), 2.15 (quint, 2H, J=2 Hz), 1.86 (quint, 2H), 1.51
6

13
(quint, 2H), 1.19 (sex, 2H), 0.96 (t, 3H, J=1Hz); C-NMR (DMSO-d6); 75.5 MHz): 181.6,
174.4, 148.9, 142.3, 135.4, 130.5, 38.2, 32.3, 27.3, 24.6, 21.5, 16.2, Anal. Calcd. For
C14H19BrN2OS, C, 48.96; H, 5.45; N, 8.57; S 9.35; Found: C, 48.69; H, 5.35; N, 8.45; S, 9.24.
2.2.4 1-Heptanoyl-3-(3-nitrophenyl) thiourea (4d)
o
Yellow solid; Rf (n-hexane: ethylacetate 1:1); 0.53; m.p: 170 C; IR (neat, cm-1): 3229 (N-H),
1
1670 (C=O), 1543 (NH), 1260 (C=S). H-NMR (DMSO-d6), 300 MHz): 12.22 (s, 1H, NH),
11.26 (s, 1H, NH), 7.69 (d, 1H, J = 7.10, Ar-H), 7.56 (d, 1H, J = 7.61 Hz, Ar-H), 7.42 (d, 1H, J =
7.10 Hz, Ar-H), 7.26 (d, 1H, J = 7.61 Hz, Ar-H), 2.91 (t, 2H, J=3Hz), 2.15 (quint, 2H, J=2Hz),
1.85 (quint, 2H), 1.38 (quint, 2H), 1.12 (sex, 2H), 0.98 (t, 3H, J=1Hz);13C-NMR (DMSO-d6);
75.5 MHz): 179.6, 170.4, 152.1, 139.2, 132.5, 129.6, 124.7, 121.5, 36.5, 31.5, 28.5, 25.7, 22.7,
15.1, Anal. Calcd. For C14H19N3O3S, C, 54.34; H, 6.18; N, 13.57; S 10.35; Found: C, 54.22; H,
599; N, 13.43; S, 10.24.
2.2.5 1-Heptanoyl-3-(2-chlorophenyl) thiourea (4e)
Light yellow solid; Rf (n-hexane: ethyl acetate 1:1); 0.56; m.p: 195oC; IR (neat, cm-1): 3219 (N-
1
H), 1680 (C=O), 1534 (N-H), 1268 (v C=S). H-NMR (DMSO-d6), 300 MHz): 12.65 (s, 1H,
NH), 12.03 (s, 1H, NH), 7.41 (d, 1H, J = 6.3Hz, Ar-H), 7.35 (d, 1H, J = 5.7Hz, Ar-H), 7.25 (d,
1H, J = 4.7 Hz, Ar-H), 6.99 (d, 1H, J = 4.7 Hz, Ar-H), 2.78 (t, 2H, J=3Hz), 2.11 (quint, 2H,
13
J=2Hz), 1.93 (quint, 2H), 1.32 (quint, 2H), 0.92 (sex, 2H), 0.86 (t, 3H, J=1Hz); C-NMR
(DMSO-d6);75.5 MHz): 179.6, 170.4, 142.19, 137.2, 133.5, 128.6, 125.3, 123.7, 35.4, 32.3, 29.4,
24.6, 21.5, 13.5, Anal. Calcd. For C14H19ClN2OS, C, 56.26; H, 6.42; N, 9.38; S 10.74; Found: C,
56.15; H, 6.29; N, 9.28; S, 10.63.
2.2.6 1-Heptanoyl-3-(2,4,6-trimethylphenyl) thiourea (4f)
7

Light yellow solid; Rf (n-hexane: ethyl acetate 1:1); 0.56; m.p: 195 oC; IR (neat, cm-1): 3219 (N-
H), 1680 (C=O), 1534 (N-H), 1268 (C=S). 1H-NMR (DMSO-d6), 300 MHz): 12.65 (s, 1H, NH),
12.03 (s, 1H, NH), 7.48 (d, 1H, J = 4.3Hz, Ar-H), 7.28 (d, 1H, J = 3.3 Hz, Ar-H), 2.78 (t, 2H,
J=3Hz), 2.15 (quint, 2H, J=2Hz), 1.85 (quint, 2H), 1.35 (quint, 2H), 1.08 (sex, 2H), 0.99 (t, 3H,
J=1Hz);13C-NMR (DMSO-d6);75.5 MHz): 179.6, 170.4, 145.16, 134.5, 127.4, 122.8, 37.1, 29.4,
24.3, 21.7, 18.7, 12.5, Anal. Calcd. For C17H26N2OS, C, 66.61; H, 8.54; N, 9.15; S 10.45; Found:
C, 66.48; H, 8.39; N, 9.02; S, 10.34.
2.2.7 1-Heptanoyl-3-(4-nitrophenyl) thiourea (4g)
Yellow solid; Rf (n-hexane:ethylacetate 1:1); 0.53; m.p: 160 oC; IR (neat, cm-1): 3229 (m, N-H),
1
1685 (C=O), 1540 (N-H), 1279 (C=S). H-NMR (DMSO- d6), 300 MHz): 11.61 (s, 1H, NH),
11.43 (s, 1H, NH), 7.67 (dd, 1H, J= 5.3 Hz, J=4.6 Hz, Ar-H), 7.50 (dd, 1H, J = 7.2 Hz, J=4.3 Hz
Ar-H), 2.51 (t, 2H, J=3Hz), 2.81 (t, 2H, J=3Hz), 2.17 (quint, 2H, J=2Hz), 1.88 (quint, 2H), 1.41
13
(quint, 2H), 1.13 (sex, 2H), 1.06 (t, 3H, J=1Hz); C-NMR (DMSO-d6); 75.5 MHz): 182.5,
173.7, 145.4, 141.3, 134.6, 129.4, 34.5, 31.5, 26.5, 23.8, 20.9, 17.4, Anal. Calcd. For
C14H19BrN2OS, C, 48.96; H, 5.45; N, 8.57; S 9.35; Found: C, 48.69; H, 5.35; N, 8.45; S, 9.24.
2.2.8 1-Heptanoyl-3-(4-bromo-2-flourophenyl) thiourea (4h)
White solid; Rf (n-hexane: ethylacetate 1:1); 0.58; m.p: 201oC; IR (neat, cm-1): 3230 (m, N-H),
1
1676 (C=O), 1547 (N-H), 1269 (C=S). H-NMR (DMSO-d6), 300 MHz): 12.11 (s, 1H, NH),
11.68(s, 1H, NH), 7.93 (d, 1H, J = 7.10 Hz, Ar-H), 7.56 (d, 1H, J = 7.61 Hz, Ar-H), 2.85 (t, 2H,
J=3Hz), 2.18 (quint, 2H, J=2Hz), 1.83 (quint, 2H), 1.22 (quint, 2H), 1.12 (sex, 2H), 0.89 (t, 3H,
13
J=1Hz); C-NMR (DMSO-d6); 75.5 MHz): 179.6, 170.4, 152.19, 142.5, 135.8,133.1, 128.5,
121.8, 34.8, 32.8, 26.3, 24.8, 22.7, 12.4, Anal. Calcd. For C14H18BrFN2OS, C, 46.53; H, 5.04; N,
7.76; S 8.84; Found: C, 46.41; H, 4.96; N, 7.59; S, 8.74.
8

2.2.9 1-Heptanoyl-3-(2,6-dibromo-4-flourophenyl) thiourea (4i)
Light yellow solid; Rf (n-hexane: ethyl acetate 1:1); 0.56; m.p: 195oC; IR (neat, cm-1): 3219 (N-
H), 1680 (C=O), 1534 (N-H), 1268 (C=S). 1H-NMR (DMSO-d6), 300 MHz): 12.65 (s, 1H, NH),
12.03 (s, 1H, NH), 7.48 (d, 1H, J = 4.3 Hz, Ar-H), 7.28 (d, 1H, J = 3.3, Ar-H), 2.78 (t, 2H,
J=3Hz), 2.15 (quint, 2H, J=2Hz), 1.85 (quint, 2H), 1.35 (quint, 2H), 1.08 (sex, 2H), 0.99 (t, 3H,
J=1Hz);13C-NMR (DMSO-d6);75.5 MHz): 179.6, 170.4, 145.16, 134.5, 127.4, 122.8, 37.1, 29.4,
24.3, 21.7, 18.7, 12.5, Anal. Calcd. For C14H17Br2FN2OS, C, 38.20; H, 3.89; N, 6.35; S 7.29;
Found: C, 38.12; H, 3.39; N, 9.02; S, 10.34.
2.2.10 1-Heptanoyl-3-benzylthiourea (4j)
White solid; Rf (n-hexane: ethylacetate 1:1); 0.58; m.p: 221oC; IR (neat, cm-1): 3230 (s, N-H),
1
1676 (C=O), 1547 (v, N-H), 1250 (C=S). H-NMR (DMSO-d6), 300 MHz): 12.11 (s, 1H, NH),
11.68 (s, 1H, NH), 7.93 (dd, 2H, J = 5.1 Hz, J= 3.6 Hz, Ar-H), 7.56 (dd, 2H, J = 7.61Hz Ar-H),
7.23 (d, 1H, J= 5.3Hz), 2.82 (t, 2H, J=3Hz), 2.11 (quint, 2H, J=2Hz), 1.93 (quint, 2H), 1.32
(quint, 2H), 0.92 (sex, 2H), 0.86 (t, 3H, J=1Hz);13C-NMR (DMSO-d6); 75.5 MHz): 179.6,
170.4, 143.19, 135.2, 133.7, 132.3, 36.7, 31.5, 28.5, 25.7, 22.7, 15.1, Anal. Calcd. For
C15H22N2OS, C, 64.69; H, 7.94; N, 10.06; S 11.51; Found: C, 64.57; H, 7.86; N, 9.92; S, 11.41.
2.3 Biological activity, kinetics and molecular docking methods and procedure
2.3.1 Urease inhibition assay
The Jack bean urease activity was determined by measuring amount of ammonia produced with
indophenols method described by Weatherburn [21]. The reaction mixtures, comprising 20 µL of
enzyme (Jack bean urease, 5 U/mL) and 20 μL of compounds in 50 μL buffer (100 mM urea,
0.01 M K2HPO4, 1 mM EDTA and 0.01 M LiCl, pH 8.2), were incubated for 30 min at 37 ºC in
96-well plate. Briefly, 50 μL each of phenol reagents (1%, w/v phenol and 0.005%, w/v sodium
9

nitroprusside) and 50 μL of alkali reagent (0.5%, w/v NaOH and 0.1% Sodium hypochlorite
NaOCl) were added to each well. The absorbance at 625 nm was measured after 10 min, using a
microplate reader (OPTI Max, Tunable). All reactions were performed in triplicate. The urease
inhibition activities were calculated according to the following formula:
Urease inhibition activity (%) = (ODcontrol – ODsample × 100) / ODcontrol
Where ODcontrol and ODsample represents the optical densities in the absence and presence of
sample, respectively [11]. Thiourea was used as the standard inhibitor for urease.
2.3.2 Kinetic study
Kinetic analysis was carried out to determine the mode of inhibition. Two inhibitors (4a and 4c)
were selected on the basis of most potent IC50 values. Kinetics were carried out by varying the
concentration of urea in the presence of different concentrations of compound H1 (0.0, 0.0195,
0.039, 0.078 and 0.312), compound 4c (0.00, 0.02, 0.04, and 0.08 µM). Briefly the urea
concentration was changed from 100, 50, 25, 12.5, 6.25, 3.12 mM for urease kinetics studies and
remaining procedure was same for all kinetic studies as describes in urease inhibition assay
protocol. Maximal initial velocities were determined from initial linear portion of absorbances up
to 10 minutes after addition of enzyme at per minute’s interval. The inhibition type on the
enzyme was assayed by Lineweaver-Burk plot of inverse of velocities (1/V) versus inverse of
substrate concentration 1/ [S] mM-1. The EI dissociation constant Ki was determined by
secondary plot of 1/V versus inhibitor concentration. Urease activity was determined by
measuring ammonia production using the indophenol method as reported previously [22]. The
results (change in absorbance per min) were processed by using SoftMaxPro.
2.3.3 Free radical scavenging assay
10

Radical scavenging activity was determined by modifying already reported method by 2,2-
diphenyl-1 picrylhydrazyl (DPPH) assay [22]. The assay solution consisted of 100 µL of DPPH
(150 µM), 20 µL of increasing concentration of test compounds and the volume was adjusted to
200 µL in each well with DMSO. The reaction mixture was then incubated for 30 minutes at
room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The assay
measurements were carried out by using a micro plate reader (OPTI Max, Tunable) at 517 nm.
The reaction rates were compared and the percent inhibition caused by the presence of tested
inhibitors was calculated [23]. Each concentration was analyzed in three independent
experiments run in triplicate.
2.4 Computational Methodology
2.4.1 Retrieval of Jack bean urease structure from PDB
The Jack bean urease structure was retrieved from Protein Data Bank (PDB) (www.rcsb.org)
with PDBID 4H9M. The selected crystal structure of urease was minimized by using UCSF
Chimera 1.10.1 tool [24]. Furthermore, the stereo-chemical properties of urease structure and
Ramachandran plot and values were generated by Molprobity server and Protparam, respectively
[25]. The Discovery Studio 4.1 Client was used to generate the hydrophobicity graph of target
protein [26]. The protein architecture and statistical percentage values of receptor proteins
helices, beta-sheets, coils and turn were predicted from online server VADAR 1.8 [27].
2.4.2 Molecular docking simulation
The synthesized chemical structures (4a-j) were sketched in ACD/ChemSketch tool and
minimized by UCSF Chimera 1.10.1 tool [28]. The molecular docking experiments were
performed using PyRx docking through VINA wizard. The grid box center values were adjusted
as for X=10.48, Y=-55.22 and Z=-26.48), respectively. While, the size parameters values
11

(X=66.60, Y=60.08, and Z=56.84) were also focused to get the better conformational binding
poses. The default exhaustiveness value was used to maximize the binding conformational
analysis. All the synthesized ligands were docked separately against urease receptor. The
Autodock VINA scoring function equation is mentioned supplementary data. The predicted
docked complexes were evaluated on the basis of lowest binding energy (Kcal/mol) values and
structure activity relationship (SAR) analyses. The three dimensional (3D) graphical depictions
of all the docked complexes were accomplished by Discovery Studio (2.1.0).
3. Results
3.1 Synthetic chemistry
The designed compounds were synthesized by using heptanoic acid (1) as starting material.
Heptanoic acid was treated with thionyl chloride to convert carboxylic acid functionality into
heptanoyl chloride. Next, the acid was treated with easily available potassium thiocyanate in dry
acetone to form key reactive intermediate isothiocyanate (2) which was not isolated (no isolation
required for next step) [23,29]. In last step, substituted aromatic amines were reacted with
isothiocyanate to afford 1-heptanoyl-3-arylthioureas in excellent yield (84-90%). The synthesis
of new 1-heptanoyl-3-arylthioureas (4a-4j) is outlined in the Scheme 1.
12

Scheme-1 Synthetic pathway along with the obtained yield of 1-heptanoyl-3-arylthioureas (4a-
4j)
3.1 Urease Inhibitory activity
13

The urease enzyme inhibition activity results are summarized in Table 1. All the compounds
showed significant potential of inhibition against Jack bean urease enzyme with IC50 values
ranging in micromolar range. Moreover, all the compounds showed higher potential than the
thiourea standard. The compound 4a and 4c showed significant level of inhibition compared to
other derivatives in the series. The compound 4a bears two nitro groups at ortho- and para-
position which perturbs electron density and further pull apart electronic cloud. Compound 4c
bear halogen atom, bromine at para position; hence, due to large atomic size, lipophilic character
of the derivative enhances. The structure activity of relationship of the most potent derivatives is
depicted in Figure 1
Figure 1 Structure activity relationship of most potent derivatives 4a and 4c derivatives.
Table.1 Urease inhibitory activity of 1-Heptanoyl-3-arylthioure derivatives (4a-4j)
Compound
Urease(Jack bean)
14

IC50 ± SEM (µM)
4a
0.0313±0.0021
0.0445±0.0036
0.0396±0.0038
0.0621±0.0041
0.0644±0.0062
0.1246±0.0091
0.1551±0.021
0.1554±0.024
0.0934±0.0082
0.1112±0.081
18.482±0.461
4b
4c
4d
4e
4f
4g
4h
4i
4j
Thiourea
Values are expressed as mean ± SEM
SEM= Standard Error of Mean
4. Discussion
4.1 Chemistry
The synthesized compounds were characterized through FT-IR, CHNS analysis, melting point,
1H NMR and 13C NMR spectroscopy. The appearance of peak at 3400-3300 cm-1 was assigned to
N-H moiety. The characteristic peak for thiocarbonyl appear at 1255-1220 cm-1. The C-H sp2
1
stretching for benzene ring appeared at 1500-1400 cm-1. The H-NMR spectrum also revealed
the formation of compounds. The most deshielded signal appeared for N-H at 12-11 ppm. As
thioureas contains two distinct N-H groups so these two hydrogens appeared as distinct singlets.
The appearance of signal at 7-8 ppm was attributed to aromatic part. The alkyl part appeared at
15

2-3 ppm value. The 13C-NMR spectroscopy also showed formation of title compounds. The
characteristic peak for thiocarbonyl appeared at 180 ppm value while carbonyl appeared at 170
ppm value. The appearance of signals at 140-120 was assigned to aromatic part. The sp3 carbons
appeared at around 30-20 ppm.
4.2 Kinetic study
Based on our results we selected the most potent compounds, namely 4a and 4c, to determine
their inhibition type and inhibition constant on jack bean urease. The potential of these
compounds to inhibit free enzyme and enzyme substrate complex was determined in term of EI
and ESI constants respectively. The kinetic studies of the enzyme by the Lineweaver-Burk plot
of 1/V versus 1/[S] in the presence of different compounds concentrations gave a series of
straight lines (Figure 2). The results of both 4a and 4c showed that compound intersected within
the second quadrant. The analysis showed that Vmax decreased to new increasing doses of
inhibitors on the other hand Km remains the same. This behavior indicates that both compounds
inhibit the urease non-competatively to form enzyme inhibitor complex. Secondary plot of slope
against the concentration of inhibitors showed enzyme inhibitor dissociation constant (Ki)
(Figure 3)
The kinetic results are presented in the Table. 2.
16

Figure 2. Lineweaver–Burk plots for inhibition of Urease in the presence of inhibitor H1. (A)
Concentrations of 4a were 0.00, 0.0195, 0.039, 0.078 and 0.312 µM. (B) The insets represent the
plot of the slope or the vertical versus inhibitor 4a concentrations to determine inhibition
constants. The lines were drawn using linear least squares fit.
17

Figure 3. Lineweaver–Burk plots for inhibition of Urease in the presence of inhibitor H1. (A)
Concentrations of 4c were 0.00, 0.020.004, and 0.08 µM. (B) The insets represent the plot of the
18

slope or the vertical versus inhibitor 4c concentrations to determine inhibition constants. The
lines were drawn using linear least squares fit.
Table 2 Kinetic parameters of the jack bean urease for urea activity in the presence of different
concentration of 4a and 4c
Code Dose
(µM)
Vmax
Km
Inhibition
Type
Ki
(ΔA /Min)
(mM)
(µM)
0.00
0.0151
0.002
3.546
3.546
3.546
3.546
3.546
4a
0.0195
0.039
0.078
0.312
Non-Competitive
0.015
0.0017
0.0014
0.0004
0.00
0.02
0.04
0.08
0.0168
5.208
5.208
5.208
5.208
4c
0.00145
0.00108
0.00077
Non-Competitive
0.0145
V max is the reaction velocity, Km is the Michaelis-Menten constant, Ki is the EI dissociation
constant.
4.3 Structural and physiochemical evaluation of Jack bean urease
Jack bean urease is a class of hydrolase protein which consists of four domains having different
numbers of amino acids. Two nickel atoms are also present in the active region of target protein.
The overall statistical VADAR analysis showed the protein architecture which contains 27%
helices, 31% β sheets and 41% coils. Moreover, Ramachandran plots indicated that 97.5% of
19

residues were present in favored regions which show the precision of phi (φ) and psi (ψ) angles
among the coordinates of jack bean urease.
4.4 Chemo-informatics properties and Lipinski’s rule of synthesized compounds
The predicted chemo-informatic properties such as molar volume and refractivity, density,
polarizability and surface tension were evaluated by computational approaches. Kadam and his
colleagues have depicted some standard value for molar refractivity (40 to 130) and molecular
weight (160 to 480), respectively [30]. Results show that compounds 4a-j have better predicted
values compared to standard values. Moreover, the Lipinski’s rule of five (RO5) results showed
that compounds (4a-j) possess good HBA, HBD and logP values which are significantly justified
its drug like behavior [31]. Moreover, their molecular weight of all compounds was also
comparable with standard value (<5000 g/mol). The RO5 justifies that molecules with poor
absorption are more likely to have more than 5 HBD, MWT over 500, logP over 5 and more than
10 HBA. However there are plenty of examples available for RO5 violation amongst the existing
drugs [32]. The overall predicted values for all the compounds are mentioned in Table. 3.
Table. 3 Chemo-informatic properties of ligands
Properties
MW
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
356.12 332.05 342.04 310.12 298.09 306.18 310.12 360.03 437.94 264.13
HBA
6
4
2
2
2
2
4
3
2
2
2
2
4
3
2
2
2
2
2
2
HBD
LogP
2.84
108
4.96
4.62
3.36
4.36
4.73
3.36
4.77
5.50
3.77
33.30
PSA (A2)
32.60 33.30 71.56 32.60 31.90 71.56 32.60 31.90
Mol Vol.(A3) 342.5 312.99 302.79 311.21 297.33 343.73 311.14 308.81 331.37 280.94
20

Drug Score
RO5
-1.55
Yes
0.10
Yes
-0.15
Yes
-1.17
Yes
-0.30
Yes
-0.18
Yes
-0.86
Yes
-0.70
Yes
-0.16
No
-0.69
Yes
4.5 Pharmacokinetic properties of screened compounds
The development of novel candidate molecule requires high attention rate with good
pharmacokinetic properties. Most probably the absorption, distribution, metabolism, excretion
and toxicity (ADMET) properties assessment were considered as major hallmark to confirm the
efficacy of lead molecules. To check the pharmacokinetic behavior of our synthesized
compounds (4a-j), pkCSM online server was employed to predict the ADMET properties (Table
4). The general evaluation in ADMET, absorption properties such as water (log mol/L) and
intestinal solubility (% absorbed) and skin permeability (logKp) values justified the strong
therapeutic potential of lead compounds. Literature study showed that compounds with good
absorption results have more potency to cross gut barrier by passive penetration to reach the
target molecule [33]. The water solubility results justified that compounds 4a-j showed good
absorption prediction. All compounds (4a-j) displayed good intestinal solubility results
compared to standard value in literature (>30 %abs) [33]. Literature study showed that a
compound with less than 30% absorbance value is considered as poorly absorbed compound
[34]. The skin permeability values of all compounds were also greater than standard value (-2.5
logKp) which showed their significance as a good lead structures and their drug likeness
behavior. Moreover, blood brain barrier (BBB) and central nervous system (CNS) permeability
values of all screened compounds were also comparable with the standard values (>0.3 to <-1
log BB and >-2 to <-3 logPS) respectively. It has been observed that compounds with greater
than 0.3 log BB value have potential to cross BBB, while with less than -1 value are poor
21

distributed to brain. Similarly, the compounds have >-2 logPS value are considered to penetrate
the CNS, while with <-3 are difficult to move in the CNS. Results showed that compounds
displayed a significant potential to cross the barriers and may be target directly to receptor
molecule. Moreover, their computational metabolic behavior was confirmed by CYP3A4, which
is isoform of cytochrome P450 [35]. The excretion and toxicity predicted values were also
justified the drug likeness behavior of these compounds on the basis of total clearance (log
ml/min/kg), AMES toxicity, maximum tolerated dose (MTD) and LD50 values. The non-
mutagenic and non-toxic behavior was also observed from AMES toxicity prediction. The
hepatotoxicity negative behavior also showed their non-toxic and less sensitive effects. No
compounds showed hepatotoxic behavior while most of compounds were depicts skin sensitive
behavior except 4a, 4h and 4i. Skin sensitization character was also observed among all
compounds and predicted results showed their negative behavior in the skin sensitization
induction. These predicted ADMET properties results justified that these novel synthesized
compounds showed good lead like potential for further evaluation.
Table 4. Pharmacokinetic assessment of synthesized compounds
ADMET Properties
WS (log mol/L)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
-4.52 -4.59 -4.40 -4.09 -3.59 -4.26 -4.08 -4.50 -4.30 -3.29
78.3 86.9 88.2 87.1 87.7 89.7 85.4 88.6 86.7 89.5
IS(%abs)
SP(log Kp)
Absorption
-2.7 -2.7 -2.7 -2.7 -2.7 -2.7 -2.7 -3.0 -2.9
-2.7
BBBP (Log BB)
CNSP (LogPS)
-0.81 0.03 0.07 -0.43 0.13 -0.04 -0.41 -0.07 -0.09 0.004
-2.89 -1.15 -1.37 -2.64 -1.49 -1.35 -2.64 -1.46 -1.64 -1.14
Distribution
CYP3A4 inhibitor Yes Yes
No
Yes Yes
No
Yes
No
Yes
Yes
Metabolism
Excretion
TC (log ml/min/kg) 0.20 0.19 -0.15 0.08 0.13 -0.14 0.01 -0.04 -0.3 -0.005
22

AMES toxicity
Max. dose
ORAT(LD50)
HT
Yes
No
No
Yes
No
No
Yes
No
No
No
-0.46 0.24 0.52 -0.04 0.41 0.46 0.02 0.55 0.79 0.18
2.83 2.69 2.68 2.76 2.68 2.42 2.80 2.69 2.74 2.23
Toxicity
No
No
No
No
No
No
No
No
No
No
No
No
No
SS
Yes Yes Yes Yes Yes Yes
Yes
*Abbrevation: WS=Water solubility, IS=intestinal solubility, SP=skin permeability,
BBBP=blood brain barrier permeability, CNSP=CNS permeability, TC=Total clearance,
ORAT=Oral Rat Acute Toxicity, HT=Hepatotoxicity, SS=Skin Sensitization.
4.6 Molecular docking analysis
4.6.1 Docking energy evaluation of synthesized compounds
To predict the best fitted conformational position within the active region of target protein all the
synthesized compounds (4a-j) were docked against urease. The generated docked complexes
were examined on the basis of minimum energy values (kcal/mol) and bonding interaction
pattern. Docking results justified that compound 4a, 4c, 4f, 4h and 4i exhibited a good binding
energy values (-7.0, -7.1, -7.2, -7.0, and -7.0 kcal/mol), respectively compared to others
compounds. The standard error for Autodock is reported as 2.5 kcal/mol [36]. However,
compounds have no large docking energy value difference more than standard value. Since the
basic nucleus of all the synthesized compounds were similar, most of ligands possess efficient
energy values and have no significant energy fluctuations. The comparative binding energy
analysis reveals that 4c and 4f was most active compound (Figure. 4). Our computational
docking analysis also showed a good association with in-vitro study.
23

Figure 4. The graphical depiction of docking energy values.
4.6.2 Binding pocket analysis of urease docked complexes
The docked complexes were further analyzed on the basis of binding interactions. However, the
best conformational pose and energy valued docked complex (4c) is illustrated in Figure 5. In
detail, the structure activity relationship (SAR) study showed that four hydrogen bonds were
observed in 4c docked complex. The amino group of carbonyl chain is directly form hydrogen
bond with His593 having bonding distance 3.15 Å. Whereas, oxygen group of benzene ring also
form hydrogen bond with Met637 with bonding distances 2.12 Å, respectively. Similarly,
carbonyl oxygen interacts with His519 and Asp494 by hydrogen interactions with binding
distance 2.98 Å and 2.59 Å, respectively. Literature data also ensured the importance of these
residues in bonding with other urease inhibitors which strengthen our docking results [37]. The
comparative binding energy and SAR analysis showed the significance of 4e compound and may
consider as potent inhibitors by targeting jack bean urease.
24

Figure 5. Docking complex of 4c. The ligand structure is displayed in grey color, while the
functional groups such as amino, sulfur and oxygen are highlighted in light blue, yellow and red
colors, respectively. The protein interacted amino acids are mentioned in maroon color in wire
format. Two nickel atoms are also present. The black dotted lines represent the hydrogen binding
and bond distances measured in angstrom (Å).
5. Conclusions
A new family of 1-heptanoyl-3-arylthioureas (4a-4j) was synthesized in straightforward way in
excellent yield. The compounds were evaluated for Jack bean urease inhibition. All the
compounds showed excellent activity against urease inhibition. The most potent derivative was
25

4a containing two nitro group at ortho and para positions while the other potent derivative 4c
bore bromine at para position. The kinetic studies were performed to undermine the mode of
inhibition of enzyme. Both derivatives were found to be non-competitive inhibitors. The Ki
values for 4a and 4c were found to be 0.015 µM and 0.0145 µM. The chemo-informatics also
revealed that these low-molecular weight target molecules can be used in drug designing. From
the results of biological activity, kinetics and molecular docking it can be inferred that these
molecules serve as structural templates for the designing potent drugs against Jack bean urease
enzyme.
Acknowledgment
Supported by University Hospital in Hradec Kralove (No. 00179906).
Disclosure Statement
No potential conflict of interest was reported by authors.
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