Synthesis of 4-thiazolidinone analogs as potent in vitro anti-urease agents

Article abstract of DOI:10.1016/j.bioorg.2015.10.005

4-Thiazolidinone analogs 1-20 were synthesized, characterized by ¹H NMR and EI-MS and investigated for urease inhibitory activity. All twenty (20) analogs exhibited varied degree of urease inhibitory potential with IC₅₀ values 1.73-69.65 μM, if compared with standard thiourea having IC₅₀ value of 21.25 ± 0.15 μM. Among the series, eight derivatives 3, 6, 8, 10, 15, 17, 19, and 20 showed outstanding urease inhibitory potential with IC₅₀ values of 9.34 ± 0.02, 14.62 ± 0.03, 8.43 ± 0.01, 7.3 ± 0.04, 2.31 ± 0.002, 5.75 ± 0.003, 8.81 ± 0.005, and 1.73 ± 0.001 μM, respectively, which is better than the standard thiourea. The remaining analogs showed good to excellent urease inhibition. The binding interactions of these compounds were confirmed through molecular docking studies.

Full text of DOI:10.1016/j.bioorg.2015.10.005

Accepted Manuscript
Synthesis of 4-Thiazolidinone Analogs as Potent in Vitro Anti-urease Agents
Fazal Rahim, Khalid Zaman, Hayat Ullah, Muhammad Taha, Abdul Wadood,
Muhammad Tariq Javed, Wajid Rehman, Muhammad Ashraf, Reaz Uddin,
Imad uddin, Humna Asghar, Aftab Ahmad Khan, Khalid M. Khan
PII:
S0045-2068(15)30029-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.10.005
YBIOO 1852
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 September 2015
19 October 2015
20 October 2015
Please cite this article as: F. Rahim, K. Zaman, H. Ullah, M. Taha, A. Wadood, M.T. Javed, W. Rehman, M. Ashraf,
R. Uddin, I. uddin, H. Asghar, A.A. Khan, K.M. Khan, Synthesis of 4-Thiazolidinone Analogs as Potent in Vitro
Anti-urease Agents, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.10.005
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Synthesis of 4-Thiazolidinone Analogs as Potent in Vitro Anti-urease Agents
Fazal Rahima*, Khalid Zamana, Hayat Ullaha, Muhammad Tahab,c, Abdul Wadoodd, Muhammad
Tariq Javeda, Wajid Rehmana, Muhammad Ashraf e, Reaz Uddinf, Imad uddin a , Humna Asghar
e , Aftab Ahmad Khana, Khalid M. Khanf
aDepatment of Chemistry, Hazara University, Mansehra-21120, Pakistan
bAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia
cFaculty of Applied Science Universiti Teknologi MARA, 40450 Shah Alam, Malaysia
dDepartment of Biochemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.
eDepartment of Biochemistry and Biotechnology, The Islamia University of Bahawalpur,
Bahawalpur-63100, Pakistan
f HEJ, Research Institute of Chemistry, ICCBS, University of Karachi, Karachi-75270, Pakistan
Abstract:
1
4-Thiazolidinone analogs 1-20 were synthesized, characterized by HNMR and EI-MS and
investigated for urease inhibitory activity. All twenty (20) analogs exhibited varied degree of
urease inhibitory potential with IC50 values 1.73-69.65 µM, if compared with standard thiourea
having IC50 value of 21.25 0.15 µM. Among the series, eight derivatives 3, 6, 8, 10, 15, 17, 19,
and 20 showed outstanding urease inhibitory potential with IC50 values of 9.34 0.02, 14.62
0.03, 8.43 0.01, 7.3 0.04, 2.31 0.002, 5.75 0.003, 8.81 0.005, and 1.73 0.001 µM,
respectively, which is better than the standard thiourea. The remaining analogs showed good to
excellent urease inhibition. The binding interactions of these compounds were confirmed through
molecular docking studies.
Keywords: 4-Thiazolidinone, Synthesis, Urease inhibition, Docking studies.
*Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343
1

1.0. Introduction:
Urease enzymes catalyze the hydrolysis of urea into carbamate and ammonia [1-3]. The
carbamate decomposes readily into another molecule of ammonia. These reactions cause
substantial rise in pH which may cause adverse effects of urease activity on agriculture, animal
and human health [4]. Clinically this enzyme is used as indicative to fix manifestation of
pathogen in urinary and gastrointestinal tracts. The bacterial urease results in stomach ulcer,
infectious stones, peptic ulcer and damaging infections both in animals and human [5]. It also
causes the pathogenesis of pyelonephritis, hepatic coma urolithiasis, hepatic encephalopathy,
ammonia and urinary catheter encrustation [2]. Lot of microorganisms uses this enzyme to give
cradle of nitrogen for growth. In germination process this enzyme has key role in metabolism of
plants [2, 6].
Heterocyclic compounds are important for medicinal chemists [7]. Thiazole analogs have
medical claims such as bacteriostatic, antibiotics [8], diuretics [9], local anesthetics [10, 11],
anti-inflammatory [12], analgesics [13, 14], and anti-HIV [15, 16].
4-Thiazolidinones are the derivatives of thiazole that have been extensively studied and found it
to be a part of vitamin-B, Penicillins and antibacterial thiazoles. Reduced thiazole occurs as
structural units in compounds of biological importance that serves in the study of polypeptide
and proteins [17]. 4-Thiazolidinones have been reported to possess a varied range of biological
activities including antitubercular [18], antibacterial [19], antitumor [20], antihistaminic [21],
anti-inflammatory [22] and anticonvulsant activity [23].
Our research group is continuously doing effort in search of biologically potent scaffolds [24],
which are easy to synthesize and have no tedious chemistry. Previously our group has reported
thiobarbituric acid analogs as potent urease inhibitors [25]. In a continued effort in search for
biologically active molecules, here we are going to report synthesis of 4-thiazolidinone
derivatives and their urease inhibition activity that has not been published earlier.
2

2. Results and Discussion:
2.1. Chemistry
Synthesis of 4-thiazolidinone analogs was done in two steps [26]. First, substituted
benzaldehyde/ acetophenone (1 mmol) was reacted with thiosemicarbazide and 2-3ml of HCl in
ethanol and reflux for 3-4 h. The color change showed reaction completion which was monitored
by TLC. On completion, reaction mixture was filtered, washed with n-hexane to get Schiff base
intermediate.
In step-2, Schiff base intermediate product was reacted with chloro acetic acid (1 mmol) and
sodium acetate (1 mmol) in acetic acid and reflux for 6 h. Reaction completion was monitored by
TLC. On completion, mixture was filtered, washed with n-hexane/ethanol to obtain 4-
thiazolidinone analogs.
Insert Scheme-1 Here
Insert Table-1 Here
2.2. Biological Activity
The urease inhibitory effects of newly synthesized 4-thiazolidinone analogs were measured, for
Bacillus pasteurii the results are summarized in Table-2 and compared with urease well known
inhibitor thiourea (IC50 value 21.25 0.15 µM) as reference compound. Out of these twenty
analogs, eight analogs 3, 6, 8, 10, 15, 17, 19, and 20 showed outstanding urease inhibitory
potential with IC50 values 9.34 0.02, 14.62 0.03, 8.43 0.01, 7.3 0.04, 2.31 0.002, 5.75
0.003, 8.81 0.005, and 1.73 0.001 µM respectively, which were many folds better than the
standard. The remaining analogs also showed good to excellent urease inhibition. Structure-
activity relationship suggested that the urease activity of a particular molecule is seemingly
directed by the substitution pattern present at aromatic residues. In particular, the analog 20
having methoxy group at 2,5 position and bromine at 4-position on phenyl part was an excellent
urease inhibitor with IC50 value 1.73
0.001 which was better than the standard inhibitor
thiourea. Compound 20 was when compared with analogs 7 and 9 having two methoxy groups
with bromo and hydroxyl group respectively at phenyl part. The inhibitory potential of
3

compound 20 was greater than 7 which were greater than 9. The difference in potential was seem
to be due difference in position of substituents. The compound 15 was found to be the second
most active analog among the series having Br at position-2 while nitro at position-4 on the
phenyl part. If we comparing compound 15 with analog 1 and 2 having only nitro group on
phenyl ring, the compound 15 showed greater potential that is mainly because of extra bromo
group. Similarly analog 17 the third most active compound among the series having methoxy
group at position-3, while ethoxy at position-4 on the phenyl part. The greater potential of analog
17 then other disubstituted (EDG) analogs like 4 and 18 was mainly because of position
difference of substituents. Similar affect was also observed in other analogs. In conclusion we
observed here that both electrons donating as well as electron withdrawing groups on phenyl ring
play role in the inhibition but the electron donating groups are superior up to some extent. The
binding interactions were confirmed through molecular docking studies.
Insert Table-2 Here
Molecular docking
In order to explore the binding mode of newly synthesized 4-thiazolidinone derivatives in the
active site of Bacillus pasteurii urease (PDB Code: 4UBP) [32] docking study was carried out
using MOE (Molecular Operating Environment) software package. The docking results showed
that all the compounds well accommodated in the binding pocket of Bacillus pasteurii urease.
The docked conformation of the most active compound 20, showed that the carbonyl oxygen of
thiazolidin ring of the compound coordinates with both nickel ions like the hydroxyl oxygen in
the acetohydroxamate (a potent urease inhibitor) [32]. In addition to nickel chelation, two
hydrogen bonds and several hydrophobic interactions between the active site residues and the
compound were also observed (Figure 1a). The carbonyl oxygen of thiazolidin ring and nitrogen
atom of imino moiety established hydrogen bonds with active site residues His222 and His323
respectively. If we compare the structures of compound 20, 4, 7 and 9 all the compounds have
about similar structures the only difference in their structures is the presence or absence of
bromine moiety and the position of methoxy moiety. The biological activities of these
compounds showed that bromine moiety and the position of methoxy play a key role in the
inhibitory activities of these compounds (Table 1). For example in case of compound 18,
although the compound have methoxy moieties but bromine is not present and the activity of this
4

compound is less as compare to compound 20. The binding mode of compound 18 showed poor
interaction as compare to compound 20. As shown in Figure 1b, although compound 18
established coordinate bond with nickel ions but only one hydrogen bond was observed whereas
in case of compound 20 two hydrogen bonds were observed. Furthermore, the distance between
the nickel ions and carbonyl oxygen is more as compare to compound 20 (Figure 1a and 1b).
Insert Figure-1 Here
Similarly, the positions of methoxy moiety play a key role in the biological activities as well as
binding interactions. The docking results showed that the compounds having adjacent methoxy
moiety showed poor interactions with active site residues of urease and less biological activities
(compound 4 and 7). About similar experimental and docking results were observed for
compounds 15 and 2. In these compounds the bromine play the same role as in the above
mentioned compounds. When bromine is present, for example compound 15, good biological
activity and strong bonding network was observed (Table 1 and Figure 2a). But when bromine
is absent from the structure (compound 2), lower biological activity and poor interactions were
observed (Tabel 1 and Figure 2b).
Insert Figure-2 Here
In case of compounds having hydroxy phenyl ring in their structures (compound 3, 5 and 6), it
was observed that the presence and absence of chlorine moiety and the position of hydroxyl
group play a pivotal role in the biological activities as well as the binding interactions of these
compounds. For example when chlorine is present, compound 3, the inhibitory activity and
binding interactions are good (Table 1). But when chlorine is absent from the structure
(compound 6) both biological activity and binding interactions were poor (Figure 3a). Like
bromine and chlorine a key role was observed for benzoyloxy-4-methylbenzine and indole
moieties. For example when these moieties present (compound 11, 12 and 18) lower biological
activities as well as poor interactions were observed for these compounds (Table 1 and Figure
3b). These lower activities and poor interactions might be due to the steric clash produce by
these bulky hydrophobic groups. The docking conformation of compound 12 showed that
although the carbonyl oxygen of the thiazolidin ring coordinate with the nickel ions of the
5

enzyme but the distance is more as compare to compound lacking benxoyl-4-methylbenzine ring
(Figure 3b).
Insert Figure-3 Here
Overall the docking results showed that bromine and chlorine moieties play a key role in the
biological activities of these compounds whereas the presences of bulky hydrophobic groups are
detrimental for the biological activities and binding interactions.
3.0. Conclusion
In conclusion we have synthesized twenty analogs of 4-thiazolidinone and evaluated for urease
inhibition. All compounds showed varied degree of urease inhibition ranging in between 1.73
0.001 to 69.65 0.12 µM when compared with the standard inhibitor thiourea having IC50 value
21.25 0.15 µM. Among the series, analogs 3, 6, 8, 10, 15, 17, 19, and 20 showed outstanding
urease inhibitory potential with IC50 values 9.34 0.02, 14.62 0.03, 8.43 0.01, 7.3 0.04,
2.31 0.002, 5.75 0.003, 8.81 0.005 and 1.73 0.001 respectively which is better than the
standard thiourea. The remaining analogs also showed good to excellent urease inhibition.
4. Experimental Section
4.1. General Methods
The NMR spectra of concerned analogs of 4-thiazolidinone were recorded by on Avance Av-
300, 500 MHz NMR spectrometers in which DMSO-d6/Acetone-d6 were used as a solvent.
Chemical shift values of spectra are assigned in δ (ppm). Tetramethylsilane act as internal
standard. Jeol JMS-600H instrument were used to obtained Electron Ionization Mass Spectra.
The probability of reactions was checked by TLC on precoated silica gel aluminum plates
(kieselgel 60,254, E. Merck, Germany). UV lamp at range of 254 and 365nm were used to
observe the spots on TLC plate.
4.2. Methodology for synthesis of 4-thiazolidinone (1-20)
Synthesis of 4-thiazolidinone analogs was done in two steps [26]. First, substituted
benzaldehyde/ acetophenone (1 mmol) was reacted with thiosemicarbazide and 2-3ml of HCl in
6

ethanol and reflux for 3-4 h. The color change showed reaction completion which was monitored
by TLC. On completion, reaction mixture was filtered, washed with n-hexane to get Schiff base
intermediate.
In step-2, Schiff base intermediate product was reacted with chloro acetic acid (1 mmol) and
sodium acetate (1 mmol) in acetic acid and reflux for 6 h. Reaction completion was monitored by
TLC. On completion, mixture was filtered, washed with n-hexane/ethanol to obtain 4-
thiazolidinone analogs.
4.2.1. (E)-2-((E)-(2-Nitrobenzylidene) hydrazono) thiazolidin-4-one (1)
Yield: 81%, 1H-NMR: (500 MHz, DMSO-d6): δ 11.2 (s, 1H, NH), 8.6 (s, 1H, HC=N), 8.0 (dd, J
= 1, J = 8 Hz, 1H, H-3), 7.9 (dd, J = 1.5 Hz, J6,4 = 8 Hz, 1H, H-6), , 7.88 (dt, J = 0.5 Hz, J = 7.5
Hz, 1H, H-4 ), 7.6 (dt, J = 1.5 Hz, J = 8.5 Hz, 1H, H-5), 3.9 (s, 2H, CH2). EI-MS: m/z (rel. int.
%): 264 (M+, 100), 219 (56), 157 (77), 101 (69) 77 (81).
4.2.2. (E)-2((E)-(4-Nitrobenzylidene) hydrazono) thiazolidine-4-one (2)
Yield: 70%, 1H-NMR: (500 MHz, DMSO-d6): δ 11.2 (s, 1H, NH), 8.5 (s 1H, CH=N), 8.3 (d, J =
8.5 Hz, 2H, H-3/5), 7.9 (d, J = 8.3 Hz, 2H, H-2/6) 3.9 (s, 2H, CH2). EI-MS: m/z (rel. int. %): 264
(M+, 100), 219 (56), 157 (77), 101 (69) 77 (81).
4.2.3. (E)-2-((E)-(3-Chloro-4-hydroxybenzylideno) thiazolidin-4-one (3)
1
Yield: 72%, H-NMR: (300 MHz, Acetone-d6): δ 11.2 (s, 1H, NH), 8.2 (s, 1H, HC=N), 7.8 (d,
J2, 6= 2.1 Hz, 1H, H-2), 7.6 (dd, J = 1.8 Hz, J6, 5 = 8.4 Hz, 1H, H-6). 7.0 (d, J = 8.4 Hz, 1H, H-5),
3.8 (s, 2H, CH2). EI-MS: m/z (rel. int. %): 271 (M+ +2, 30), 269 (M+, 100), 105 (52), 140 (64),
154 (76) 77 (47).
4.2.4. (E)-2-((E)-(2, 3-Dimethoxybenzylidene) hydrazono) thiazolidin-4-one) (4)
7

Yield: 64%,.1H-NMR: (500 MHz, DMSO-d6): δ 11.2 (s, 1H, NH), 8.5 (s, 1H, HC=N), 7.4 (dd,
J6, 4 = 3 Hz, J = 6.5 Hz, 1H, H-6), 7.1 (m, 2H, H-4/5 ), 3.8 (s, 2H, CH2), 3.7 (s, 6H, OMe). EI-
MS: m/z (rel. int. %): 279 (M+, 100), 263 (37), 157 (44), 101 (39) 77 (57).
4.2.5. (E)-2-((E)-(3, 5-Dichloro-2-hydroxybenzylidene) hydrazono) thiazolidin-4 one (5)
1
Yield: 81%, H-NMR: (300 MHz, Acetone-d6): δ 11.2 (s, 1H, NH), 8.5 (s, 1H, HC=N), 7.9 (s,
1H, H-4), 7.4 (d, J6, 4 = 2.4 Hz, 1H, H-6), 3.8 (s, 2H, CH2). EI-MS: m/z (rel. int. %) 307 (M+ +2,
8), 305 (M+, 18), 263 (34), 187 (55), 124 (120), 97 (48).
4.2.6. (E)-2((E)-(4-Hydroxybenzylidin) hydrazono) thiazolidine-4-one (6)
Yield: 68%, 1H-NMR (300 MHz, Acetone-d6), δ 10.2 (s, 1H, NH), 8.0 (s, 1H, HC=N), 7.6, (d, J
= 8.7 Hz, 2H, H-3/5), 6.8 (d, J = 8.4 Hz, 2H, H-2/6), 3.7 (s, 2H, CH2). EI-MS: m/z (rel. int. %)
235 (M+, 100), 195 (22), 135 (14), 120 (92), 106 (51).
4.2.7. (E)-2((E)-(3-Bromo-4, 5-dimethoxybenzylidene) hydrazono) thiazolidine 4-one (7)
1
Yield: 65%, H-NMR (300 MHz, Acetone-d6): δ 10.4 (s, 1H, NH), 8.2 (s, 1H, HC=N), 8.0 (s,
1H, H-2), 7.9 (d, J = 3.9 Hz, 1H, H-6), 3.9 (s, 6H, 2xOMe), 3.8 (s, 2H, CH2). EI-MS: m/z (re lint
%) 358 (M+ +2, 83), 356 (M+, 85), 278 (2), 228 (20), 169 (10), 78 (29).
4.2.8. (E)-2((E)-(3-nitrobenzylidene) hydazono) thiazolidin-4-one (8)
Yield: 75%, 1H-NMR(300 MHz, Acetone-d6), δ 10.4 (s,1H, NH), 8.6 (s, 1H, HC=N), 8.2 (s, 1H,
H-2), 8.2 (d, J = 8.1 Hz, 1H, H-6), 7.7 (t, J = 8.1 Hz, 2H, H-4/5), 3.8 (s, 2H, CH2). EI-MS: m/z
(rel int%) 224 (M+, 69), 118 (58), 89 (53), 76 (100), 60 (71).
4.2.9. (E)-2-((E)-(4-Hyrazoxy-3, 5-dimethoxybenzylidene) hydrazono) thiazolidin-4-one (9)
1
Yield: 70%, H-NMR (300 MHz, Acetone-d6), δ 10.4 (s, 1H, NH), 8.2 (s, 1H, HC=N), 8.0 (s,
1H, OH), 7.12 (d, J = 4.8Hz, 2H, H-2/6), 3.8 (s, 2H, CH2), 3.5 (s, 6H, 2 x OMe). EI-MS: m/z (rel
int%) 295 (M+, 95), 166 (29), 151 (13), 95 (27), 68 (18).
4.2.10. (E)-2-((E)-(4-(Benzyloxy)-2-methoxybenzylidene) hydrazono) thiazolidin-4-one (10)
Yield: 68%, (1H-NMR (300 MHz, Acetone-d6), δ 10.2 (s, 1H, NH), 8.2 (s, 1H, HC=N), 7.55 (d,
J3, 5 =1.8 Hz, 1H, H-3), 7.50 (m, 3H, H-5’/6’/2’), 7.3 (m, 3H, H-3’/4’/6), 7.1 (dd, J = 1.8 Hz, J =
8.4 Hz, 1H, H-5), 5.1 (s, 2H, OCH2). 3.87 (s, 2H, CH2), 3.85 (s, 3H, OMe). EI-MS: m/z (rel. int.
%) 355 (M+, 13), 263 (23) 163 (7) 135 (9).
4.2.11. 4-((E)-((E)-(4-Oxothiazolidin-2-ylidene) hydrazono) methyl) benzaldehyde (11)
8

Yield: 60%, (1H-NMR (300 MHz, Acetone-d6), δ 10.2 (s, 1H, NH), 8.2 (s, 1H, HC=N), 8.1 (s,
1H, CHO aldehydic), 7.9 (d, J = 8.7 Hz, 2H, H-2/3) 7.5 (d, J = 8.4Hz, 2H, H-5/6), 3.8 (s, 2H,
CH2). EI-MS: m/z (rel. int. %) 247 (M+, 20), 129 (25), 178 (78), 115 (36), 76 (46).
4.2.12. (Z)-2-((Z)-(4-(Dimethyl amino) benzylodine) hydrazono) thiazolidine-4-one (12)
Yield: 63%, (1H-NMR (300 MHz, Acetone-d6), δ 10.3 (s, 1H, NH), 8.2 (s, 1H, HC=N), 7.9, (d, J
= 7.2 Hz, 2H, H-3/5), 6.9 (d, J = 7.4 Hz, 1H, H-2), 3.7 (s, 2H, CH2), 3.06 (s, 6H, 2 x OMe). EI-
MS: m/z (rel. int. %) 262 (M+, 100), 219 (67), 157 (47), 115 (21), 77 (58).
4.2.13. (E)-2-((E)-(4-(Benzyloxy) benzylidene) hydrazono) thiazolidine-4-one (13)
Yield: 68%, 1H-NMR (300 MHz, Acetone-d6) δ 10.1 (s, 1H, NH), 8.0 (s, 1H, HC=N), 7.5 (dd, J
= 1.6 Hz, J = 7.4 Hz, 2H, H-2/6), 7.4 (m, 3H, H-5’/6’/2’), 7.2 (m, 2H, H-3’/4’), 7.0 (dd, J = 1.8
Hz, J = 8.4 Hz, 2H, H-3/5), 5.1 (s, 2H, OCH2). 3.9 (s, 2H, CH2). EI-MS: m/z (rel. int. %) 325
(M+, 66), 263 (75), 219 (45), 101 (100), 77 (81).
4.2.14. (2Z)-2, 2’ (Z)-(1, 4-Phenylenebis (methane-1-yl-ylidene)) bis (hydrazine-2, 1-diylidene))
dithiazolidin-4-one (14)
1
Yield: 55%, H-NMR (300 MHz, Acetone-d6), δ 10.2 (s, 2H, 2 x NH), 8.0 (s, 2H, 2 x HC=N’),
7.41 (dd, J = 1.6 Hz, J = 8.1 Hz, 2H, H-3/5), 7.40 (dd, J = 1.6 Hz, J2, 3/6, 5 = 8.0 Hz, 2H, H-3/5),
3.8 (s, 4H, 2-CH2). EI-MS: m/z (rel. int. %) 360 (M+, 72), 273 (61), 233 (41), 157 (36), 101
(100).
4.2.15. (Z)-2-((Z)-(1-(2-Bromo-4-nitrophenyl) ethylidene) hydrazono) thiazolidine-4-one (15)
Yield: 58%, (1H-NMR (300 MHz, Acetone-d6), δ 10.1 (s, 1H, NH), 8.2 (s, 1H, H-3), 8.1 (d, J5, 6
= 8.1 Hz, 1H, H-5), 7.9 (d, J6, 5 = 8.0 Hz, 1H, H-6), 3.7 (s, 2H, CH2), 2.7 (s, 3H, CH3). EI-MS:
m/z (rel. int. %) 359 (M+ +2, 98), 357 (M+, 100), 270 (51), 146 (44), 101 (378), 77 (19).
4.2.16 (E)-2-(E)-((1H-Indole-3-yl) methylene) hydrazono) thiazolidine-4-one (16)
Yield: 58%, 1H-NMR (300 MHz, Acetone-d6), δ 10.5 (s, 1H, NH), 10.3 (s, 1H, NH), 8.1 (s, 1H,
HC=N), 7.5 (d, J = 7.2 Hz, 2H, H-4/7), 7.3 (s, 1H, H-2) 7.1 (m, 2H, H-5/6) 3.8 (s, 2H, CH2). EI-
MS: m/z (rel. int. %) 258 (M+, 100), 157 (76), 116 (35), 101 (56), 70 (18).
4.2.17. (Z)-2-((Z)-(4-Ethoxy-3-methoxybenzoylidine) hydrazono) thiazolidine-4-one (17)
9

Yield: 77%, 1H-NMR (300 MHz, Acetone-d6), δ 10.1 (s,1H, NH), 8.1 (s, 1H, HC=N), 7.2 (s, 1H,
H-2), 7.1 (m, 2H, H-5/6) 3.7 (m, 2H, OCH2), 3.6 (s, 3H, OMe) 1.34 (t, 3H, CH3). EI-MS: m/z
(rel. int. %) 293 (M+, 75), 219 (92), 157 (38), 101 (100), 77 (23).
4.2.18. (Z)-2-((Z)-(2, 4-Dimethoxybenzoylidine) hydrazono) thiazolidine-4-one (18)
1
Yield: 56%, H-NMR (300 MHz, Acetone-d6), δ 10.1 (s,1H, NH), 8.2 (s, 1H, HC=N), 7.6 (m,
2H, H-5/6) 7.3 (s, 1H, H-3), 3.8 (s, 2H, CH2), 3.6 (s, 6H-OMe).EI-MS: m/z (rel. int. %) 279 (M+,
100), 219 (51), 100 (31), 77 (56), 77 (37).
4.2.19. (Z)-2-((Z)-(Napthalen-1-ylmethylene) hydrazono) thiazolodin-4-one (19)
1
Yield: 56%, H-NMR (300 MHz, Acetone-d6), δ 10.4 (s,1H, NH), 8.1 (s, 1H, HC=N),7.5 (m,
2H, H-2/4), 7.4 (d, J = 7.2 Hz, 1H, H-8), 7.3 (m, 4H, H-5/67/3), 3.8 (s, 2H, CH2). EI-MS: m/z
(rel. int. %) 289 (M+, 60), 157 (80), 127 (100), 101 (33), 70 (16).
4.2.20. (Z)-2-((Z)-(4-Bromo-2, 5-dimethoxybenzoylidine) hydrazono)thiazolidine-4-one (20)
Yield: 45%, 1H-NMR (300 MHz, Acetone-d6), δ 10.4 (s,1H, NH), 8.2 (s, 1H, HC=N), 7.4 (s, 1H,
H-3) 7.2 (s, 1H, H-6), 3.84 (s, 2H, CH2), 3.83 (s, 6H, 2xOMe); EI-MS: m/z (rel. int. %) 360 (M+
+2, 99), 358 (M+, 100), 328 (52), 219 (37), 157 (28), 77 (62).
4.3. Urease inhibition Assay
This assay was modified from Berthelot assay and was employed for the determination of urease
activity [27]. The assay is based on the hydrolysis of urea into ammonia which reacts with
phenol-hypochlorite to form light blue colored complex measured at 625 nm. A total volume of
85 µl assay mixture contained 10 µl of 50 mM phosphate buffer, pH 7.0, 10 µl of sample
solution and 25 µl of Bacillus pasteurii urease (Sigma) solution (0.015 units). The contents were
pre-incubated at 37ºC for 10 min. Then, 40 µl of urea stock solution (20 mM) was added to each
well and incubation continued at 37ºC for further 10 min and preread at 625nm using the 96-well
plate reader Synergy HT (Biotek Inc.). Phenol hypochlorite (115 µl) reagent was added in each
well (freshly prepared by mixing 45 µl phenol reagent with 70 µl of alkali reagent). For color
development, incubation was done at 37oC for another 10 min. Absorbance was again measured
at 625 nm. The percentage enzyme inhibition was calculated by the following formula:
Inhibition (%) = 100 – [(Abs. of test sample / Abs. of control) × 100]
IC50 values (concentration at which enzyme inhibition is 50%) of active compounds were
determined by measuring activities at further dilutions and the data was computed by using EZ-
10

Fit Enzyme software (Perrella Inc, USA). The absorbance units of uninhibited enzyme were
between 1.0-1.2.
Molecular docking
The three dimensional (3D) X-ray structure of BP urease with the resolution 1.55 Å was
retrieved from the protein data bank (http://www.rscb.org./pdb; code 4UBP) [28]. All the water
molecules were removed from the structure and hydrogen atoms were added. This structure was
then energy minimized with amber99 force field in the MOE Software packages
(http://www.chempcomp.com). The three dimensional structure of the compounds were modeled
using Builder software implemented on MOE. All the structures were then energy minimized
using mmff94 force field in MOE prior to molecular docking studies.
Acknowledgements: This work was economically sustained by the Higher Education
Commission (HEC), Pakistan, Project No. 20-1910 under the National Research Program for
Universities.
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Captions
Scheme-1: Synthesis of 4-thiazolidinone analogs 1-20
Table-1: Different substituent 4-thiazolidinone analogs 1-20
Table-2: Urease inhibitory activities and molecular docking score of compounds 1-20.
Figure-1: Docking conformation of compounds (a) 20 and (b) 18 in the active site of urease
enzyme.
Figure-2: Docking conformation of compounds (a) 15 and (b) 2 in the active site of urease
enzyme.
Figure-3: Docking conformation of compounds (a) 6 and (b) 12 in the active site of urease
enzyme.
14

Scheme-1:
15

Figure-1:
16

Figure-2
17

Figure-3:
18

Table-1: Different substituent of 4-thiazolidinone analogs 1-20
S.No
1
R1
R2
H
H
H
2
3
H
H
H
4
5
6
H
H
H
7
8
9
OCH3
OH
OCH3
H3C
H
H
10
11
N
CH3
19

H
H
12
13
H
14
15
CH3
H
H
16
17
H
H
18
19
H
20
Table-2: Urease inhibitory activities and molecular docking score of compounds 1-20.
Compound No.
IC50(µM)
47.52 0.06
26.91 0.05
9.34 0.02
52.53 0.08
26.71 0.06
14.62 0.03
Docking Score
-10.9123
1
2
3
4
5
6
-10.8194
-15.2562
-10.6082
-10.1501
-11.0493
20

36.64 0.06
8.43 0.01
37.91 0.07
7.3 0.04
7
-9.1254
-17.5732
-10.4323
-20.8895
-8.3454
-8.3095
-7.5423
-7.1573
-21.1516
-7.0901
-21.0824
-6.9867
-14.2858
-23.8513
8
9
10
48.54 0.08
68.92 0.02
39.21 0.09
43.93 0.08
2.31 0.002
51.42 0.09
5.75 0.003
69.65 0.12
8.81 0.005
1.73 0.001
21.25 0.15
11
12
13
14
15
16
17
18
19
20
Thiourea
21

Synthesis of 4-Thiazolidinone Analogs as Potent in Vitro Anti-urease Agents
Fazal Rahima*, Khalid Zamana, Hayat Ullaha, Muhammad Tahab,c, Abdul Wadoodd, Muhammad Tariq
Javeda, Wajid Rehmana, Muhammad Ashraf e, Reaz Uddinf, Imad uddin a , Humna Asghar e ,Aftab Ahmad
Khana, Khalid M. Khanf
aDepatment of Chemistry, Hazara University, Mansehra-21120, Pakistan
bAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia
cFaculty of Applied Science Universiti Teknologi MARA, 40450 Shah Alam, Malaysia dDepartment of
Biochemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.
eDepartment of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur-
63100, Pakistan
f HEJ,Research Institute of Chemistry, ICCBS, University of Karachi, Karachi-75270, Pakistan
Br
Compound 20
H
O
H3CO
OCH3
N
u
(IC50 = 1.73 ± 1.001 M)
N
N
Potent Inhibitor of Urease
S
Standard Inhibitor Thiourea
u
(IC50 = 21.25 ± 0.15 M)
22

*Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343
23