Synthesis, biological evaluation and docking studies of some novel isatin-3-hydrazonothiazolines

Article abstract of DOI:10.1039/c6ra10043k

A new series of thirty nine 5-trifluoromethoxy/fluoro/chloro-isatin 3-hydrazonothiazolines 5a-n, 6a-o and 7a-j were synthesized by cyclization of the corresponding intermediate N⁴-aryl-substituted isatin-3-thiosemicarbazones 3 (prepared by cond

Full text of DOI:10.1039/c6ra10043k

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Synthesis, biological evaluation and docking studies of some novel isatin-3-
hydrazonothiazolines
a
a
b
a
Maqbool Ahmad , Humayun Pervez *, Sumera Zaib , Muhammad Yaqub , Muhammad Moazzam
c
b
b
Naseer , Shafi Ullah Khan , Jamshed Iqbal *
a
Institute of Chemical Sciences, Organic Chemistry Division, Bahauddin Zakariya University,
Multan 60800, Pakistan
b
Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad
2
2060, Pakistan
c
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Eꢀmail addresses: pdhpervez@hotmail.com (H. Pervez); drjamshed@ciit.net.pk (J. Iqbal)
Abstract
New series of thirty nine 5ꢀtrifluoromethoxy/fluoro/chloroꢀisatin 3ꢀhydrazonothiazolines 5a-n, 6a-o
4
and 7a-j were synthesized by cyclization of the corresponding intermediate N ꢀarylꢀsubstituted
4
isatinꢀ3ꢀthiosemicarbazones 3 (prepared by condensation of appropriate isatin 1 with appropriate N ꢀ
arylꢀsubstituted 3ꢀthiosemicarbazides 2) with 4ꢀchlorophenacyl bromide 4 in absolute ethanol or
ethanolꢀbenzene mixture and screened for their cytotoxicity, phytotoxicity, antifungal and urease
inhibitory potential. All the synthesized compounds were found to be almost inactive in the brine
ꢀ4
ꢀ4
shrimp (Artemia salina) bioassay, demonstrating IC50 values >1.62 x 10 − 2.17 x 10 M. In
phytotoxicity assay, out of thirty nine compounds tested, six i.e. 5i, 6h, 6i, 6k, 7c and 7h proved to
be active, showing weak or nonꢀsignificant (5ꢀ30%) activity at the highest tested concentration (500
µg/mL). Similarly, in antifungal assay, twenty six compounds i.e. 5a, 5b, 5d-f, 5hꢀj, 5m, 6a, 6b, 6d,
6
j, 6lꢀo, 7a, 7b and 7d-j were found to be active against one, two, or three selected fungal strains,
exhibiting weak or nonꢀsignificant inhibition (10ꢀ30%). Of these, 6d and 6o displayed relatively
better activity profile in terms of the number of organisms inhibited. On the other hand, in urease
inhibition bioassay, all the synthesized hydrazonothiazolines proved to be potent enzyme inhibitors,
demonstrating inhibitory activity with IC50 values ranging from 3.70 ± 0.62 to 849 ± 2.26 ꢁM.

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Compounds 5c, 5g-i, 5k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o, 7a, 7e, 7i and 7j were, however, found to be
relatively very much potent, displaying outstanding enzymatic activity (IC50 = 3.70 ± 0.62 – 20.9 ±
0
.57 ꢁM) even better than the reference inhibitor, thiourea (IC50 = 22.3 ± 1.12 ꢁM) and may thus act
as valid leads for further studies. Molecular docking studies of the synthesized isatinꢀthiazolines 5a-
n, 6a-0 and 7a-j were also carried out to elucidate their relationship with the binding pockets of the
enzyme. This study offers the first example of exhibition of urease inhibitory potential by isatinꢀ
thiazolines and as such provides a solid basis for further research on these compounds to develop
more potent antiurease compounds of medicinal /agricultural interest.
Keywords: Cytotoxicity, Hydrazones, Hydrazonothiazolines, Isatin, Phytotoxicity, Thiazoline,
Urease inhibition, Urease inhibitors
Introduction
The literature survey indicates that isatin and its derivatives possess a wide spectrum of biological
1ꢀ20
properties.
Among isatin derivatives, isatinꢀthiosemicarbazones are reported to possess diverse
1
1,7
pharmacological activities, including antiulcer,
analgesic and antiꢀinflammatory,
1
,4,7,12,21,22
1,2,3,5,7,22
23ꢀ25
1,2,5,7,12,25
anticancer,
antimicrobial,
antituberculosis,
antiviral,
and enzymatic
1
,4,11,17
inhibition.
Stimulated by this and as a part of our research work aiming to the synthesis of
isatinꢀbased organic compounds of medicinal/agricultural interest, we recently reported the synthesis
4
of a number of 5ꢀ(un)ꢀN ꢀarylꢀsubstituted isainꢀ3ꢀthiosemicarbazones as antimicrobial, cytotoxic,
2
6ꢀ33
phytotoxic and more importantly antiurease compounds.
Investigation of the structureꢀactivity
relationship (SAR) studies in the synthesized compounds revealed that in certain cases, the type and
4
position of different substituents about the phenyl ring attached to N of the thiosemicarbazone
2
6,27,29
part
positionꢀ5 of the isatin moiety
different activities. Moreover, it has been reported by some other authors that certain 5ꢀbromoisatinꢀ
and/or the presence of an inductively electronꢀwithdrawing group (NO
2
, F
3
CO, F, or Cl) at
28,30ꢀ33
played a key role in the inducement and/ or increment of

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3
ꢀhydrazonothiazolines, prepared by cyclization of the respective thiosemicarbazones with phenacyl
bromides, exhibited high cytotoxicity at low concentrations when tested for antiviral activity against
MDCK cells. The presence of more than one lipophilic functions in these compounds was considered
34
responsible for such
a
display of activity.
Likewise, 5ꢀbromo isatinꢀderived 3ꢀ
hydrazonothiazolidinones, obtained by cyclization of the corresponding thiosemicarbazones with
21
ethyl 2ꢀbromopropionate, have been found to show favourable cytotoxicity. Also, certain 5ꢀ
fluoroisatinꢀderived 3ꢀhydrazonothiazolidinones, obtained by condensation of 5ꢀfluoroisatin with
35
different thiazolidinꢀ4ꢀones, have been claimed to possess antimicrobial activity. In view of these
points, it was envisioned that the simultaneous presence of different lipophilic groups at positionꢀ5 of
the isatin scaffold and at the N atom of thiazoline moiety in the isatinꢀthiazoline hybrids would result
in an increase in the selected activities. Thus, the present work to synthesize three new series of
4
thirty nine title hydrazonothiazolines (derived by cyclization of avariety of N ꢀarylꢀsubstituted 5ꢀ
trifluoromethoxy/fluoro/chloroꢀisatin 3ꢀthiosemicarbazones with 4ꢀchlorophenacyl bromide) and
screen them for their cytotoxicity, phytotoxicity, antifungal and specially urease inhibitory potential
was accomplished. It is pertinent to mention here that such a category of isatin derivatives has been
scarcely investigated before by other researchers for their antiurease potential. The work presented
herein illustrates the influences of the type of aryl groups (modified by placing one, two, or three
functionalities on the phenyl ring) attached to N atom of the thiazoline moiety as well as the presence
of inductively electronꢀattracting/lipophilic trifluoromethoxy, fluoro and chloro groups at positionꢀ5
of the isatin part on the cytotoxic, phytotoxic, antifungal and urease inhibitory properties of the
synthsized isatinꢀthiazoline hybrids. This study furnishes some motivating and attentionꢀgrabbing
results, which are reported herein.
Results and discussion

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The present study describes the synthesis and in vitro evaluation of cytotoxic, phytotoxic, antifungal
3
6,37
and urease inhibitory potential of thirty seven currently and two previously reported
isatinꢀ
hydrazonothiazolines 5a-n, 6a-d, 6f-h, 6j-o, 7a-j and 6e, 6i, respectively.
Chemistry of compounds 5a-n, 6a-o and 7a-j
The designed isatinꢀhydrazonothiazolines 5-7 were prepared in 73ꢀ91% yield from 5ꢀsubstituted
4
isatins 1 by first reacting them with equimolar quantities of appropriate N ꢀsubstituted 3ꢀ
thiosemicarbazides 2 in aqueous ethanol containing a catalytic amount of glacial acetic acid, and
then by treating the resultant intermediate thiosemicarbazones 3 with 4ꢀchlorophenacyl bromide 4 in
absolute ethanol or ethanolꢀbenzene mixture at reflux temperature (Scheme 1).
The structures of the synthesized isatinꢀ3-hydrazonothiazolines 5a-n, 6a-o and 7a-j were established
1
on the basis of elemental (CHN) and spectral (IR, HNMR, EIMS) analyses. Satisfactory elemental
analyses were obtained for all the compounds synthesized in this study. Also, the spectral data were
21,34,38
in ageement with the relevant literature.
The IR spectra of isatinꢀthiazolines 5a-n, 6a-o and 7a-j
ꢀ1
showed a single band resulting from NH stretching of indole in the 3261ꢀ3008 cm region. The
ꢀ1
lactam C=O and azomethine C=N were observed in the 1739ꢀ1693 and 1631ꢀ1558 cm regions,
2
1,34,38
1
respectively.
of thiosemicarbazone N ꢀH and N ꢀH,and displayed one or two separate singlets at δ 10.39ꢀ10.75
and δ 10.35ꢀ10.77, respectively, for indole NH. The thiazoline ring C ꢀH resonated either as a singlet
The HꢀNMR spectra of 5a-n, 6a-o and 7a-j did not show the down field signals
4
2
5
21,34,38
at δ 7.01ꢀ7.22 or two separate singlets at δ 6.90ꢀ7.12 or a multiplet at δ 6.95ꢀ7.13.
Furthermore,
1
the HꢀNMR spectra of compounds 6c, 6d, 6f, 6o, 7a, 7c, 7d and 7f-j exhibited duplicate signals,
confirming the presence of two isomers. It is suggested that in these compounds, the restricted
rotation about the azomethine (C=N) linkage and the partial double bond character of the lactam CꢀN
bond, induced by delocalization of the nitrogen lone pair of electrons onto the carbonyl oxygen, led
21,38
to the formation of E and Z isomers.
The EI mass spectra of 5a-n, 6a-o and 7a-j showed

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molecular ions of different intensity peculiar to the isatin and thiazoline moieties. The key
38
fragmentation pathways involved the NꢀN, NꢀC, and NꢀCO bonds fission. The proposed
fragmentation patterns of compounds 5c, 6g and 7f, representing each series of isatinꢀthiazoline
hybrids, are illustrated in Figures 1, 2 and 3, respectively. Xꢀray structures of two representative
examples 6e and 6i were determined in order to prove the allocated structures and to substantiate
conformations of the synthesized isatinꢀhydrazonothiazolines. The related crystallographic data and
3
6,37
refinement details of 6e and 6i have been reported somewhere else.
R1
NH
S
R1
O
NNH
C
R
R
NH
S
EtOHꢀHOAc
Reflux, 2h
O
H NHN C
O
2
N
N
H
H
1
2
3
Cl
R1
Cl
N
S
N N
C
O
CH Br
2
R
4
O
N
EtOH (abs.)
Reflux, 12ꢀ15h
or
H
5-7
EtOHꢀBenzene
Reflux, 12ꢀ15h
5
a
b
c
d
e
f
R=OCF
R=OCF
R=OCF
R=OCF
R=OCF
R=OCF
R=OCF
3
3
3
3
3
3
3
; R
; R
; R
; R
; R
; R
; R
1
=2ꢀCH
=3ꢀCH
3
3
5n
6a
6b
6c
6d
6e
6f
R=OCF
3
; R
1
=2,4,6ꢀ(Cl)
3
6m
6n
60
7a
7b
7c
R=F ; R
R=F ; R
R=F ; R
1
1
1
=2,5ꢀ(Cl)
2
5
5
5
5
5
5
1
1
1
1
1
1
R=F ; R
R=F ; R
R=F ; R
R=F ; R
R=F ; R
R=F ; R
1
1
1
1
1
1
=2ꢀCH
=3ꢀCH
=4ꢀCH
3
3
3
=2,6ꢀ(Cl)
2
=4ꢀ CH
3
=2,4,6ꢀ(Cl)
3
=2ꢀO CH
3
R=Cl ; R
R=Cl ; R
R=Cl ; R
R=Cl ; R
1
1
1
1
=2ꢀCH
=3ꢀCH
=4ꢀCH
3
3
3
=3ꢀOCH
=4ꢀOCH
=2ꢀF
3
=2ꢀOCH
=3ꢀOCH
=4ꢀOCH
3
3
3
3
g
7d
=2ꢀOCH
3

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5
5
5
5
5
5
h
R=OCF
R=OCF
R=OCF
R=OCF
R=OCF
R=OCF
3
3
3
3
3
3
; R
; R
; R
; R
; R
; R
1
1
1
1
1
1
=3ꢀF
6g
6h
6i
R=F ; R
R=F ; R
R=F ; R
R=F ; R
R=F ; R
R=F ; R
1
1
1
1
1
1
=2ꢀF
7e
7f
7g
7h
7i
R=Cl ; R
R=Cl ; R
R=Cl ; R
R=Cl ; R
R=Cl ; R
R=Cl ; R
1
1
1
1
1
1
=3ꢀOCH
=4ꢀOCH
=2ꢀF
3
i
=4ꢀF
=3ꢀF
3
j
=2,4ꢀ(Cl)
=2,5ꢀ(Cl)
=2,6ꢀ(Cl)
=3,4ꢀ(Cl)
2
2
2
2
=4ꢀF
k
l
6j
=2,4ꢀ(F)
2
2
=3ꢀF
6k
6l
=2,6ꢀ(F)
=4ꢀF
m
=2,4ꢀ(Cl)
2
7j
=2,4ꢀ(F)
2
Scheme 1. Synthesis of title hydrazonothiazolines 5a-n, 6a-o and 7a-j.

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CH3
CH3
N
S
N
S
Cl
Cl
F3CO
N N
O
N
N
ꢀ
CO
F3CO
H
NH
m/z 500
m/z 528 (M+)
N
H
CH3
N
F3CO
N
S
Cl
O
N
N
H
m/z 229
m/z 299
ꢀ
N2
F3CO
CH3
N
NH
H
m/z 201
N
S
Cl
H
CH3
m/z 286
F3CO
ꢀH
O
N
C S
N
m/z 149
H
m/z 215
CH3
ꢀCO
F3CO
NH
S
N
CH
Cl
m/z 118
m/z 187
ꢀ
HCN
Cl
ꢀHNC
m/z168
ꢀHC C
Cl
CH3
F3CO
HC C
m/z 136
m/z 160
m/z 111
m/z 91
Figure 1. The proposed fragmentation pattern of compound 5c.

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F
NH
NH
N
F
O
F
N
H
m/z 136
H
m/z 163
N
Cl
N
S
m/z 303
F
F
N
Cl
N
N N
O
Cl
F
F
ꢀ
CO
N
N
S
H
S
NH
N
H
+
m/z 438
Cl
m/z 466
H
(
M )
ꢀ
N2
F
N
S
F
Cl
H
O
m/z 290
N
H
ꢀH
CH
C
m/z 149
m/z 136
2
H
ꢀ
HC C
ꢀ
HNCO
F
ꢀ
CO
Cl
F
N
C S
m/z 108
m/z 153
m/z 111
F
F
NH
ꢀHCN
m/z 121
F
ꢀHNC
CH
N
m/z 95
ꢀ
F
m/z 122
F
S
Cl
m/z 76
m/z 168
m/z 94
Figure 2. The proposed fragmentation pattern of compound 6g.

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OCH3
Cl
NH
NH
N
Cl
O
N
H
m/z 152
H
m/z 179
N
Cl
N
S
OCH3
OCH3
m/z 315
N
S
Cl
N
S
N N
O
Cl
Cl
Cl
ꢀ
CO
N
N
H
OCH3
NH
m/z 494 (M+)
m/z 466
N
H
ꢀN2
H
OCH
3
N
S
ꢀH
Cl
Cl
H
O
m/z 302
N
S
ꢀ
N
C S
H
OCH3
m/z 165
m/z 165
OCH3
ꢀ
HC C
Cl
ꢀ
CO
CH
N
m/z 136
N
m/z 244
S
Cl
Cl
Cl
ꢀ HC C
NH
ꢀ
m/z 168
m/z 137
Cl
ꢀHNC
Cl
m/z 111
OCH3
OCH3
Cl
ꢀHCN
ꢀHCHO
HC
N
m/z 110
m/z 134
m/z 107
m/z 77
Figure 3. The proposed fragmentation pattern of compound 7f.

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Biology
Cytotoxicity (in vitro) of compounds 5a-n, 6a-o and 7a-j
The synthesized isatinꢀ3-hydrazonothiazolines 5a-n, 6a-o and 7a-j were screened for their cytotoxic
effects by a brine shrimp (Artemia salina) lethality bioassay using etoposide, a standard anticancer
drug as a reference point for comparison to the trial compounds. All the compounds of this series
ꢀ4
ꢀ4
gave LD50 values >1.62 x 10 − 2.17 x 10 M in the present assay when tested at 100, 10 and 1
µg/mL concentrations and thus can be considered to be almost inactive.
Phytotoxicity (in vitro) of compounds 5a-n, 6a-o and 7a-j
The synthetic compounds 5a-n, 6a-o and 7a-j were further screened for their phytotoxicity potential
at 500, 50 and 5 µg/mL concentrations. Out of thirty nine compounds tested, only six i.e. 5i, 6h, 6i,
6
k, 7c and 7h appeared to be active, exhibiting weak or nonꢀsignificant (5ꢀ30%) plant growth
inhibition at the highest tested concentration (500 µg/mL) compared to the standard drug, paraquat,
which displayed 100% inhibition at 0.015 µg/mL concentration (Table 1).
Table 1. Growth inhibition of Lemna aequinocitalis by compound 5a-n, 6a-o and
,
7
a-j* ** at different concentrations
Cl
R1
N
N
N
S
R
O
N
H
Compound
R
R
1
% Growth inhibition
5
00 µg/mL
05
50 µg/mL
00
5 µg/mL
00
5i
OCF
3
4ꢀF

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6
6
h
F
F
3ꢀF
05
05
15
18
30
00
00
00
00
00
00
00
00
00
00
6i
4ꢀF
k
F
2,6ꢀ(F)
2
7
c
Cl
Cl
4ꢀCH
3ꢀF
3
7
h
*The reference compound, paraquat, shows 100% growth inhibition at 0.015 µg/mL
Antifungal activity (in vitro) of compounds 5a-n, 6a-o and 7a-j
Antifungal activity of the synthesized isatinꢀhydrazonothiazolines 5a-n, 6a-o and 7a-j was
determined against five fungal cultures i.e. Candida albicans, Aspergillus flavus, Microsporum
canis, Fusarium solani and Candida glabrata at 200 µg /mL in DMSO (Table 2). Of the thirty nine
compounds tested, twenty six i.e. 5a, 5b, 5d-f, 5hꢀj, 5m, 6a, 6b, 6d, 6j, 6lꢀo, 7a, 7b and 7d-j were
found to be active against one, two, or three selected pathogens, displaying varied but low or nonꢀ
significant inhibition (10ꢀ30%). Among these, 6d and 6o bearing fluoro group at positionꢀ5 of the
isatin scaffold and methoxy and chloro functions, respectively, at positionsꢀ2 ( ortho) and ꢀ2,4,6 (
ortho, para, ortho) of the phenyl ring attached to N atom of the thiazoline moiety exhibited relatively
better activity profile in terms of the number of organisms affected.
,
Table 2. Antifungal activity (in vitro) of compounds 5a-n, 6a-o and 7a-j* ** (% inhibition)

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Cl
R1
N
N
N
S
R
O
N
H
Microbial species
C. albicans A. flavus M. canis F. solani C. glabrata
Compound
R
R1
5
a
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
F
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3
3
3
3
3
3
3
3
3
2ꢀCH
3ꢀCH
2ꢀOCH
3ꢀOCH
4ꢀOCH
3ꢀF
4ꢀF
2,4ꢀ(Cl)
3,4ꢀ(Cl)
3
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
A
00
00
00
00
00
00
00
00
00
00
00
20
00
20
00
00
10
00
00
20
00
00
00
00
00
00
B
20
20
00
20
00
25
00
00
00
00
20
20
10
30
10
30
20
20
20
00
20
25
25
20
25
00
C
00
25
10
00
10
00
30
25
10
25
00
20
00
00
00
00
25
00
00
00
00
00
00
00
00
20
D
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
E
5
5
b
d
3
3
3
3
5
5
e
f
5
h
5
5
i
j
2
5m
2
6
a
b
d
2ꢀCH
3ꢀCH
3
6
6
3
2ꢀOCH
2,4ꢀ(F)
2
2,4ꢀ(Cl)
2,5ꢀ(Cl)
2,6ꢀ(Cl)
3
6
6
j
l
2
2
2
6m
6
n
6
7
o
a
2,4,6ꢀ(Cl)
3
2ꢀCH
3ꢀCH
3
7
7
b
d
3
2ꢀOCH
3ꢀOCH
4ꢀOCH
2ꢀF
3ꢀF
4ꢀF
2,4ꢀ(F)
2
3
3
3
7
7
e
f
7
g
7
h
7
7
i
j
Standard
Drug**

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*
Concentration used 200 µg/mL; ** Standard drugs (MIC in µg/mL): A = Miconazole (110.8
µg/mL); B= Amphotericin B (20 µg/mL); C = Miconazole (98.4 µg/mL); D = Miconazole (73.25
µg/mL); E= Miconazole (110.8 µg/mL); 00: absence of measurable inhibitory action
Urease inhibition (in vitro) of compounds 5a-n, 6a-o and 7a-j
The synthesized 5ꢀtrifluoromethoxy/fluoro/chloroꢀisatin 3ꢀhydrazonothiazolines 5a-n, 6a-o and 7a-j,
respectively, were tested for their antiurease activity against Jack bean urease. Thiourea served as
reference standard in this assay. All the compounds proved to be potent inhibitors of the enzyme,
exhibiting inhibitory activity with IC50 values of 3.70 ± 0.62 – 849 ± 2.26 ꢁM. Compounds 5c, 5g-i,
5
k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o, 7a, 7e, 7i and 7j were, however, found to be relatively very much
potent, displaying excellent activity (IC50 values 3.70 ± 0.62 – 20.9 ± 0.57 ꢁM) even better than the
reference inhibitor, thiourea (IC50 = 22.3 ± 1.12 ꢁM) and may thus act as compelling leads for further
studies. The remaining compounds i.e. 5a, 5b, 5d-f, 5j, 5l, 5m, 6a, 6d-h, 6j, 6m, 7b-d and 7f-h
showed a varying degree of activity with IC50 values of 24.6 ± 2.14 – 849 ± 2.26 ꢁM (Table 3).
The structureꢀactivity relationship (SAR) studies in the case of 5ꢀtrifluoromethoxyisatin derivatives
5
aꢀn revealed that compound 5k having chloro substituents at positionsꢀ2 and ꢀ5 of the phenyl ring
attached to N atom of the thiazoline moiety was the most potent urease inhibitor of the series,
demonstrating several fold more activity (IC50 = 3.70 ± 0.62 ꢁM) than the reference inhibitor,
thiourea (IC50 = 22.3 ± 1.12 ꢁM). Comparison of the urease inhibitory potential of compound 5k
with that of closely related dichloroꢀsubstituted compounds 5j, 5l and 5m indicated that chloro
substitution at positionsꢀ2,5 (ortho ,meta) of the phenyl ring was more favourable than at positionsꢀ
2
,4 (ortho, para), ꢀ2,6 (ortho, ortho) and ꢀ3,4 (meta, para), respectively. The dichloro–substituted
derivatives 5j, 5l and 5m displayed relatively much lower activity (IC50 = 44.5 ± 3.15, 129 ± 5.91
and 134 ± 2.21 ꢁM, respectively) in the present assay. This clearly showed that compound 5k
compared to 5j, 5l and 5m interfered with the enzyme in a different fashion. Interestingly, the only
trichloro–substituted compound tested in this assay i.e. 5n bearing the substituents at positionsꢀ2,4,6

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(
ortho, para, ortho) of the phenyl ring demonstrated much higher activity in contrast to the dichloroꢀ
substituted compounds 5j and 5l with the substituents at positionsꢀ2,4 (ortho, para) and ꢀ2,6 (ortho,
ortho) of the phenyl ring, respectively (IC50 value 14.1 ± 1.31 vs. 44.5 ± 3.15 and 129 ± 5.91 ꢁM).
Chlorine is both electronꢀdonating by mesomeric effect (+M) and electronꢀwithdrawing by inductive
effect (ꢀI). Much higher enzyme inhibitory activity presented by compound 5n in the present assay
clearly indicated that the ultimate or overall electronꢀwithdrawing effect of the three chloro functions
increased its inhibitory potential, though not exclusively. The next most potent urease inhibitor was
compound 5g having a fluoro group at positionꢀ2 of the phenyl ring, showing inhibitory activity with
IC value of 7.27 ± 0.33 ꢁM. This compound was found to be three fold more active than the
5
0
reference inhibitor, thiourea, but two fold less active than the most potent urease inhibitor 5k. The
other highly active monofluoroꢀsubstituted derivatives 5h and 5i, possessing the substituent at
positionsꢀ3 and ꢀ4 of the phenyl ring exhibited enzymatic inhibition with IC50 values of 12.0 ± 0.83
and 11.7 ± 0.07 ꢁM, respectively. This clearly indicated that compound 5g in comparison to 5h and
5
i meddled with the enzyme differently and more competently. The remaining relatively very much
potent urease inhibitor was compound 5c with a methyl substituent at positionꢀ4 of the phenyl ring
attached to N atom of the thiazoline moiety. This compound was found to be two fold more active
than the reference inhibitor (thiourea) but three times less active than the most potent urease inhibitor
of the series i.e. compound 5k (IC50 value 10.3 ± 0.51 vs. 22.3 ± 1.12 and 3.70 ± 0.62 ꢁM,
respectively). The other methylꢀsubstituted compounds 5a and 5b bearing the substituent at
positionsꢀ2 and ꢀ3 of the phenyl ring displayed relatively much lower inhibitory activity (IC values
50
3
36 ± 5.45 and 427 ± 2.89 ꢁM, respectively). These results showed that steric hindrance played a
significant role in reducing the inhibitory potential of the compounds.
Among 5ꢀfluoroisatin derivatives 6aꢀo, compounds 6i and 6l having fluoro and chloro substituents at
positionsꢀ4 (para) and ꢀ2,4 (ortho, para) of the phenyl ring attached to N atom of the thiazoline
moieties were the most potent urease inhibitors of the series, presenting excellent and almost the

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same inhibitory activity (IC50 values 8.20 ± 0.01 and 8.40 ± 0.23 ꢁM, respectively). Comparison of
the urease inhibitory potential of compound 6i with that of closely related fluoroꢀsubstituted
derivatives 6g and 6h showed that fluoro substitution at positionꢀ4 of the phenyl ring was more
favourable than at positionsꢀ2 and ꢀ3. The fluoroꢀsubstituted compounds 6g and 6h exhibited
relatively much lower activity (IC50 values 26.1 ± 1.11 and 32.6 ± 3.42 ꢁM, respectively) in the
present assay. Also, comparison of the antiurease potential of compound 6l with that of 6m and 6n
indicated that chloro substitution at positionsꢀ2,4 (ortho, para) was more favourable than at ꢀ2,5
(ortho, meta) and ꢀ2,6 (ortho, ortho). The dichloroꢀsubstituted derivative 6n compared to 6l
displayed lower but still exciting activity (IC50 value 12.7 ± 0.62 vs. 8.40 ± 0.23 ꢁM), while 6m
demonstrated relatively much lower activity with IC50 value of 33.1 ± 2.88 ꢁM. Similarly, the
trichloroꢀsubstituted compound 6o having the chloro substituents at positionsꢀ2,4,6 (ortho, para,
ortho) of the phenyl ring exhibited lower but still stimulating inhibitory activity (IC50 = 14.3 ± 0.12
ꢁM) in comparison to the corresponding dichloroꢀsubstituted compounds 6l and 6n, showing enzyme
inhibition with IC50 values of 8.40 ± 0.23 and 12.7 ± 0.62 ꢁM, respectively. In case of difluoroꢀ
substituted compounds tested in this assay, compound 6k bearing the fluoro substituents at positionsꢀ
2
,6 (ortho, ortho) of the phenyl ring was found to show much higher inhibitory potential compared to
compound 6j having the substituents at positionsꢀ2,4 (ortho, para) (IC50 value 11.0 ± 0.86 vs. 66.6 ±
.40 ꢁM). This indicated that compound 6k in contrast to 6j intermingled with the enzyme in a
5
different manner, resulting into much marked enhancement in enzyme inhibitory potential. Amongst
methylꢀsubstituted compounds 6a-6c, compound 6c possessing the substituent at positionsꢀ4 of the
phenyl ring was found to be the most active one, displaying enzyme inhibition with IC50 value of
1
1.4 ± 1.51 ꢁM. The next most active derivative was 6b with the substituent at positionꢀ3 of the ring,
which showed inhibitory potential with IC50 value of 15.0 ± 1.92 ꢁM. The remainder derivative 6a
having the substituent at positionꢀ2 of the phenyl ring exhibited much lower inhibitory activity with
IC value of 275 ± 4.99 ꢁM. This indicated that in case of 6a, steric hindrance played a pivotal role
5
0

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in decreasing the inhibitory potential of the compound. The above results demonstrated that
compounds 6g and 6h in contrast to 6i, and 6m in comparison to 6l, 6n and 6o, interacted with the
enzyme differently, resulting into reduction in the inhibitory potential to a smaller or greater extent.
The results given in the Table revealed that compared to compounds 5a-n having trifluoromethoxy
substituent at positionꢀ5 of the isatin part, substitution of fluoro group at the same position in the case
of 6a-o caused either a decrement or an increment in the enzymatic activity in certain cases. For
example, compound 6c having a methyl substituent at positionꢀ4 of the phenyl ring attached to N
atom of the thiazoline moiety showed inhibition of the enzyme with IC50 value of 11.4 ± 1.51 ꢁM,
whereas the respective compound 5c bearing trifluoromethoxy function at positionꢀ5 of the isatin
moiety displayed more inhibitory activity (IC50 = 10.3 ± 0.51 ꢁM). Similarly, compounds 6f-h
possessing methoxy and fluoro substituents at positions ꢀ4 and ꢀ2, ꢀ3 of the phenyl ring, respectively,
were found to demonstrate reduced activity (IC50 values 849 ± 2.26, 26.1 ± 1.11 and 32.6 ± 3.42 ꢁM)
in comparison to the corresponding 5ꢀtrifluoromethoxyisatin derivatives 5f-h, which exhibited
inhibitory activity with IC50 values of 102 ± 5.82, 7.27 ± 0.33 and 12.0 ± 0.83 ꢁM, respectively.
Also, compound 6m possessing chloro functions at positions ꢀ2 and ꢀ5 of the phenyl ring was found
to show decreased activity (IC50 = 33.1 ± 2.88 ꢁM) when compared with the respective compound
5
k having trifluoromethoxy substituent at positionꢀ5 of the isatin moiety, which displayed enzyme
inhibition with IC50 value 3.70 ± 0.62 ꢁM. Much marked decrement in enzyme inhibitory activity
was found to occur in case of compounds 6g, 6h and 6m, respectively, when compared with the
corresponding 5ꢀtrifuoromethoxyisatinꢀderived hydrazonothiazolines 5g, 5h and 5k (IC50 values 7.27
±
0.33 → 26.1 ± 1.11 ꢁM, 12.0 ± 0.83 → 32.6 ± 3.42 ꢁM and 3.70 ± 0.62 → 33.1 ± 2.88 ꢁM,
respectively). On the contrary, compounds 6a and 6b bearing methyl function at positions ꢀ2 and ꢀ3
of the phenyl ring, respectively, displayed increased inhibitory activity (IC50 values 275 ± 4.99 and
1
5.0 ± 1.92 ꢁM, respectively) in contrast to the respective compounds 5a and 5b, showing enzyme
inhibition with IC50 values of 336 ± 5.45 and 427 ± 2.89 ꢁM. Similarly, compounds 6d and 6e

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having methoxy substituent at positionsꢀ2 and ꢀ3 of the phenyl ring displayed increased enzyme
inhibition with IC50 values 40.1 ± 5.32 and 29.3 ± 1.17 ꢁM, respectively, when compared with the
corresponding compounds 5d and 5e bearing trifluoromethoxy group at positionꢀ5 of the isatin
scaffold, demonstrating inhibition of the enzyme with IC values of 42.9 ± 4.67 and 42.6 ± 1.40 ꢁM.
50
Furthermore, the monofluoroꢀsubstituted derivative 6i possessing the substituent at positionꢀ4 of the
phenyl ring showed enhanced enzyme inhibition with IC50 value of 8.20 ± 0.01 ꢁM in comparison to
the corresponding 5ꢀtrifluoromethoxyisatinꢀderived hydrazonothiazoline 5i, which displayed
inhibitory activity with IC50 value of 11.7 ± 0.07 ꢁM. Also, the dichloroꢀsubstituted compounds 6l
and 6n having the substituents at positionsꢀ2,4 (ortho, para) and ꢀ2,6 (ortho, ortho) exhibited
enhanced enzyme inhibition (IC values 8.40 ± 0.23 and 12.7 ± 0.62 ꢁM, respectively) in contrast to
50
the respective compounds 5j and 5l bearing trifluoromethoxy residue at positionꢀ5 of the isatin
moiety, demonstrating inhibitory activity with IC50 values of 44.5 ± 3.15 and 129 ± 5.91 ꢁM.
Relatively, marked increment in enzyme inhibitory activity (IC50 values 427 ± 2.89 → 15.0 ± 1.92
ꢁM, 44.5 ± 3.15 → 8.40 ± 0.23 ꢁM and 129 ± 5.91 → 12.7 ± 0.62 ꢁM, respectively) occurred in case
of compounds 6b, 6l and 6n. Among the remaining compounds of the series i.e. 6j and 6k,
compound 6j having fluoro substituents at positionsꢀ2 and ꢀ4 of the phenyl ring exhibited inhibitory
activity with IC50 value of 66.6 ± 5.40 ꢁM. In contrast, 6k possessing the same substituents at
positions ꢀ2 and ꢀ6 of the phenyl ring showed much enhanced activity (IC50 = 11.0 ± 0.86 ꢁM).
These results showed that the presence of fluoro group (exercising both –ve inductive and +ve
mesomeric effects) at positionꢀ5 of the isatin scaffold as well as the nature, number and position of
different functions present in the phenyl ring caused the molecules to meddle with the enzyme in a
different fashion.
In case of 5ꢀchloroisatinꢀderived hydrazonothiazolines 7a-j, 7a bearing methyl group at positionꢀ2 of
the phenyl ring attached to N atom of the thiazoline moiety was found to be the most potent
antiurease compound, showing enzymatic activity (IC50 = 9.20 ± 0.77 ꢁM) much higher than the

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reference inhibitor, thiourea (IC50 = 22.3 ± 1.12 ꢁM). Comparison of the antiurease activity of
compound 7a with that of closely related methylꢀsubstituted compounds 7b and 7c having the
substituent at positionsꢀ3 and ꢀ4 of the phenyl ring, respectively, revealed that methyl substitution at
positionꢀ2 of the phenyl ring was more favourable than at ꢀ3 and ꢀ4. Compound 7b was found to be >
two fold less active than 7a but slightly more active than 7c (IC value 24.6 ± 2.14 vs. 9.20 ± 0.77
50
and 25.3 ± 3.20 ꢁM, respectively). This showed that compounds 7b and 7c compared to 7a interfered
with the enzyme much less efficiently, resulting in a decrement in their inhibitory potential. The next
most potent antiurease compound was 7e possessing methoxy substituent at positionꢀ3 of the phenyl
ring. This compound exhibited slightly less inhibitory activity (IC =10.3 ± 0.38 ꢁM) than the most
50
potent inhibitor 7a (IC = 9.20 ± 0.77 ꢁM) but much more than the reference inhibitor, thiourea
5
0
(IC50 = 22.3 ± 1.12 ꢁM). The other potent methoxyꢀsubstituted compounds 7d and 7f having the
substituent at positionꢀ2 and ꢀ4 of the phenyl ring attached to N atom of the thiazoline moiety,
however, displayed relatively much lower activity with IC50 values of 38.6 ± 1.50 ꢁM and 81.3 ±
7
.99 ꢁM, respectively. This clearly demonstrated that compound 7e intermingled with the enzyme
differently and much more competently. The remaining relatively much more potent urease inhibitor
was compound 7i bearing a fluoro substituent at positionꢀ4 of the phenyl ring. This compound was
found to be slightly less active than the most potent urease inhibitor of the series i.e. compound 7a
but two fold more active than the reference inhibitor, thiourea (IC50 value 11.3 ± 0.32 vs. 9.20 ± 0.77
and 22.3 ± 1.12 ꢁM, respectively). The other fluoroꢀsubstituted compound 7g possessing the
substituent at positionꢀ2 of the phenyl ring
Table 3. Inhibition of Jack been urease by compounds 5a-n, 6a-o and 7a-j

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Cl
R1
N
N
N
S
R
O
N
H
Compound
R
R
1
IC50 ± SEM
µM)
Compound
R
R
1
IC50 ± SEM
(µM)
(
5
a
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
OCF
F
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2ꢀCH
3ꢀCH
4ꢀCH
3
336 ± 5.45
427 ± 2.89
10.3 ± 0.51
42.9 ± 4.67
42.6 ± 1.40
102 ± 5.82
7.27 ± 0.33
12.0 ± 0.83
11.7 ± 0.07
44.5 ± 3.15
3.70 ± 0.62
129 ± 5.91
134 ± 2.21
14.1 ± 1.31
275 ± 4.99
15.0 ± 1.92
11.4 ± 1.51
40.1 ± 5.32
29.3 ± 1.17
849 ± 2.26
6g
F
F
2ꢀF
3ꢀF
26.1 ± 1.11
32.6 ± 3.42
8.20 ± 0.01
5
b
3
3
6h
5
c
6i
F
4ꢀF
5
d
2ꢀOCH
3ꢀOCH
4ꢀOCH
2ꢀF
3
3
3
6j
F
2,4ꢀ(F)
2,6ꢀ(F)
2
2
66.6 ± 5.40
11.0 ± 0.86
8.40 ± 0.23
33.1 ± 2.88
12.7 ± 0.62
14.3 ± 0.12
9.20 ± 0.77
24.6 ± 2.14
25.3 ± 3.20
38.6 ± 1.50
10.3 ± 0.38
81.3 ± 7.99
25.6 ± 2.91
32.0 ± 0.69
11.3 ± 0.32
20.9 ± 0.57
22.3 ± 1.12
5
e
6k
F
5
f
6l
F
2,4ꢀ(Cl)
2,5ꢀ(Cl)
2,6ꢀ(Cl)
2
2
2
5
g
6m
F
5
h
3ꢀF
6n
F
5
i
4ꢀF
6o
F
2,4,6ꢀ(Cl)
3
5
j
2,4ꢀ(Cl)
2,5ꢀ(Cl)
2,6ꢀ(Cl)
3,4ꢀ(Cl)
2
2
2
2
7a
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
2ꢀCH
3ꢀCH
4ꢀCH
3
3
3
5
k
7b
5
l
7c
5
m
7d
2ꢀOCH
3ꢀOCH
4ꢀOCH
2ꢀF
3
3
3
5
n
2,4,6ꢀ(Cl)
3
7e
6
a
2ꢀCH
3ꢀCH
4ꢀCH
3
3
3
7f
6
b
F
7g
6
c
F
7h
3ꢀF
6
d
F
2ꢀOCH
3ꢀOCH
4ꢀOCH
3
3
3
7i
7j
4ꢀF
6
e
F
2,4ꢀ(F)
2
6
f
F
Thiourea*
*
Reference inhibitor of urease enzyme
showed inhibitory activity with IC value of 25.6 ± 2.91 ꢁM. On the contrary, compound 7h with
50
the fluoro substituent at positionꢀ3 of the phenyl ring displayed reduced enzymatic activity with IC50
value of 32.0 ± 0.69 ꢁM. Finally, the only difluoroꢀsubstituted compound tested in the present assay

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i.e. 7j having fluoro substituents at positionsꢀ2,4 (ortho, para) of the phenyl ring, though found to be
markedly less active than the monofluoroꢀsubstituted compound 7i (20.9 ± 0.57 vs. 11.3 ± 0.32 ꢁM),
but was still more active than the monofluoroꢀsubstituted compound 7g (IC50 = 25.6 ± 2.91 ꢁM) as
well as the reference inhibitor, thiourea (IC = 22.3 ± 1.12 ꢁM). Fluorine is both strongly electronꢀ
5
0
attracting by inductive effect (ꢀI) and electronꢀdonating by mesomeric effect (+M). Noticeably higher
inhibitory activity shown by compounds 7g, 7i and 7j compared to 7h in the present assay is
attributed, though not exclusively, to the overall or ultimate electronꢀdonating influences of the
fluoro functions present at positionꢀ2 (ortho) and ꢀ4 (para) of the phenyl ring attached to N atom of
the thiazoline moiety.
The results given in the Table further revealed that compared with the 5ꢀfluoroisatinꢀderived
thiazolines 6a-o, thiazolines of this series exhibited either increased or decreased enzyme inhibition
in certain cases. For example, compound 7a possessing a methyl group at positionꢀ2 of the phenyl
ring attached to N atom of the thiazoline moiety showed inhibitory activity with IC50 value 9.20 ±
0
.77 ꢁM, whereas the respective compound 6a displayed enzymatic inhibition with IC value of 275
50
±
4.99 ꢁM. Similarly, compounds 7d-f bearing methoxy functions at positionꢀ2, ꢀ3 and ꢀ4 of the
phenyl ring, respectively, showed enhanced activity (IC50 values 38.6 ± 1.50, 10.3 ± 0.38 and 81.3 ±
.99 ꢁM) compared to the corresponding compounds 6d-f, demonstrating enzyme inhibition with
7
IC50 values of 40.1 ± 5.32, 29.3 ± 1.17 and 849 ± 2.26 ꢁM, respectively. Also, the monofluoroꢀ
substituted compounds 7g and 7h having the substituent at positionꢀ2 and ꢀ3 of the phenyl ring,
respectively, displayed enhanced activity (IC50 values 25.6 ± 2.91 and 32.0 ± 0.69 ꢁM) in
comparison to the respective compounds 6g and 6h, exhibiting enzyme inhibitory activity with IC50
values of 26.1 ± 1.11 and 32.6 ± 3.42 ꢁM, respectively. Furthermore, the difluoroꢀsubstituted
compound 7j with the substituents at positionsꢀ2 and ꢀ4 of the phenyl ring showed increased activity
(
IC = 20.9 ± 0.57 ꢁM) in contrast to the corresponding compound 6j, displaying inhibition of the
50
enzyme with IC50 value of 66.6 ± 5.40 ꢁM. Marked increment was found to occur in case of

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compounds 7a, 7e, 7f and 7j. To the contrary, compounds 7b and 7c possessing the methyl
substituent at positionꢀ3 and ꢀ4 of the phenyl ring, respectively, exhibited decreased enzyme
inhibitory activity (IC50 values 24.6 ± 2.14 and 25.3 ± 3.20 ꢁM) compared to the corresponding
compounds 6b and 6c, showing activity with IC values of 15.0 ± 1.92 and 11.4 ± 1.51 ꢁM. Also,
50
compound 7i bearing the fluoro function at positionꢀ4 of the phenyl ring showed reduced inhibitory
activity (IC50 value 11.3 ± 0.32 ꢁM) in contrast to the respective compound 6i, exhibiting enzyme
inhibition with IC50 value of 8.20 ± 0.01 ꢁM. Relatively, pronounced reduction in the enzymatic
activity was observed in the case of 7c.
The above results showed that the simultaneous presence of varied inductively electronꢀwithdrawing
groups at positionꢀ5 of the isatin scaffold and the variously substituted aryl functions at N atom of
the thiazoline moiety caused the isatinꢀthiazoline hybrids to meddle with the enzyme differently and
sometimes relatively much more competently.
Molecular docking studies of compounds 5a-n, 6a-o and 7a-j
Molecular docking studies of isatinꢀhydrazonothiazolines 5a-n, 6a-o and 7a-j were performed using
LeadIT to investigate the binding modes of inhibitors as well as interactions in the active site. The
structures were docked to the crystallographic structure of Jack bean urease PDB ID: 3LA4 having
biꢀnickel center in its active site. After reproducing the coꢀcrystallized reference ligand into the
active site of receptor, all the derivatives were docked inside the active site. Most of the compounds
were found interacting deep inside with the active site residues and showed coordinated interactions
with the nickel ions in the active site of the receptor. The active site of the receptor contains the
amino acids residues His409, Ala436, Arg439, Ala440, His492, Asp494, His519, His593, His594,
Arg609, Ala636 and Met637.
Overall, the structures of the compounds are too large to be accommodated in the active pocket of
the enzyme; therefore, some part lies within the bottom, while the rest interacts with the mid gorge

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area. The putative binding modes of the most active compounds among the three series i.e. 5a-n, 6a-
o and 7a-j are given below:
Binding mode analysis of compounds 5a-n, 6a-o and 7a-j

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Among 5ꢀtrifluoromethoxyisatinꢀhydrazonothiazolines 5a-n, 5k was found to be the most potent
inhibitor of Jack bean urease. It was directed deep towards the nickel ion present inside the active
site of the receptor, as shown in Figure 4. Its 5ꢀtrifluoromethoxyisatin part was located towards
bottom of the binding pocket, whereas the 4ꢀchlorophenyl and 2,5ꢀdichlorophenyl substituents
attached to C and N atoms of the thiazoline moiety, respectively, were directed more towards
entrance of the active site. In all the compounds studied, the 5ꢀtrifluoromethoxy isatin scaffold did
not form direct bond with the Ni center. The 2,5ꢀdichlorophenyl substituent attached to C atom of the
thiazoline moiety showed πꢀπ interactions with the imidazole ring of His593. Additionally, nonꢀ
covalent molecular interactions were found between 5ꢀtrifluoromethoxy isatin moiety and Arg609 in
the active site, while the hydrogen bonding was noticed between imidazole ring of His492 and the
carbonylꢀO of the 5ꢀtrifluoromethoxy isatin part of compound 5k. Figure 4 reports a prioritized
binding pose of the potent inhibitor 5k, and it is selected after Hyde assessment and visualization.

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Figure 4. The putative binding mode of compound 5k bound to the active site of Jack bean urease.
Carbon atoms of the compound are golden coloured and that of protein are blue. Dark green spheres
are two nickel ions.
Binding mode analysis of compounds 6a-o
As evident from the in vitro results, amongst 5ꢀfluoroisatinꢀhydrazonothiazolines 6a-o, 6i was the
most active inhibitor of Jack bean urease. The docking studies revealed that it adopts best orientation
inside the active pocket by forming hydrogen bonding with the amino acid residues of His492,
His593 and His594. The 5ꢀfluoroisatin part of the compound was directed into the pocket towards
the nickel ions. Furthermore, the 4ꢀchlorophenyl substituent attached to C and 4ꢀfluorophenyl one to
N atoms of the thiazoline moiety was aligned towards His594 and Asp494, respectively.
Additionally, πꢀcharge interactions were found with amino acid Asp494, which stabilized the pose.
The thiazoline part of the compound showed πꢀπ interactions with the imidazole ring of His594. The
putative binding pose of compound 6i was selected after Hyde assessment and visualization, and is

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depicted in Figure 5.
Figure 5. The putative binding mode of compound 6i bound to the active site of Jack bean urease.
Carbon atoms of the compound are magenta coloured and that of protein are blue. Dark green
spheres are two nickel ions.
Binding mode analysis of compounds 7a-j
Docking studies supported the experimental results that potent inhibitors inhibit the catalytic site of
the enzyme. The docking studies revealed the stable hydrogen bonding interactions between the
oxygen and hydrogen atoms of the inhibitors and the amino acid residues Arg439, His593 and
Arg609. Figure 6 reports the prioritized binding mode of the most potent inhibitor 7a; the pose was
selected after Hyde assessment and visualization of 30 lowest energy docked conformations. The 5ꢀ
chloroisatin part of the molecule was oriented towards amino acids His409, Ala436, Arg439, Ala440
and His593 where πꢀπ interactions were observed with His593 in the active pocket; the 4ꢀ
chlorophenyl and 2ꢀmethylphenyl substituents attached to C and N atoms of the thiazoline moiety,
respectively, were inclined towards Asp494.

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Figure 6. The putative binding mode of compound 7a bound to the active site of Jack bean urease.
Carbon atoms of the compound are light green coloured and that of protein are blue. Dark green
spheres are two nickel ions.
(5k)
(6i)
(7a)
Figure 7. 2D Interaction diagrams of the selected docked conformations for most potent inhibitors.
Hydrogen bond interactions are indicated with dotted lines and hydrophobic interactions are shown
with green lines. Metal interactions were observed by Nickel ions.

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Conclusions
Conclusively, three new series of thirty nine isatinꢀ3ꢀhydrazonothiazolines have been synthesized in
this study and evaluated for their in vitro cytotoxic, phytotoxic, antifungal and urease inhibitory
effects. All the compounds proved to be almost inactive in the brine shrimp (Artemia salina) lethality
bioassay. However, in phytotoxicity bioassay, six compounds were found to be active, exhibiting
weak or nonꢀsignificant (5ꢀ30%) activity at the highest tested concentration (500µg/mL). Similarly,
in antifungal assay, out of thirty nine compounds tested, twenty six proved to be active, showing
weak or nonꢀsignificant activity (10ꢀ30%) against one, two, or three fungal species. On the other
hand, the urease inhibition testing results have demonstrated that all the thirty nine compounds tested
appeared as potent urease inhibitors; seventeen of these i.e. 5c, 5g-i, 5k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o,
7
a, 7e, 7i and 7j proved to be relatively highly potent ones and may act as convincing leads for
further studies. These compounds, being non toxic, could be potent candidates for orally effective
therapeutic agents used for the treatment of certain clinical conditions induced by microbial ureases.
Also, by demonstrating weak or nonꢀsignificant phytotoxicity at the highes tested concentration, they
invite much attention to their utility as potent inhibitors of soil ureases, as they could be mixed with
fertilizers in small quantities to enhance the overall effectiveness of nitrogen utilization. Generally,
urease inhibitory potential of the sunthesized isatinꢀthiazolines was found to be dependent upon
electronic effects of the substituents at positionꢀ5 of the isatin scaffold and on the phenyl ring
attached to N atom of the thiazoline moiety. In order to understand the binding mechanism,
molecular docking studies were performed for compounds 5a-n, 6a-o and 7a-j, which elucidated
their relationship with the binding pockets of urease. Based on the data presented in Table 3 and in
terms of further development and SAR studies, simultaneous substitution of diverse inductively
electronꢀwithdrawing groups at positionꢀ5 of the isatin scaffold and the aryl substituents (modified
by placing one or more lipophilic functions about the phenyl ring) at N as well as Cꢀ4 atoms of the
thiazoline moiety in the hybrid molecules certainly warrants investigations.

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Experimental
General procedure
Chemical materials and solvents were obtained from MerckꢀSchuchatdt, Fluka and SigmaꢀAldrich.
Melting points were determined on a FisherꢀJohns melting point apparatus and are not corrected.
Elemental analyses were performed by using a Leco CHNSꢀ9320 (USA) elemental analyzer. The
data obtained from all the synthesized compounds were found to be satisfactory. FTꢀIR spectra
(
KBr) were run on a Shimadzu 8400 or Thermo Scientific Nicolet 6700 FTIR spectrophotometer
1
using ATR facility. HꢀNMR spectra were recorded in C
2
D
6
SO on Bruker (RhenistettenꢀForchheim,
Germany) AM 300, Bruker Avance AV300 and Bruker Spectrospin 300 spectrometers, operating at
00 MHz and using Si (CH as an internal standard. Mass spectra were obtained on MATꢀ312 and
3
3 4
)
JEOL JMSꢀ600H mass spectrometers.
Synthesis
General procedure for the synthesis of isatin-3-thiosemicarbazones 3
4
An appropriate 5ꢀsubstututed isatin 1 (0.01mol), N ꢀsubstituted thiosemicarbazide 2 (0.01mol) and
50% aqueous ethanol (40 mL) containing a catalytic amount of acetic acid were mixed and heated
under reflux for 2h. The solid obtained in each case during heating was filtered and washed
thoroughly with hot 50% aqueous ethanol to give the desired thiosemicarbazones 3, which were used
30,32,33,39,40
as such in the next step without further purification.
General procedure for the synthesis of isatin-3-hydrazonothiazolines 5-7
An appropriate isatinꢀ3ꢀthiosemicarbazone 3 (0.005 mol) was mixed with 4ꢀchlorophenacyl bromide
4
(0.005 mol) in absolute ethanol (25 mL) or in a mixture of ethanolꢀbenzene and heated under reflux
for 12ꢀ15 h.The refluxate was concentrated on a rotary evaporator and the precipitare thus formed in

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each case was filtered. Thorough washing with warm diethyl ether or nꢀhexane followed by
crystallization from ethanolꢀwater furnished the target compounds 5-7 in pure form.
The different compounds are characterized as under:
(3
Z)-5-(Trifluoromethoxy)-1
H
-indole-2,3-dione-3-{[(2
Z)-4-(4-chlorophenyl)-3-(2-
methylphenyl)-1,3-thiazol-2(3
H
)-ylidene]hydrazone} 5a
ꢀ1
Yield 82% as orange brown crystals; m.p. 248ꢀ250 °C; IR (KBr, cm ): 3122 (NH stretching), 1717
1
(
C=O), 1618 (C=N); HꢀNMR (DMSOꢀd
6
, δ, ppm): 2.08 (s, 3H, CH
ꢀH, C ꢀH), 7.17 (s, 1H, thiazoline =CH), 7.27 (d, J = 9.1
ꢀH) 7.29 (dd, J = 9.1 Hz, 1H, indole C ꢀH), 7.31ꢀ7.38 (m, 4H, Nꢀphenyl C
ꢀH, indole C ꢀH), 7.42 (d, J = 7.4 Hz, 1H, Nꢀphenyl C ꢀH), 10.66 (s, 1H, indole
NH); EIMS (70eV): m/z (%): 530 (M +2, 41), 529 (M +1, 31), 528 (M , 100), 499 (5), 301 (7), 300
19), 299 (39), 298 (40), 297 (77), 288 (8), 287 (12), 286 (48), 285 (23), 284 (81), 240 (14), 228 (7),
87 (6), 174 (6), 170 (5), 168 (12), 162 (19), 139 (5), 134 (6), 133 (7), 91 (14), 77 (5); Anal calcd. for
S (528): C 56.76, H 3.03, N 10.06; found: C 56.77, H 2.98, N 10.11.
3
), 6.84 (d, J = 9.1 Hz, 1H,
indole C
Hz, 2H, phenyl C
H, phenyl C ꢀH, C
7
ꢀH), 7.13ꢀ7.16 (m, 2H, Nꢀphenyl C
4
5
2
ꢀH, C
6
6
6
ꢀ
3
5
4
3
+
+
+
(
1
25 3 4 2
C H16ClF N O
(3
Z)-5-(Trifluoromethoxy)-1
H
-indole-2,3-dione-3-{[(2
Z)-4-(4-chlorophenyl)-3-(3-
methylphenyl)-1,3-thiazol-2(3
H
)-ylidene]hydrazone} 5b
ꢀ1
Yield 88% as orange yellow fluffy crystals; m.p. 268ꢀ270 °C; IR (KBr, cm ): 3111 (NH stretching),
1
1
709 (C=O), 1622 (C=N); HꢀNMR (DMSOꢀd
6
3
, δ, ppm): 2.28 (s, 3H, CH ), 6.83 (d, J = 8.2 Hz, 1H,
indole C
ꢀH, C
7
ꢀH), 7.05 (s, 1H, Nꢀphenyl C
2
ꢀH), 7.09 (s, 1H, thiazoline =CH), 7.13ꢀ7.37 (m, 9H, Nꢀphenyl
C
4
5
ꢀH, C
6
ꢀH, phenyl C
2
ꢀH, C
3
ꢀH, C
5
ꢀH, C
6
ꢀH, indole C
4
6
ꢀH, C ꢀH), 10.59 (s, 1H, indole NH);
+
+
+
EIMS (70eV): m/z (%): 530 (M +2, 47), 529 (M +1, 34), 528 (M , 100), 501 (6), 500 (16), 500 (7),
3
00 (6), 299 (11), 298 (6), 288 (10), 287 (7), 286 (32), 285 (6), 284 (30), 228 (6), 187 (4), 170 (5),
68 (12), 149 (13), 133 (4), 91 (9), 69 (9); Anal calcd. for C25 S (528): C 56.76 H 3.03
1
H
3 4 2
16ClF N O
N 10.06; found: C 56.70, H 3.00, N 10.00.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-