Synthesis and biological evaluation of some N 4-aryl-substituted 5-fluoroisatin-3-thiosemicarbazones

Article abstract of DOI:10.1007/s00044-013-0575-7

A series of N ⁴-aryl-substituted 5-fluoroisatin-3- thiosemicarbazones 3a-3l was synthesized and evaluated for selected biological activities. The brine shrimp lethality bioassay was carried out to study their in vitro cytotoxicity potential and

Full text of DOI:10.1007/s00044-013-0575-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0575-7
ORIGINAL RESEARCH
Synthesis and biological evaluation of some N4-aryl-substituted
5-fluoroisatin-3-thiosemicarbazones
•
Humayun Pervez Naveeda Saira Mohammad Saeed Iqbal
•
•
•
Muhammad Yaqub Khalid Mohammed Khan
Received: 20 December 2012 / Accepted: 5 March 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of N4-aryl-substituted 5-fluoroisatin-3-
thiosemicarbazones 3a–3l was synthesized and evaluated
for selected biological activities. The brine shrimp lethality
bioassay was carried out to study their in vitro cytotoxicity
potential and besides, their antifungal, phytotoxic and
urease inhibitory effects were also investigated. Seven
compounds i.e. 3a, 3d, 3f, 3g, 3h, 3j and 3k proved to be
active in the brine shrimp assay, displaying promising
Keywords: Antifungal ꢀ Cytotoxicity ꢀ 5-Fluoroisatin ꢀ
Phytotoxicity ꢀ Thiosemicarbazones ꢀ Urease inhibitors
Introduction
It is evident from the literature that isatin and its derivatives
are associated with a wide variety of biological activities like
analgesic, anticonvulsant, antimicrobial, anti-inflammatory,
antiglycation, antineoplastic, antiplasmodial, antituberculo-
sis and antiviral (Aboul-Fadl and Bin-Jubair, 2010; Bal et al.,
2005; Banerjee et al., 2011; Beauchard et al., 2006; Chen
et al., 2005; Chiyanzu et al., 2005, 2003; Da Silva et al., 2001;
Guzel et al., 2008; Hall et al., 2011, 2009; Hyatt et al., 2007;
Jarrahpour et al., 2007; Karali et al., 2007; Khan et al., 2009;
Pandeya et al., 2005; Patel et al., 2006; Pirrung et al., 2005;
Quenelle et al., 2006; Ravichandran et al., 2007; Singh
et al., 2010; Smitha et al., 2008; Terzioglu et al., 2006; Vine
et al., 2009). Among isatin derivatives, isatin-derived
thiosemicarbazones have been reported to exhibit diverse
chemotherapeutic activities, including antimicrobial, anti-
tuberculosis, antiulcer, cytotoxicity and enzymatic inhibi-
tion (Aboul-Fadl and Bin-Jubair, 2010; Bal et al., 2005;
Chiyanzu et al., 2005, 2003; Da Silva et al., 2001; Hall et al.,
2011, 2009; Karali et al., 2007; Pandeya et al., 2005; Pirrung
et al., 2005; Quenelle et al., 2006; Terzioglu et al., 2006;
Vine et al., 2009). In view of these facts and as a part of our
synthetic work on the potential biologically active isatin
derivatives, we have recently synthesized a number of
5-(un)-substituted N4-aryl-substituted isatin-3-thiosemi-
carbazones as antibacterial, antifungal, cytotoxic, phyto-
toxic and antiurease compounds (Pervez et al., 2007, 2008,
2009, 2010, 2011a, b, 2012a, b). Structure–activity rela-
tionship (SAR) studies revealed that in certain cases, the
nature and position of the substituents about phenyl ring
cytotoxicity
(LD50 = 6.89 9 10-5–2.79 9 10-4 M).
Amongst these, 3a and 3h were found to be the most active
ones (LD50 = 6.89 9 10-5 and 9.79 9 10-5 M, respec-
tively). Compounds 3i, 3j and 3 k displayed moderate
(40 %) antifungal activity against one or two fungal strains
i.e. A. flavus and/or M. canis. In phytotoxicity assay, all the
synthesized compounds, including the reference point 2m
showed weak-to-moderate (15–70 %) activity at the high-
est tested concentration (500 lg/mL). In urease inhibition
assay, compounds 3f, 3g and 3j proved to be the most
potent inhibitors, demonstrating relatively a higher degree
of enzymatic inhibition with IC50 values ranging from 37.7
to 47.3 lM.
H. Pervez (&) ꢀ N. Saira ꢀ M. Yaqub
Organic Chemistry Division, Institute of Chemical Sciences,
Bahauddin Zakariya University, Multan 60800, Pakistan
e-mail: pdhpervez@hotmail.com
M. S. Iqbal
Department of Chemistry, FC College, Lahore 54600, Pakistan
K. M. Khan
H.E.J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
123

Med Chem Res
attached to N4 of the thiosemicarbazone moiety and the
presence of inductively electron-withdrawing groups (nitro,
trifluoromethoxy, chloro) at position-5 of the isatin scaffold
played an important role in the inducement and/or
enhancement of different activities. Moreover, it has been
reported by some other workers that certain N4-aryl-substi-
tuted 5-bromo- and 5-chloro-isatin 3-thiosemicarbazones
were found to exhibit favourable cytotoxic and antifungal
activities (Karali et al., 2002; Pandeya et al., 1999). In view
of this and in continuation of our work in search of new
isatin-based bioactive compounds with improved efficacy,
the study of the combination of halogen substitution at
position-5 of the isatin scaffold with attachment of a variety
of aryl substituents to N4 of the thiosemicarbazone moiety
was considered worth pursuing. The present work, therefore,
deals with the synthesis and biological evaluation of a series
of twelve N4-aryl-substituted 5-fluoroisatin-3-thiosemica-
bazones. It describes the effects of the type of aryl functions
(modified by placement of one or two substituents about the
phenyl ring) at N4 of the thiosemicarbazone moiety as well as
the presence of fluoro substituent at position-5 of the isatin
scaffold on the cytotoxic, antifungal, phytotoxic and urease
inhibitory properties of these compounds.
good-to-excellent yields (72–93 %). The structures of all
the synthesized compounds were confirmed by IR, 1H
NMR, 13C NMR, EIMS and elemental (C, H, N) analyses.
The IR spectra of 3a–3l showed the absorption bands of
NH stretching in the 3460–3200 and 3195–3072 cm-1
regions. The absorption bands of lactam C=O, azomethine
C=N and thioamide C=S stretchings appeared in the
1691–1684, 1619–1539 and 1279–1244 cm-1 regions,
respectively (Karali, 2002; Naumov and Anastasova, 2001;
Omar et al., 1984; Petrov et al., 1986). The 1H NMR
spectra of 3a–3l displayed three separate singlets at d
10.63–11.01, d 11.26–11.31 and d 12.68–12.84 attributed
to thiosemicarbazone N4–H, indole NH and thiosemicar-
bazone N2–H, respectively (Karali, 2002; Laatsch et al.,
1984; Naumov and Anastasova, 2001; Nizamuddin et al.,
1999; Omar et al., 1984; Petrov et al., 1986). The 13C
NMR spectra of 3a–3l supported the IR and 1H NMR
findings (Guzel et al., 2008; Karali et al., 2007). Further-
more, the electron impact mass spectra (EIMS) of the
synthesized thiosemicarbazones 3a–3l demonstrated
molecular ions of different intensities, confirming their
molecular weights. The major fragmentation pathway
involved the rupture of exocyclic N–N, NH–CS and
endocyclic NH–CO bonds. The proposed fragmentation
pattern of 3f is illustrated in Fig. 1.
Results and discussion
Biology
This study illustrates the synthesis and in vitro determi-
nation of the cytotoxic, antifungal, phytotoxic and urease
inhibitory potential of twelve N4-aryl-substituted 5-fluor-
oisatin-3-thiosemicarbazones 3a–3l.
In vitro cytotoxicity
All the synthesized thiosemicarbazones 3a–3l were
screened for their cytotoxic activity by a brine shrimp
(Artemia salina) lethality bioassay. Compound 3m
i.e. 2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-phenyl-1-
hydrazinecarbothioamide, the synthesis of which has been
reported elsewhere (Pervez et al., 2007), served as refer-
ence point (without a substituent in the isatin scaffold as
well as on the phenyl ring) to assess the influence of
Chemistry
5-Fluoroisatin 1 was reacted with appropriate N-substituted
thiosemicarbazides 2 in aqueous ethanol (50 %) containing
a few drops of glacial acetic acid to give the corresponding
5-fluoroisatin-3-thiosemicarbazones 3a–3l (Scheme 1) in
Scheme 1 Synthesis of title
compounds 3a–3l
NNHCSNHR
O
RNHCSNHNH2
F
F
2
O
O
N
H
N
H
50% EtOH(aq.)-AcOH
Reflux, 2h
3
1
123

Med Chem Res
Fig. 1 The proposed
fragmentation pattern of 3f
F
.
F
+
.
NNHCSNH
O
+
NNHCSNH
+
F
F
NCS
CO
N
N
m/z 332 (M+)
H
m/z 304
H
m/z 134
.
F
HF
S
.
+
.
N
F
+
.
NNH2
+
.
NCS
+
F
HN
N
F
+
O
O
N
N
H
m/z 153
m/z 150
m/z 179
m/z 163
N2
H
HNCO
.
.
F
+
CS
.
2H
CO
F
+
O
N
NNH2
.
F
+
.
m/z 151H
NH2
+
NH
F
m/z 136
CO
H
H
F
N
F
NH2
m/z 111
m/z 135
N
HF
CH2N2
HCN
m/z 122
H
NH3
m/z 93
NH
CH2
F
F
.
.
+
+
NH
F
F
F
.
+
N
N
m/z 91
m/z 108
m/z 108
m/z 94
m/z 96 m/z 95
of the isatin scaffold and on the phenyl ring attached to N4
of the thiosemicarbazone moiety, respectively, caused
either elimination or reduction in the activity. This infer-
ence was supported by the results obtained in our earlier
studies (Pervez et al., 2008, 2009). For example, compound
3a having inductively electron-withdrawing fluoro sub-
stituent at position-5 of the isatin scaffold displayed
promising cytotoxic activity (LD50 = 6.89 9 10-5 M),
whereas the corresponding compound 3m i.e. the reference
point possessing no fluoro group in the isatin moiety was
found to be almost inactive (LD50 = [3.38 9 10-4 M)
(Pervez et al., 2008). On the contrary, compounds 3b, 3c,
3e and 3i with 2-trifluoromethyl, 3-trifluoromethyl, 4-tri-
fluoromethoxy and 2,4-difluoro substituents about the
phenyl ring showed almost no cytotoxic effects
(LD50 = [5.24 9 10-5,[5.24 9 10-5,[5.03 9 10-5 and
[5.71 9 10-5 M, respectively), whereas the correspond-
ing compounds without fluoro group at position-5 of the
Table 1 Brine shrimp bioassay for compounds 3a–3l
Compounds
LD50(M)
3a
6.89 9 10-5
[5.24 9 10-5
[5.24 9 10-5
1.76 9 10-4
[5.03 9 10-5
2.79 9 10-4
1.21 9 10-4
9.79 9 10-5
[5.71 9 10-5
1.15 9 10-4
1.07 9 10-4
[4.91 9 10-5
[3.38 9 10-4
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m (Pervez et al., 2007, 2008)
isatin scaffold gave LD50 values of 1.36 9 10-4
,
substituents about the isatin moiety as well as the phenyl
ring attached to N4 of the thiosemicarbazone part of the test
compounds on their cytotoxicity potential. The results
presented in Table 1 revealed that compared with the ref-
erence compound 3m, compound 3a having fluoro group
alone in the isatin scaffold displayed induction of cytotoxic
activity. However, in the rest cases, combined substitution
of fluoro and some other substituents in the aromatic ring
2.20 9 10-5, 1.80 9 10-5 and 2.00 9 10-5 M, respec-
tively (Pervez et al., 2009). Also, compared with
compound 3f (LD50 = 2.79 9 10-4 M), the dihalogeno-
substituted compound 3l possessing fluoro and bromo
substituents at positions-2 and -4 of the phenyl ring,
respectively, exhibited no cytotoxic effect (LD50
=
[4.91 9 10-5 M) in the present assay. Similarly,
123

Med Chem Res
compounds 3d, 3f, 3g, 3j and 3k with 4-trifluoromethyl,
2-fluoro, 3-fluoro, 2,6-difluoro and 3,5-difluoro substituents
about the phenyl ring showed reduced cytotoxic activity
(LD50 = 1.76 9 10-4, 2.79 9 10-4, 1.21 9 10-4, 1.15 9
10-4 and 1.07 9 10-4 M, respectively) in comparison to
the corresponding compounds (without fluoro group at
position-5 of the isatin moiety), which gave LD50 values of
1.17 9 10-4, 3.10 9 10-5, 4.60 9 10-5, 2.00 9 10-5 and
2.10 9 10-5 M (Pervez et al., 2009). Relatively, much
pronounced reduction in the cytotoxic activity was
varied inhibition (10–40 %). Compound 3a with no sub-
stituent on the phenyl ring attached to N4 of the thiosemi-
carbazone moiety was found to be active against M. canis
only, displaying 20 % inhibition. To the contrary, com-
pounds 3b–3d having trifluoromethyl substituents at differ-
ent positions of the phenyl ring were active against more than
one fungal strains. Of these, compound 3c with a meta sub-
stituent was found to be the most active one, showing 30, 30
and 25 % inhibition of A. flavus, M. canis and F. solani,
respectively. Next active to it was compound 3d having a
para substituent, demonstrating 20, 25 and 5 % inhibition of
the same fungal strains. However, the ortho-substituted
compound 3b was found to be active against A. flavus and
M. canis only, exhibiting 15 and 20 % inhibition. Similarly,
compound 3e having trifluoromethoxy substituent at para
position of the phenyl ring was found to be active against
these two fungi, showing 10 and 30 % inhibition, respec-
tively. Amongst the mono-halogenated compounds 3f–3h,
compound 3f possessing fluoro substituent at ortho position
of the phenyl ring was found to be active against two fungal
strains i.e. M. canis and F. solani, demonstrating 30 and
20 % inhibition, respectively. Also, the para-substituted
compound 3h was found to be active against these fungi,
displaying 15 and 10 % inhibition. However, the meta-
substituted compound 3g was found to be the most active
one, exhibiting 10, 25 and 15 % inhibition of three fungal
strains i.e. A. flavus, M. canis and F. solani, respectively.
Similarly, in the cases of di-halogenated compounds 3i–3l,
compound 3i having fluoro substituents at the ortho and para
positions was found to be the most active one, showing
inhibitory activity (40, 35 and 15 %, respectively) against A.
flavus, M. canis and F. solani. Compared to this, compound
observed in the case of 3k (LD50 = 2.10 9 10-5
?
1.07 9 10-4 M). This showed that the simultaneous pres-
ence of inductively electron-attracting groups in the isatin
scaffold as well as the phenyl ring substituted at N4 of the
thiosemicarbazone moiety caused either elimination or
reduction in the cytotoxicity potential. However, compared
with monofluoro-substituted compounds 3f and 3g, com-
pound 3h having fluoro substituent at position-4 of the
phenyl ring displayed promising activity (LD50 = 9.79 9
10-5 M) in the present assay.
The exact mechanism of cytotoxic activity exhibited by
the test thiosemicarbazones 3a–3l is not known. However, it
is generally accepted that the mode of cytotoxic action of this
class of agents involves the inhibition of ribonucleoside
diphosphate reductase (RDR), an enzyme containing a
diferric centre involved in the rate-limiting step of DNA
synthesis. Notably, it is the capability of these compounds to
act as transition metal chelating agents that escorts to their
cytotoxic activity. The chelation of iron (Fe) from intracel-
lular Fe pools results in the inhibition of RDR. This was
thought to be the major mechanism involved in the inhibition
of RDR by thiosemicarbazones, in which the diferric Fe
centreisrequiredtostabilizethetyrosylradicalandisdeemed
essential for the enzymatic activity (Danuta et al., 2009).
On the whole, out of twelve compounds tested for cyto-
toxic activity, seven i.e. 3a, 3d, 3f–3h, 3j and 3k proved to be
active, exhibiting promising cytotoxicity (LD50 = 6.89 9
10-5–2.79 9 10-4 M) against Artemia salina. The remain-
ing compounds i.e. 3b, 3c, 3e, 3i and 3l gave LD50 values
ranging from [4.91 9 10-5 to [5.71 9 10-5 M in the
present assay and, therefore, can be considered as inactive.
These preliminary results indicate that structural modi-
fications may lead to the development of certain novel
cytotoxicants with improved efficacy.
Table 2 In vitro antifungal activity of compounds 3a–3l* (%
inhibition)
Compounds Microbial species
T.
C.
A.
M.
F.
longifusus albicans flavus canis solani glabrata
C.
3a
3b
3c
3d
3e
3f
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
15
30
20
10
00
10
00
40
40
00
00
20
20
30
25
30
30
25
15
35
40
40
20
00
00
25
05
00
20
15
10
15
00
00
10
00
00
00
00
00
00
00
00
00
00
00
00
In vitro antifungal activity
3g
3h
3i
The synthetic compounds 3a–3l were also screened for their
antifungal activity against six fungal cultures i.e. Tricho-
phyton longifusus, Candida albicans, Aspergillus flavus,
Microsporum canis, Fusarium solani and Candida glabrata
(Table 2) at 200 lg/mL in DMSO. All the compounds were
found to be active against one or more fungi, exhibiting
3j
3k
3l
* Concentration used 200 lg/mL of DMSO
123

Med Chem Res
3j with fluoro substituents at ortho positions was active
against A. flavus and M. canis, displaying 40 % inhibition,
whereas compound 3k possessing fluoro substituents at meta
positions showed 40 % inhibition against M. canis only. The
remainder di-halogenated compound 3l with fluoro and
bromo substituents at ortho and para positions of the phenyl
ring, respectively, exhibited 20 and 10 % inhibition of
M. canis and F. solani. Although there is no ready explana-
tion for this variation in activity, it may be related to the
ability of the compounds to enter the cell or their ability to
react at the unknown target site(s) in the microorganisms.
According to Overtone’s concept of cell permeability, the
lipid membrane that surrounds the cell favours the perme-
ation of only lipid-soluble compounds. So, liposolubility/
lipophilicity is an important factor controlling the antifungal
activity. Delocalization of electrons over the whole molecule
increased the lipophilicity. The increased lipophilicity in
turn enhanced penetration of the compounds into the cell
membranes, thus further restricting proliferation of the
microorganisms (Al-Amiery et al., 2012). Furthermore, like
cytotoxicity, antifungal activity of the present compounds
could be based on the deactivation of ribonucleoside
diphosphate reductase (RDR). Importantly, it is the ability of
the test thiosemicarbazones 3a–3l to act as transition metal
chelators coordinating with ferrous ions of the RDR, thus
inhibiting its activity (Pandeya et al., 1999).
mL concentrations. Here too, compound 3m i.e. 2-(2-oxo-
1,2-dihydro-3H-indol-3-ylidene)-N-phenyl-1-hydrazine-
carbothioamide served as reference point to evaluate the
effects of fluoro function at position-5 of the isatin scaffold
as well as other substituents about the phenyl ring attached
to N4 of the thiosemicarbazone moiety on the plant growth
inhibitory potential of these compounds. All compounds
except 3a displayed weak or non-significant (15–35 %)
plant growth inhibition at the highest tested concentration
(500 lg/mL). Compound 3a having flluoro substituent at
position-5 of the isatin scaffold, however, exhibited mod-
erate (70 %) inhibition at this concentration (Table 3). On
the other hand, three compounds i.e. 3c, 3j and 3k, out of
twelve compounds tested, showed no plant growth inhibi-
tion, whereas the rest, including reference point displayed
non-significant (5–20 %) inhibition at the lowest tested
concentration (5 lg/mL).
From the results obtained in this assay, it may be con-
cluded that our compounds, in general, exhibited either no
or non-significant plant growth inhibition at much higher
levels of concentration compared with the standard para-
quat, which displayed 100 % inhibition at a concentration
of 0.015 lg/mL. Furthermore, compared with the reference
compound 3m having no substituent in the isatin scaffold
as well as about the phenyl ring attached to N4 of the
thiosemicarbazone moiety and displaying moderate (40 %)
plant growth inhibition at the highest tested concentration
(500 lg/mL), the percent inhibition values exhibited by our
test compounds revealed that the nature, number and
Conclusively, compound 3a having no substituent about
its phenyl ring displayed 20 % inhibition of M. canis only,
whereas the remainder compounds displayed relatively
better activity profile in terms of percentage of inhibition
and/or the number of fungal strains inhibited. This clearly
indicates that the effects of different substituents about the
phenyl ring attached to N4 of the thiosemicarbazone moiety
played an important role in enhancing the capability of the
compounds to exhibit either induced or increased inhibi-
tory activity. Notably, three of the six fungi tested i.e.
T. longifusus, C. albicans and C. glabrata were resistant to
all the tested compounds, while the two others i.e. A. flavus
and F. solani showed resistance to some of them. Fur-
thermore, none of the compounds of the present series
exhibited significant antifungal activity; however, the
negative findings or display of weak or moderate activities
against certain selected fungal strains in the present assay
do not prevent from further investigation of these com-
pounds against other fungal strains. Also, further investi-
gations on the SAR and appropriate chemical modifications
among the synthesized thiosemicarbazones is likely to
provide more effective compounds with improved efficacy.
Table 3 Percentage growth inhibition of Lemna aequinocitalis by
compounds 3a–3l at different concentrations
Compounds
500 (lg/mL) 50 (lg/mL) 5 (lg/mL)
(% G.I.)
(% G.I.)
(% G.I.)
3a
3b
3c
3d
3e
3f
70
25
30
25
35
35
30
35
30
30
15
30
40
15
10
10
15
20
25
25
25
20
10
10
25
25
10
05
00
10
10
20
15
10
10
00
00
10
15
3g
3h
3i
3j
3k
3l
3m (Pervez et al., 2007,
2012b)
In vitro phytotoxicity
The reference compound paraquat shows 100 % growth inhibition at
a concentration of 0.015 g/mL
The synthesized thiosemicarbazones 3a–3l were further
screened for their phytotoxic effects at 500, 50 and 5 lg/
G.I. growth inhibition
123

Med Chem Res
position of the substituents did not affect the phytotoxic
inhibitory potential to a great extent.
3g and 3k possessing 4-trifluoromethyl, 3-fluoro and 3,5-
difluoro substituents about the phenyl ring exhibited
enhanced activity (53.4 % with IC50 value 90.40, 79.8 %
with IC50 value 45.3 and 80.2 % with IC50 value 150.70,
respectively) as compared to the corresponding compounds
bearing no fluoro substituent at position-5 of the isatin
scaffold, which exhibited 44.6 %, 57.3 % with IC50 value
50.6 and 38.8 % inhibition at the tested concentration
(100 lM). Much pronounced enhancement in the enzymatic
activity was observed in the cases of 3d and 3k
(44.6 ? 53.4 % with IC50 value 90.40 and 38.8 ? 80.2 %
with IC50 value 150.70, respectively). To the contrary,
compounds 3b, 3c, 3e, 3f, 3i and 3j having 2-trifluoromethyl,
3-trifluoromethyl, 4-trifluoromethoxy, 2-fluoro, 2,4-difluoro
and 2,6-difluoro functions on the phenyl ring were found to
display reduced activity (77.3 % with IC50 value 50.53,
92.6 % with IC50 value 59.86, 18.9 %, 94.6 % with IC50
value 37.7, 22.1 % and 72.5 % with IC50 value 47.3,
respectively), when compared with the respective com-
poundswithout fluoro groupatposition-5 ofisatin part, which
demonstrated 63.1 % with IC50 value 33.1, 78.2 % with IC50
value 20.6, 49.6 %, 84.9 % with IC50 value 20.6, 71.9 % with
IC50 value 47.6 and 92.0 % with IC50 value 33.1 inhibition of
the enzyme at the tested concentration (100 lM). Much
pronounced reduction occurred in the cases of 3c, 3f and 3i
(78.2 % with IC50 value 20.6 ? 92.6 % with IC50 value
59.86, 84.9 % with IC50 value 20.6 ? 94.6 % with IC50
value 37.7 and 71.9 % with IC50 value 47.6 ? 22.1 %,
respectively). This indicated that the simultaneous presence
of diverse inductively electron-attracting groups in the isatin
moiety as well as on the phenyl ring attached to N4 of the
thiosemicarbazone part caused the molecules to intermingle
with the enzymatic activity differently, resulting into incre-
ment or decrement in their inhibitory potential. Overall, all
the synthesized compounds 3a–3l tested for their urease
inhibitory potential displayed enzymatic inhibition. Of these,
eight i.e. 3a–3d, 3f, 3g, 3j and 3k were found to be potent
inhibitors. Compounds 3f, 3g and 3j having one or two fluoro
substituents at positions-2, -3 and -2,6 of the phenyl ring were
found to be the most potent ones, showing IC50 values 37.7,
45.3 and 47.3, respectively. The remainder compounds i.e.
3a–3d and 3k exhibited varying degree of activity with IC50
values ranging from 50.53 to 342.93 lM.
In vitro urease inhibitory activity
All the synthetic thiosemicarbazones 3a–3l were also sub-
jected to urease inhibition studies. Thiourea and compound
3m i.e. 2-(2-oxo-1,2-dihydro-3H-indole-3-ylidine)-N-phe-
nyl-1-hydrazinecarbothioamide served as reference points in
this assay. The results given in Table 4 demonstrated that
compared with compound 3 m having no substituent in the
isatin part as well as on the phenyl ring, substitution of fluoro
function at position-5 of the isatin scaffold alone or in
combination with different aryl groups (possessing one or
two substituents about the phenyl ring) at N4 of the thio-
semicarbazone moiety caused either induction or enhance-
ment of enzymatic activity in certain cases at the tested
concentrations (200 or 500 lM). This inference receives
support from the results obtainedby usin early investigations
(Pervez et al., 2008, 2009). For example, compound 3a
bearing fluoro substituent at position-5 of the isatin scaffold
displayed induced activity (53 % with IC50 value 342.93) at
500 lM concentration as compared to the corresponding
compound 3m having no fluoro substituent at position-5 of
the isatin moiety, which showed no inhibitory activity at the
tested concentration(100 lM). Furthermore, compounds 3d,
Table 4 Inhibition of Jack bean urease by compounds 3a–3l
Compounds
Percentage (%) Percentage (%) IC
of inhibition at of inhibition at (lM)
SEM
50
200 lM
500 lM
3a
3b
3c
3d
3e
3f
53.00
342.93 1.58
50.53 0.31
59.86 0.24
90.4 0.66
NA
77.3
53.4
92.6
18.9
94.6
37.7 0.16
45.3 0.16
NA
3g
3h
3i
79.8
45.6
22.1
72.5
80.2
NA
3j
47.3 0.08
150.7 1.17
NA
3k
3l
All ureases, regardless of their source, contain in addi-
tion to two nickel ions (Benini et al., 1998, 2001, 2004,
2000; Ciurli et al., 1999; Krajewska and Zaborska, 2007a,
2007b), one to three protein subunits present in varying
stoichiometric ratios (Mobley et al., 1995). Thus, an anti-
urease compound can intermingle with the enzymatic
activity by interacting either with the nickel ions or the
protein constituent. b-Mercaptoethanol, hydroxamic acids
and phosphorodiamidates, for example, are the synthetic
inhibitors, which interact with the urease activity by
21.9
97.1
3m (Pervez
et al., 2007,
2008)a
Thioureab
99.0
21.0 0.11
NA No inhibitory activity
a
Reference thiosemicarbazone screened against human urease at
100 lM concentration
b
Reference inhibitor of urease enzyme
123

Med Chem Res
binding to the nickel ions present at its active site [Ciurli
et al., 1999; Kuhler et al., 1995; Nagata et al., 1993;
Zaborska et al., 2007), whereas sulphenamide, quinones
and heavy metal ions have been reported to inhibit its
activity by intermingling with the sulphhydryl (S–H)
functions present in the protein component (Krajewska,
2008; Krajewska and Zaborska, 2007a, 2007b; Krajewska
et al., 2004; Kuhler et al., 1995; Nagata et al., 1993 Kra-
jewska and Zaborska, 2007a). Although the actual mech-
anism of urease inhibitory activity exhibited by our trial
compounds 3a–3l is not documented, it is intriguing to
carry out investigations as regards detailed kinetics of such
interaction. Apparently, these compounds seem to be
mechanism-based urease inhibitors and inhibit the enzyme
through a chelate interaction, which binds to its active site
in a normal substrate-like mode. This process appears to be
similar to charge-transfer transitions, which occur between
thiolate of b-mercaptoethanol and nickel (II) of the
enzyme, suggesting that the thiolate binds directly to the
nickel ion(s). Diverse non-covalent interactions, including
hydrogen bonds and hydrophobic contacts may stabilize
such enzyme-inhibitors chelate interactions and thus con-
tribute towards their inhibitory potential (Amtul et al.,
2002). Detailed kinetic investigations to get an insight into
the mechanism of inhibition are underway, the results of
which will be reported in due course of time.
well as different inductively electron-attracting substituents
about the phenyl ring attached to N4 of the thiosemicar-
bazone moiety.
Experimental section
Chemistry
All chemicals and solvents were purchased from Aldrich,
Fluka and Merck-Schuchatdt. Melting points were deter-
mined on cover slips using a Fisher-Johns melting point
apparatus and are uncorrected. Elemental (C, H, N) anal-
yses were performed on a Leco CHNS-9320 (USA) ele-
mental analyser and were in full agreement with the
proposed structures within 0.4 % of the theoretical limits,
except where noted otherwise. Infrared (IR) spectra (KBr
discs) were run on Shimadzu Prestige-21 FT-IR spec-
trometer. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded in DMSO-d6 on Bruker (Rhenistet-
ten-Forchheim, Germany) AM 300 spectrometer, operating
at 300 MHz and using TMS as an internal standard. The
chemical shifts (d) are reported in parts per million (ppm)
and coupling constants in Hz. Carbon-13 nuclear magnetic
resonance (13C NMR) spectra were recorded at 75 MHz
with the same internal standard. The electron impact mass
spectra (EI MS) were determined with MAT-312, JEOL
MSRoute and JEOL JMS 600 mass spectrometers. The
progress of the reaction and purity of the products were
checked on TLC plates coated with Merck silica gel 60
GF254, and the spots were visualized under ultraviolet light
at 254 and 366 nm and/or spraying with iodine vapours.
In summary, a series of twelve 5-fluoroisatin-3-thiose-
micarbazones 3a–3l has been synthesized and character-
ized by analytical and spectral data. The synthesized
thiosemicarbazones were screened for their in vitro cyto-
toxic, antifungal, phytotoxic and urease inhibitory effects.
Seven out of twelve compounds tested proved to be active
in the brine shrimp bioassay, displaying promising cyto-
toxicity. In antifungal assay, all the compounds showed
weak-to-moderate activity against one or more fungi.
However, the negative findings or demonstration of weak
or moderate activity against the selected fungal strains do
not preclude from further investigations of these com-
pounds against some other pathogens. In phytotoxicity
bioassay, the synthesized thiosemicarbazones showed
weak-to-moderate activity at the highest tested concentra-
tion. The urease inhibition screening results have sub-
stantiated that eight out of twelve compounds tested
appeared as potent urease inhibitors; three of these i.e. 3f,
3g and 3j proved to be the most potent ones and may act as
leads for further studies. These compounds, exhibiting no
significant phytotoxic activity at the highest tested con-
centration, invited attention to their usefulness as potent
soil ureases inhibitors, as they could be mixed with fertil-
izers in small quantities to increase the overall efficacy of
nitrogen utilisation. In general, the urease inhibitory
activity was found to be dependent upon electronic effects
of the fluoro function at position-5 of the isatin scaffold as
General procedure for the preparation of
5-fluoroisatin-thiosemicarbazons (3a–3l)
To a solution of 5-fluoroisatin 1 (2.5 mmol) in 50 %
aqueous ethanol (10 mL) containing a catalytic amount of
glacial acetic acid, the appropriate thiosemicarbazide 2
(2.5 mmol) dissolved in ethanol (10 mL) was added under
stirring. The reaction mixture was then heated under reflux
for 2 h. The crystalline or amorphous solid formed during
heating was collected by suction filtration. Thorough
washing with hot aqueous ethanol (50 %) furnished the
target compounds 3a–3l in pure form.
N-Phenyl-2-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-
1-hydrazinecarbothioamide (3a) Yield 93 % as yellow
powder; m.p. 218–220 °C (lit. (Karali et al., 2007)
m.p. 218 °C); IR (KBr, cm-1): 3460, 3195 (NH), 1691
(C=O), 1595 (C=N), 1265 (C=S); 1H NMR (300 MHz,
DMSO-d6) d, ppm, 6.95 (dd, J = 8.7, 4.2 Hz, 1H, indole
C7–H), 7.22 (td, J = 8.7, 2.7 Hz, 1H, indole C6–H), 7.29
123

Med Chem Res
(t, J = 7.5 Hz, 1H, phenyl C4–H), 7.45 (t, J = 7.5 Hz, 2H,
phenyl C3–H, C5–H), 7.61 (d, J = 8.1 Hz, 2H, phenyl C2–
H, C6–H), 7.65 (dd, J = 8.4, 2.7 Hz, 1H, indole C4–H),
10.85 (s, 1H, CSNH), 11.26 (s, 1H, indole NH), 12.68 (s,
1H, NNH); 13C NMR (75 MHz, DMSO-d6) d, ppm,
108.11, 108.46, 112.10, 112.21, 117.47, 117.79, 121.41,
125.63, 126.21, 128.43, 138.27, 138.74, 156.67, 159.82,
162.76, 176.30; EI MS (70 eV) m/z (%), 314 (M?, 34), 286
(100), 254 (3), 221 (14), 197 (6), 179 (40), 163 (15), 162
(29), 151 (17), 15 (20), 149 (23), 136 (53), 135 (71), 122
(36), 108 (25), 96 (6), 95 (7), 91 (16), 77 (48), 65 (14).
Anal calcd. for C15H11FN4OS: C, 57.31; H, 3.53; N, 17.82.
Found: C, 57.20; H, 3.52; N, 17.79.
(8), 75 (17). Anal calcd. for C16H10F4N4OS: C, 50.26; H,
2.64; N, 14.65. Found: C, 50.20; H, 2.63; N, 14.60.
2-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-[4-
(trifluoromethyl)phenyl]-1-hydrazinecarbothioamide (3d)
Yield 82 % as yellow fluffy crystals; m.p. 249–250 °C; IR
(KBr, cm-1): 3285, 3187 (NH), 1694 (C=O), 1608 (C=N),
1
1271 (C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.94
(dd, J = 8.7, 4.2 Hz, 1H, indole C7–H), 7.25 (td, J = 9.3,
2.7 Hz, 1H, indole C6–H), 7.63 (dd, J = 8.1, 2.7 Hz, 1H,
indole C4–H), 7.81 (d, J = 8.7 Hz, 2H, phenyl C2–H, C6–
H), 7.95 (d, J = 8.4 Hz, 2H, phenyl C3–H, C5–H), 11.01
(s, 1H, CSNH), 11.30 (s, 1H, indole NH), 12.80 (s, 1H,
NNH); 13C NMR (75 MHz, DMSO-d6) d, ppm, 108.28,
108.46. 112.22, 112.27, 117.79, 117.95, 121.14, 121.20,
123.25, 125.47, 125.51, 125.54, 125.93, 126.14, 132.26,
132.28, 138.90, 141.99, 157.46, 159.04, 162.78, 176.28; EI
MS (70 eV) m/z (%), 382 (M?, 32), 354 (100), 218 (5), 204
(19), 179 (22), 168 (28), 163 (9), 151 (17), 150 (34), 145
(49), 136 (23), 135 (25), 122 (61), 108 (31), 96 (12), 95
(31), 94 (12), 75 (18). Anal calcd. for C16H10F4N4OS: C,
50.26; H, 2.64; N, 14.65. Found: C, 50.13; H, 2.63; N,
14.69.
2-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-[2-
(trifluoromethyl)phenyl]-1-hydrazinecarbothioamide (3b)
Yield 75 % as yellow fluffy crystals; m.p. 247–248 °C; IR
(KBr, cm-1): 3300, 3200 (NH), 1684 (C=O), 1580 (C=N),
1
1279 (C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.94
(dd, J = 8.7,4.2 Hz, 1H, indole C7–H), 7.22 (td, J = 8.7,
2.7 Hz, 1H, indole C6–H), 7.53 (dd, J = 8.1, 2.7 Hz, 1H,
indole C4–H), 7.56–7.64 (m, 2H, phenyl C3–H, C4–H),
7.77–7.86 (m, 2H, phenyl C5–H, C6–H), 10.85 (s, 1H,
CSNH), 11.29 (s, 1H, indole NH), 12.75 (s, 1H, NNH); 13
C
NMR (75 MHz, DMSO-d6) d, ppm, 107.98, 108.32,
112.24, 112.35, 117.67, 117.99, 121.25, 121.38, 121.60,
125.23, 126.54, 126.59, 126.66, 126.94, 128.42,
132.15,133.23, 136.82, 138.95, 156.68, 159.83, 162.79,
178.56; EI MS (70 eV) m/z (%), 382 (M?, 13), 354 (65),
334 (3), 313 (2), 285 (4), 256 (13), 219 (4), 196 (11), 184
(78), 179 (23), 163 (10), 151 (30), 150 (45), 136 (27), 135
(35), 122 (100), 108 (54), 96 (20), 95 (55), 94 (21), 91 (5),
75 (37). Anal calcd. for C16H10F4N4OS: C, 50.26; H, 2.64;
N, 14.65. Found: C, 50.19; H, 2.63; N, 14.60.
2-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-[4-
(trifluoromethoxy)phenyl]-1-hydrazinecarbothioamide (3e)
Yield 80 % as light orange powder; m.p. 244–245 °C; IR
(KBr, cm-1): 3306, 3183(NH), 1695 (C=O), 1580 (C=N),
1
1250 (C=S); H NMR (300 MHz, DMSO-d6) d, ppm, d
6.94 (dd, J = 8.4, 4.2 Hz, 1H, indole C7–H), 7.21 (td,
J = 9.3, 2.7 Hz, 1H, indole C6–H), 7.45 (d, J = 8.4 Hz,
2H, phenyl C2 –H, C6–H), 7.60 (dd, J = 8.1, 2.7 Hz, 1H,
indole C4–H), 7.75 (d, J = 8.7 Hz, 2H, phenyl C3–H, C5–
H), 10.92 (s, 1H, CSNH), 11.28 (s, 1H, indole NH), 12.73
(s, 1H, NNH), 12.73 (s, 1H, NNH); 13C NMR (75 MHz,
DMSO-d6) d, ppm, 108.09, 108.43, 112.16, 112.27, 117.60,
117.92, 121.11, 121.18, 121.31, 127.37, 131.98, 132.02,
137.45, 138.83, 145.89, 145.91, 156.67, 159.82, 162.76,
176.51; EI MS (70 eV) m/z (%), 398 (M?, 26), 370 (100),
313 (3), 235 (3), 220 (18), 179 (19), 163 (9), 151 (19), 150
(38), 136 (16), 135 (20), 122 (58), 108 (36), 96 (13), 95
(42), 94 (9), 75 (11). Anal calcd. for C16H10F4N4O2S: C,
48.24; H, 2.53; N, 14.07. Found: C, 48.22; H, 2.54; N,
14.01.
2-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-[3-
(trifluoromethyl)phenyl]-1-hydrazinecarbothioamide (3c)
Yield 72 % as orange powder; m.p. 219–220 °C; IR (KBr,
cm-1): 3248, 3202(NH), 1697 (C=O), 1547 (C=N), 1244
1
(C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.94 (dd,
J = 8.7, 4.2 Hz, 1H, indole C7–H) 7.22 (td, J = 8.7,
2.7 Hz, 1H, indole C6–H), 7.63 (dd, J = 8.1, 2.4 Hz, 1H,
indole C4–H), 7.66–7.71 (m, 2H, phenyl C5–H, C6–H),
8.00–8.06 (m, 2H, phenyl C2–H, C4–H), 11.01 (s, 1H,
CSNH), 11.31 (s, 1H, indole NH), 12.78 (s, 1H, NNH); 13
C
NMR (75 MHz, DMSO-d6) d, ppm, 108.13, 108.48,
112.19, 112.30, 117.68, 118.00, 121.12, 121.25, 121.66,
121.71, 121.76, 121.81, 122.13, 122.50, 122.55, 125.74,
128.90, 129.28, 129.57, 132.17, 132.21, 138.88, 138.90,
139.10, 156.68, 159.83, 162.77, 176.43; EI MS (70 eV) m/
z (%), 382 (M?, 20), 354 (100), 218 (4), 204 (19), 184 (10),
179 (21), 168 (30), 163 (9), 151 (19), 150 (35), 145 (48),
136 (16), 135 (20), 122 (50), 108 (8), 96 (9), 95 (23), 94
2-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-(2-
fluorophenyl)-1-hydrazinecarbothioamide
(3f) Yield
80 % as yellow fluffy crystals; m.p. 260 °C; IR (KBr,
cm-1): 3300, 3169, 3101 (NH), 1696 (C=O), 1619 (C=N),
1
1264 (C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.95
(dd, J = 8.4, 4.2 Hz, 1H, indole C7–H), 7.22 (td, J = 9.3,
2.4 Hz, 1H, indole C6–H), 7.29–7.43 (m, 3H, phenyl C4–H,
C5–H, C6–H), 7.48 (d, J = 7.8 Hz, phenyl C3–H), 7.55 (dd,
123

Med Chem Res
J = 8.1, 2.4 Hz, indole C4–H), 10.78 (s, 1H, CSNH), 11.28
(s, 1H, indole NH), 12.74 (s, 1H, NNH); 13C NMR
(75 MHz, DMSO-d6) d, ppm, 107.96, 108.30, 112.18,
112.29, 112.91, 116.17, 117.59, 117.90, 121.25, 121.38,
124.43, 124.47, 126.19, 126.35, 129.13, 129.23, 130.16,
132.06, 132.11, 138.87, 138.88, 155.62, 156.66, 158.91,
159.81, 162.72, 178.00; EI MS (70 eV) m/z (%), 332 (M?,
9), 304 (100), 284 (5), 221 (4), 197 (3), 179 (12), 168 (13),
150 (16), 135 (29), 122 (46), 108 (27), 96 (11), 95 (20), 91
(3), 83 (21), 75 (18). Anal calcd. for C15H10F2N4OS: C,
54.21; H, 3.03; N, 16.86. Found: C, 54.16; H, 3.03; N,
16.84.
(62). Anal calcd. for C15H10F2N4OS: C, 54.21; H, 3.03; N,
16.86. Found: C, 54.21; H, 3.05; N, 16.84.
N-(2,4-Difluorophenyl)-2-(5-fluoro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (3i) Yield
78 % as yellow fluffy crystals; m.p. 259–260 °C; IR (KBr,
cm-1): 3300, 3217(NH), 1694 (C=O), 1539 (C=N), 1263
1
(C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.95 (dd,
J = 8.4, 4.2 Hz, 1H, indole C7–H), 7.15–7.25 (m, 2H,
indole C6–H, phenyl C6–H), 7.43 (td, J = 9.0, 2.7 Hz, 1H,
phenyl C5–H), 7.50–7.58 (m, 2H, phenyl C3–H, indole C4–
H), 10.72 (s, 1H, CSNH), 11.28 (s, 1H, indole NH), 12.76
(s, 1H, NNH); 13C NMR (75 MHz, DMSO-d6) d, ppm,
104.31, 104.65, 104.98, 107.98, 108.32, 111.39, 111.43,
111.68, 111.73. 112.24, 112.34, 117.68, 118.00, 121.24,
121.36, 122.92, 122.97, 123.09, 123.13, 131.44, 131.57,
132.28, 132.32, 138.96, 155.77, 155.94, 156.86, 159.09,
159.26, 159.34, 159.49, 159.83, 162.61, 162.75, 178.33; EI
MS (70 eV) m/z (%), 350 (M?, 31), 322(100), 179 (6), 172
(9), 163 (30), 151 (5), 150 (8), 136 (5), 135 (5), 122 (11),
108 (9), 95 (3), 75 (3). Anal calcd. for C15H9F3N4OS: C,
51.43; H, 2.59; N, 15.99. Found: C, 51.44; H, 2.58; N,
16.00.
2-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-(3-
fluorophenyl)-1-hydrazinecarbothioamide
(3g) Yield
74 % as orange fluffy crystals; m.p. 256 °C; IR (KBr,
cm-1): 3304, 3163,3103 (NH), 1697 (C=O), 1595 (C=N),
1
1261 (C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.93
(dd, J = 8.4, 4.2 Hz, 1H, indole C7–H), 7.11–7.17 (m, 1H,
phenyl C5–H), 7.24 (td, J = 8.7, 4.3 Hz, 1H, indole C6–H),
7.44–7.53 (m, 2H, phenyl C2–H, C6–H), 7.69 (dd, J = 8.4,
4.2 Hz, 2H, indole C4–H, phenyl C2–H), 10.81 (s, 1H,
CSNH), 11.29 (s, 1H, indole NH), 12.73 (s, 1H, NNH); 13
C
NMR (75 MHz, DMSO-d6) d, ppm, 108.22, 108.48,
112.10, 112.18, 112.26, 112.35, 112.68, 112.89, 117.67,
117.91, 121.18, 121.22, 121.25, 129.93, 130.02, 132.00,
132.04, 132.85, 139.90, 140.01, 157.07, 159.43, 160.40,
162.77, 162.81, 176.16; EI MS (70 eV) m/z (%), 332 (M?,
4), 304 (26), 197 (14), 179 (14), 168 (20), 163 (4), 153
(27), 151 (14), 150 (18), 136 (27), 135 (22), 122 (66), 111
(34), 108 (40), 96 (32), 95 (100), 94 (20), 84 (22), 83 (77),
75 (63), 69 (15), 57 (49). Anal calcd. for C15H10F2N4OS:
C, 54.21; H, 3.03; N, 16.86 Found: C, 54.17; H, 3.04; N,
16.84.
N-(2,6-Difluorophenyl)-2-(5-fluoro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (3j) Yield
90 % as orange powder; m.p. 274–275 °C; IR (KBr,
cm-1): 3360, 3194(NH), 1694 (C=O), 1583 (C=N), 1246
1
(C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.95 (dd,
J = 8.7, 4.2 Hz, 1H, indole C7–H), 7.20–7.30 (m, 3H,
indole C4–H, C6–H, phenyl C4–H), 7.46–7.56 (m, 2H,
phenyl C3–H, C5–H), 10.63 (s, 1H, CSNH), 11.29 (s, 1H,
indole NH), 12.84 (s, 1H, NNH); 13C NMR (75 MHz,
DMSO-d6) d, ppm, 107.94, 108.28, 111.90, 112.13, 112.21,
112.24, 112.35, 115.63, 115.85, 116.06, 117.75, 118.07,
121.16, 121.28, 129.66, 129.79, 129.92, 132.70, 132.74,
139.04, 139.06, 156.64, 156.88, 156.94, 159.79, 160.18,
160.24, 162.69, 178.66; EI MS (70 eV) m/z (%), 350 (M?,
28), 322 (100), 282 (2), 221 (4), 196 (8), 179 (20), 171
(21), 163 (9), 151 (15), 150 (32), 136 (17), 135 (23), 122
(57), 108 (61), 101 (20), 96 (9), 95 (22), 94 (13), 75 (16).
Anal calcd. for C15H9F3N4OS: C, 51.43; H, 2.59; N, 15.99.
Found: C, 51.39; H, 2.60; N, 15.91.
2-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-(4-
fluorophenyl)-1-hydrazinecarbothioamide
(3h) Yield
82 % as orange powder; m.p. 265–266 °C (lit. (Karali
et al., 2007) m.p. 246–248 °C); IR (KBr, cm-1): 3355,
3155 (NH), 1692 (C=O), 1540 (C=N), 1258 (C=S); 1H
NMR (300 MHz, DMSO-d6) d, ppm, 6.94 (dd,
J = 8.4,4.2 Hz, 1H, indole C7–H), 7.18–7.31 (m, 3H,
phenyl C2–H, C6–H, indole C6–H), 7.58–7.62 (m, 3H,
phenyl C3–H, C5–H, indole C4–H), 10.87 (s, 1H, CSNH),
11.82 (s, 1H, indole NH), 12.68 (s, 1H, NNH); 13C NMR
(75 MHz, DMSO-d6) d, ppm, 108.14, 108.31. 112.15,
112.21, 115.10, 115.25, 117.58, 117.74, 121.28, 121.34,
127.89, 127.94, 131.74, 131.76, 134.62, 134.64, 138.76,
157.45, 159.03, 159.25, 160.86, 162.75, 176.69; EI MS
(70 eV) m/z (%), 332 (M?, 20), 304 (80), 221 (4), 197 (9),
179 (20), 168 (32), 163 (10), 151 (25), 150 (28), 136 (40),
135 (35), 122 (90), 108 (48), 96 (49), 95 (100), 80 (68), 75
N-(3,5-Difluorophenyl)-2-(5-fluoro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (3k) Yield
77 % as orange powder; m.p. 259–260 °C; IR (KBr,
cm-1): 3290, 3072 (NH), 1693 (C=O), 1602 (C=N), 1265
1
(C=S): H NMR (300 MHz, DMSO-d6) d, ppm, 6.93 (dd,
J = 8.7, 4.2 Hz, 1H, indole C7–H), 7.14–7.25 (m, 2H,
indole C6–H, phenyl C4–H), 7.55–7.63 (m, 3H, indole C4 –
H, phenyl C2–H, C6 –H), 10.89 (s, 1H, CSNH), 11.31 (s,
1H, indole NH), 12.78 (s, 1H, NNH); 13C NMR (75 MHz,
123

Med Chem Res
DMSO-d6) d, ppm, 101.01, 101.22, 101.42, 107.81, 107.86,
107.98, 108.04, 108.29, 108.49, 112.22, 112.28, 117.84,
118.03, 121.03, 121.11, 132.36, 132.39, 138.95, 140.72,
140,83, 140.94, 157.29, 159.18, 160.79, 160.91, 162.75,
162.85, 175.93; EI MS (70 eV) m/z (%), 350 (M?, 30), 321
(100), 220 (2), 196 (9), 179 (20), 168 (29), 163 (4), 151
(17), 150 (27), 136 (18), 135 (19), 122 (50), 108 (24), 96
(9), 95 (18), 94 (9), 82 (26). Anal calcd. for C15H9F3N4OS:
C, 51.43; H, 2.59; N, 15.99. Found: C, 51.36; H, 2.61; N,
16.01.
shrimps were added to each vial (30 shrimps/dilution) and
the volume was adjusted with sea water to 5 mL per vial.
After 24 h, the number of survivors was counted
(McLaughlin et al., 1991; Meyer et al., 1982). Data were
analysed by a Finney computer program to determine the
LD50 values (Finney, 1971).
Antifungal activity in vitro
Antifungal activities of all the compounds were studied
against six fungal cultures. Sabouraud dextrose agar
(Oxoid, Hampshire, England) was seeded with 105 (cfu)
mL-1 fungal spore suspensions and transferred to petri
plates. Discs soaked with 20 lL (200 lg/mL in DMSO) of
all the compounds were placed at different positions on the
agar surface. The plates were incubated at 27–29 °C for
seven days. The results were recorded (Hussain et al.,
2003) as zone of inhibition (mm) and compared with
standard drugs, miconazole and amphotericin B.
N-(4-Bromo-2-fluorophenyl)-2-(5-fluoro-2-oxo-1,2-dihydro-
3H-indol-3-ylidene)-1-hydrazinecarbothioamide (3l)
Yield 80 % as orange fluffy crystals; mp 209–210 °C; IR
(KBr, cm-1): 3310, 3171(NH), 1691 (C=O),1580 (C=N),
1
1271 (C=S); H NMR (300 MHz, DMSO-d6) d, ppm, 6.94
(dd, J = 8.7, 4.2 Hz, 1H, indole C7–H), 7.22 (td, J = 9.3,
2.7 Hz, 1H, indole C6–H), 7.45–7.54 (m, 3H, phenyl C3–H,
C5–H, C6–H), 7.74 (dd, J = 9.3, 2.7 Hz, 1H, indole C4–H),
10.76 (s, 1H, CSNH), 11.30 (s, 1H, indole NH), 12.78 (s,
1H, NNH); 13C NMR (75 MHz, DMSO-d6) d, ppm,
107.99, 108.20, 112.22, 112.28, 117.73, 117.92, 119.39,
119.58, 120.20, 120.27, 121.16, 121.24, 125.97, 126.06,
127.63, 127.66, 131.64, 132.35, 132.38, 138.92, 156.17,
157.26, 158.18, 159.15, 162.68, 177.96; EI MS (70 eV) m/
z (%), 412/410 (M?, 15/14), 384/382 (100/95), 233/231
(27/26), 214 (27), 191/189 (21/23), 179 (37), 168 (68), 163
(35), 151 (31), 150 (52), 136 (40), 135 (47), 122 (97), 108
(78), 96 (17), 95 (37), 94 (45), 75 (16). Anal calcd. for
C15H9BrF2N4OS: C, 43.81; H, 2.21; N, 13.62. Found: C,
43.72; H, 2.20; N, 13.51.
Phytotoxicity in vitro
This bioassay was performed according to a modified
protocol (McLaughlin et al., 1991). The test compounds
were incorporated into sterilized E-medium at different
concentrations i.e. 5, 50 and 500 lg/mL in methanol.
Sterilized conical flasks were inoculated with compounds
of the desired concentrations prepared from the stock
solution and allowed to evaporate overnight. Each flask
was inoculated with 20 mL of sterilized E-medium and
then ten Lemna aequinocitalis Welv, each containing a
rosette of three fronds, were placed on media. Other flasks
were supplemented with methanol serving as a negative
control and the reference inhibitor i.e. paraquat as the
positive one. Treatments were replicated three times and
the flasks incubated at 30 °C in Fisons Fi-Totron 600 H
growth cabinet for 7 days, 9,000 lux light intensity,
56 10 rh (relative humidity), and 12 h day length.
Growth of Lemna aequinocitalis in compounds-containing
flasks was determined by counting the number of fronds
per dose and growth inhibition was calculated with refer-
ence to negative control.
Biology
Cytotoxicity in vitro
Brine shrimp (Artemia salina leach) eggs were hatched in a
shallow rectangular plastic dish (22 9 32 cm) filled with
artificial sea water, which was prepared with a commercial
salt mixture (Instant Ocean, Aquarium System, Inc.,
Mentor, Ohio, USA) and double-distilled water. An
unequal partition was made in the plastic dish with the help
of a perforated device. Approximately 50 mg of eggs were
sprinkled into the large compartment, which was darkened,
while the smaller compartment was opened to ordinary
light. After 2 days, nauplii were collected by a pipette from
the lighted side. A sample of the test compound was pre-
pared by dissolving 2 mg of each compound in 2 mL of
methanol. From this stock solution, 500, 50 and 5 lL were
transferred to 9 vials, three for each dilution, and one vial
was kept as control having 2 mL of methanol. The solvent
was allowed to evaporate overnight. After 2 days, when
shrimp larvae were ready, 1 mL of sea water and 10
Urease inhibitory activity in vitro
Reaction mixtures comprising 25 lL of enzyme (Jack bean
urease) solution and 55 lL of buffers containing 100 mM
urea were incubated with 5 lL of test compounds (0.2 and
0.5 mM concentrations) at 30 °C for 15 min in 96-well
plates. Urease inhibitory activity was determined by mea-
suring ammonia production using the indophenol method
as described by Weatherburn (Weatherburn, 1967). In
brief, 45 lL each of phenol reagent (1 % w/v phenol and
123

Med Chem Res
Benini S, Rypniewski WR, Wilson KS, Miletti S, Ciurli S, Mangani S
(2000) The complex of Bacillus pasteurii urease with acet-
0.005 % w/v sodium nitropruside) and 70 lL of alkali
reagent (0.5 % w/v NaOH and 0.1 % active chloride
NaOCl) were added to each well. The increasing absor-
bance at 630 nm was measured after 50 min, using a
microplate reader (Molecular Device, USA). All reactions
were performed in triplicate in a final volume of 200 lL.
The results (change in absorbance per min) were processed
using SoftMax Pro software (Molecular Device, USA). All
the assays were performed at pH 8.2 (0.01 M K2HPO4.
3H2O, 1 mM EDTA and 0.01 M LiCl). Percentage inhibi-
´
˚
ohydroxamate anion from X-ray data at 1.55 A resolution. J Biol
Inorg Chem 5:110–118
Benini S, Rypniewski WR, Wilson KS, Ciurli S, Mangani S (2001)
Structure-based rationalization of urease inhibition by phos-
phate: novel insights into the enzyme mechanism. J Biol Inorg
Chem 6:778–790
Benini S, Rypniewski WR, Wilson KS, Mangani S, Ciurli S (2004)
Molecular details of urease inhibition by boric acid: insights into
the catalytic mechanism. J Am Chem Soc 126:3714–3715
Chen L-R, Wang Y-C, Lin YW, Chou S-Y, Chen S-F, Liu LT, Wu
Y-T, Kuo C-J, Chen T-S, Juang S-H (2005) Synthesis and
evaluation of isatin derivatives as effective SARS coronavirus
3CL protease inhibitors. Bioorg Med Chem Lett 15:3058–3062
Chiyanzu I, Hansell E, Gut J, Rosenthal PJ, McKerrow JH, Chibale K
(2003) Synthesis and evaluation of isatins and thiosemicarba-
zone derivatives against cruzain falcipain-2 and rhodesain.
Bioorg Med Chem Lett 13:3527–3530
tions were calculated from the formula 100 - (ODtestwell
/
ODcontrol) 9 100. Thiourea was used as the standard
inhibitor of urease.
Conclusions
Chiyanzu I, Clarkson C, Smith PJ, Lehman J, Gut J, Rosenthal PJ,
Chibale K (2005) Design, synthesis and anti-plasmodial evalu-
ation in vitro of new 4-aminoquinoline isatin derivatives. Bioorg
Med Chem 13:3249–3261
Ciurli S, Benini S, Rypniewski WR, Wilson KS, Miletti S, Mangani S
(1999) Structural properties of the nickel ions in urease: novel
insights into the catalytic and inhibition mechanisms. Coord
Chem Rev 190–192:331–355
Da Silva JFM, Garden SJ, Pinto AC (2001) The chemistry of isatins: a
review from 1975 to 1999. J Braz Chem Soc 12:273–324
Danuta SK, Patricia Q, Desrr D (2009) Thiosemicarbazones: the new
wave in cancer treatment. Future Med Chem 1:1143–1151
Finney DJ (1971) Probit Analysis, 3rd edn. Cambridge University
Press, London
We have reported the potential of N4-aryl-substituted
5-fluoroisatin-3-thiosemicarbazones to exhibit cytotoxic
and antiurease activities. Based on the preliminary data
presented in Tables 1 and 4, and in terms of further
development and structure–activity relationship (SAR)
studies, simultaneous substitution of different inductively
electron-withdrawing groups at position-5 of the isatin
scaffold and on the phenyl ring attached to N4 of the thi-
osemicarbazone moiety certainly warrants further studies.
Work in this regard along with extended SAR studies will
be reported in the very near future.
Guzel O, Karali N, Salman A (2008) Synthesis and antituberculosis
5-methyl/trifluoromethoxy-1H-indole-2,3-dione
activity
of
3-thiosemicarbazone derivatives. Bioorg Med Chem 16:8976–
8987
Acknowledgments This work was supported by the Higher Educa-
tion Commission (HEC), Pakistan (Project No. 20-873/R&D/07/452).
Hall MD, Salam NK, Hellawell JL, Fales HM, Kensler CB, Ludwig
JA, Szakacs G, Hibbs DE, Gottesman MM (2009) Synthesis,
activity, and pharmacophore development for isatin-b-thiosemi-
carbazones with selective activity toward multidrug-resistant
cells. J Med Chem 52:3191–3204
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