Synthesis and toxicity evaluation of some N4-aryl substituted 5-trifluoromethoxyisatin-3-thiosemicarbazones

Article abstract of DOI:10.3390/molecules16086408

A series of twenty one N4-aryl substituted 5-trifluoromethoxyisatin-3- thiosemicarbazones 3a-3u was synthesized by the reaction of trifluoromethoxyisatin 1 with different arylthiosemicarbazides 2 in aqueous ethanol (50%), containing a few drops of acetic

Full text of DOI:10.3390/molecules16086408

Molecules 2011, 16, 6408-6421; doi:10.3390/molecules16086408
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Toxicity Evaluation of Some N4-Aryl Substituted
5-Trifluoromethoxyisatin-3-thiosemicarbazones
Humayun Pervez 1,*, Naveeda Saira 1, Mohammad Saeed Iqbal 2, Muhammad Yaqub 1 and
Khalid Mohammed Khan 3
1
Department of Chemistry, Bahauddin Zakariya University, Multan-60800, Pakistan
2
Department of Chemistry, FC College, Lahore-54600, Pakistan
3
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
* Author to whom correspondence should be addressed; E-Mail: pdhpervez@hotmail.com;
Tel.: +92-61-9210083; Fax: +92-61-9210138.
Received: 16 June 2011; in revised form: 13 July 2011 / Accepted: 22 July 2011 /
Published: 29 July 2011
Abstract: A series of twenty one N4-aryl substituted 5-trifluoromethoxyisatin-3-
thiosemicarbazones 3a-3u was synthesized by the reaction of trifluoromethoxyisatin 1 with
different arylthiosemicarbazides 2 in aqueous ethanol (50%), containing a few drops of
acetic acid. Their structures were established on the basis of analytical (CHN) and spectral
1
(IR, H-NMR, EIMS) data. All the synthesized compounds were evaluated for their
toxicity potential by a brine shrimp lethality bioassay. Ten compounds i.e., 3a, 3e, 3i-3l
and 3n-3q proved to be active in this assay, displaying promising toxicity
(LD50 = 1.11 × 10−5 M − 1.80 × 10−4 M). Amongst these, 3k, 3n and 3o were found to be
the most active ones (LD50 = 1.11 × 10−5 M − 1.43 × 10−5 M). Compound 3k showed the
highest activity with a LD50 value of 1.11 × 10−5 M and can, therefore, be used as a lead for
further studies. Structure-activity relationship (SAR) studies revealed that the presence of
strong inductively electron-attracting trifluoromethoxy substituent at position-5 of the
isatin moiety played an important role in inducing or enhancing toxic potentiality of some
of the synthesized compounds.
Keywords: isatin; 5-trifluoromethoxyisatin; thiosemicarbazones; 5-trifluoromethoxyisatin-
3-thiosemicarbazones; toxicity

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1. Introduction
Much interest has been shown in isatin and its derivatives due to their diversified pharmacological
properties including antimicrobial, anticonvulsant, antineoplastic, antiviral, antihypertensive,
anti-inflammatory and enzymatic inhibition activities [1-18]. Amongst isatin derivatives,
isatin-thiosemicarbazones have been found to demonstrate numerous chemotherapeutic properties such as
anticancer, antimicrobial, antituberculosis, antiulcer, antiviral, antiplasmodial, cytotoxic and enzymatic
inhibition [1-6,8,9,12,19,20]. Prompted by this and in continuation of our work on bioactive isatin
derivatives [21-25], it was of interest to synthesize some new isatin-3-thiosemicarbazones, which may
exhibit better or different types of biological properties. It is pertinent to mention that a number of
N4-aryl substituted isatin-derived thiosemicarbazones, prepared earlier in our laboratory, displayed
different biological activities such as antibacterial, antifungal, cytotoxic, phytotoxic and urease
inhibition [22-25]. Structure-activity relationship (SAR) studies in the synthesized N4-aryl substituted
isatin-3-thiosemicarbazones revealed that in certain cases, the nature and position of different
substituents about the phenyl ring attached to N4 of the thiosemicarbazone moiety [22,23,25] and/or
the presence of an inductively electron-withdrawing nitro group at position-5 of the isatin scaffold [24]
played an important role in the inducement or enhancement of different activities. Furthermore, it has
been reported by some other workers that certain N4-aryl substituted 5-nitroisatin-3-thiosemicabazones
exhibit more cytotoxic activity than their alkyl and alkenyl counterparts [26]. Also, N4-aryl substituted
5-bromoisatin-3-thiosemicabazones have been found to show favourable cytotoxicity [27]. In view
of this, it was envisaged that the combined effect of substitution of different inductively
electron-attracting groups at position-5 of the isatin part and the attachment of a variety of aryl
substituents to N4 of the thiosemicarbazone moiety would result in increased toxic activity. Thus, the
present work to synthesize some title thiosemicarbazones and screen them for their toxicity potential
by a brine shrimp (Artemia salina) lethality bioassay was accomplished. This work describes the
effects of the nature of aryl groups (modified by placement of one, two, or three substituents about
the phenyl ring) attached to N4 of the thiosemicarbazone moiety as well as the presence of
trifluoromethoxy substituent at position-5 of the isatin scaffold on the toxicity potential of these
compounds.
2. Results and Discussion
This study illustrates the synthesis and in vitro determination of the toxic effects of nineteen new
and two previously reported [15] N4-aryl substituted 5-trifluoromethoxyisatin-3-thiosemicarbazones
3b-3d, 3f-3u and 3a, 3e, respectively.
2.1. Chemistry
5-Trifluoromethoxyisatin 1 was reacted with appropriate N-substituted thiosemicarbazides 2 in
aqueous ethanol (50%) containing a catalytic amount of glacial acetic acid to give the corresponding
5-trifluoromethoxyisatin-3-thiosemicarbazones 3a-3u (Scheme 1) in moderate to excellent yields
(50–93%). The structures of all the synthesized compounds were confirmed by means of their
1
analytical (CHN) and spectral (IR, H-NMR, EIMS) data. Satisfactory elemental analysis (±0.4% of

Molecules 2011, 16
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theoretical values) was obtained for all compounds, except where noted otherwise. The IR spectra of
3a-3u showed the absorption bands of NH stretching in the 3354–3200 and 3196–3055 cm−1 regions.
The absorption bands of lactam C=O, azomethine C=N and thioamide C=S stretchings appeared in the
1705–1692, 1629–1564 and 1182–1143 cm−1 regions, respectively [26,28-30]. The 1H-NMR spectra of
3a-3u displayed three separate singlets at δ 10.45–11.07, δ 11.39–11.48 and δ 12.58–12.81 attributed
to thiosemicarbazone N4-H, indole NH and thiosemicarbazone N2-H, respectively [26,28,31,32]. The
electron impact mass spectra (EIMS) of the synthesized thiosemicarbazones demonstrated molecular
ions of different intensities, confirming their molecular weights. Compounds 3r and 3u did not display
molecular ion peaks in their mass spectra; however, the fragments corresponding to thiosemicarbazone
part of the molecules, formed by N-N and NH-CS bond rupture, confirmed their structures. X-ray
structures of two representative examples 3k and 3t were determined in order to confirm the assigned
structures and establish conformations of the synthesized thiosemicarbazones 3a-3u. Relevant crystal
data and details of structural elucidation have been reported elsewhere [33,34].
Scheme 1. The synthetic route to the title compounds 3a-3u.
NNHCSNHR
O
RNHCSNHNH2
F3CO
F3CO
2
O
O
N
H
3a-3u
50% EtOH(aq.)-AcOH
Reflux, 2h
N
H
1
3a R = C6H5
3l R = 4-F3COC6H4
3b R = 4-C2H5C6H4
3c R = 2-FC6H4
3d R = 3-FC6H4
3e R = 4-FC6H4
3f R = 4-ClC6H4
3g R = 4-BrC6H4
3h R = 4-IC6H4
3i R = 2-F3CC6H4
3j R = 3-F3CC6H4
3k R = 4-F3CC6H4
3m R = 2,4-(CH3O)2C6H3
3n R = 2,4-F2C6H3
3o R = 2,6-F2C6H3
3p R = 3,5-F2C6H3
3q R = 2-F-4-BrC6H3
3r R = 2,4-Cl2C6H3
3s R = 3,4-Cl2C6H3
3t R = 2-CH3-5-ClC6H3
3u R = 2,4,6-Cl3C6H2
2.2. Toxicity
All the synthesized thiosemicarbazones 3a-3u were screened for toxic activity by a brine shrimp
(Artemia salina) lethality bioassay. The compound 2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-
phenyl-1-hydrazinecarbothioamide, whose synthesis has been reported elsewhere [21], served as
reference compound (without a substituent in the isatin part as well as on the phenyl ring) to evaluate
the effects of substituents about the isatin scaffold as well as the phenyl ring attached to N4 of the
thiosemicarbazone moiety of the test compounds on their toxicity potential. The results given in the
Table revealed that substitution of trifluoromethoxy group alone in the isatin scaffold or in
combination with some other functions on the phenyl ring resulted into either induction or
enhancement of toxic activity in certain cases, compared with the reference compound. To the
contrary, in some other cases, such a combination caused either elimination or reduction in the activity.
This inference receives support from the results obtained by us in the earlier studies [22,23,25]. For

Molecules 2011, 16
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example, compound 3a possessing inductively electron-attracting trifluoromethoxy substituent at
position-5 of the isatin scaffold exhibited promising toxic activity (LD50 = 4.49 × 10−5 M), whereas the
corresponding compound i.e., the reference point having no trifluoromethoxy group in the isatin
moiety, was found to be inactive (LD50 = >3.38 × 10−4 M) [22]. Similarly, compound 3k having
trifluoromethyl substituent at position-4 of the phenyl ring was found to display enhanced toxicity
(LD50 = 1.11 × 10−5 M) when compared with the corresponding compound with no trifluoromethoxy
function at position-5 of the isatin scaffold, giving a LD50 value of 1.17 × 10−4 M [23]. Also,
compounds 3n and 3o possessing fluoro groups at positions-2,4 and -2,6 of the phenyl ring exhibited
increased toxic activity (LD50 = 1.43 × 10−5 M and 1.34 × 10−5 M, respectively) in comparison to the
corresponding compounds having no trifluoromethoxy substituent at position-5 of the isatin part,
giving the same LD50 values of 2.00 × 10−5 M. Much more pronounced enhancement was observed in
the case of 3k (LD50 = 1.17 × 10−4 M →1.11 × 10−5 M). However, compound 3l with trifluoromethoxy
group at position-4 of the phenyl ring attached to N4 of the thiosemicarbazone moiety and the
corresponding compound possessing no such substituent at position-5 of the isatin scaffold displayed
almost the same activity (LD50 = 1.81 × 10−5 M and 1.80 × 10−5 M, respectively) [23] indicating that
the introduction of trifluoromethoxy group at position-5 of the isatin scaffold did not significantly
affect toxicity potential of the compound having trifluoromethyl substituent at position-4 of the phenyl
ring. On the contrary, compounds 3b-3d, 3h, 3r and 3u with 4-ethyl, 2-fluoro, 3-fluoro, 4-iodo,
2,4-dichloro and 2,4,6-trichloro substituents about the phenyl ring showed almost no toxic effects
(LD50 = >2.45 × 10−4 M, >2.51 × 10−4 M, >2.51 × 10−4 M, >1.98 × 10−4 M, >2.23 × 10−4 M and
>2.07 × 10−4 M, respectively), whereas the corresponding compounds without trifluoromethoxy group
at position-5 of the isatin scaffold gave LD50 values of 5.10 × 10−5 M, 3.10 × 10−5 M, 4.60 × 10−5 M,
1.26 × 10−4 M, 1.20 × 10−4 M and 1.10 × 10−5 M, respectively [23,25]. Similarly, compounds 3i, 3j and
3p with 2-trifluoromethyl, 3-trifluoromethyl and 3,5-difluoro substituents about the phenyl ring
showed reduced toxic activity (LD50 = 1.80 × 10−4 M, 1.70 × 10−4 M and 4.32 × 10−5 M, respectively)
in comparison to the corresponding compounds (without trifluoromethoxy group at position-5 of the
isatin moiety), which gave LD50 values of 1.36 × 10−4 M, 2.20 × 10−5 M and 2.10 × 10−5 M [23].
Relatively, much pronounced reduction in the toxic activity was observed in the case of 3j
(LD50 = 2.20 × 10−5 M → 1.70 × 10−4 M). This showed that the simultaneous presence 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 toxicity potential. However,
compared with monofluoro-substituted compounds 3c and 3d, compound 3e having fluoro substituent
at position-4 of the phenyl ring displayed promising activity (LD50 = 3.72 × 10−5 M) in the present
assay. Similarly, compared with compound 3c, the dihalogeno-substituted compound 3q possessing
fluoro and bromo substituents at positions-2 and -4 of the phenyl ring, respectively, exhibited induced
toxic activity (LD50 = 1.60 × 10−4 M) in the present assay.
On the whole, out of twenty one compounds tested for toxic activity, ten i.e., 3a, 3e, 3i-3l and
3n-3q proved to be active, exhibiting promising toxicity (LD50 = 1.11 × 10−5 M − 1.80 × 10−4 M)
against Artemia salina. The remaining compounds i.e., 3b-3d, 3f-3h, 3m and 3r-3u gave LD50 values
ranging from >1.98 × 10−4 M to >2.51 × 10−4 M in the present assay and, therefore, can be considered
as inactive. Compound 3k having an inductively electron-attracting trifluoromethyl substituent at
position-4 of the phenyl ring proved to be the most potent toxicant, as it showed exciting activity

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with a LD50 value of 1.11 × 10−5 M, which was even better than the potent anticancer drug etoposide
(LD50 = 1.27 × 10−5 M) used as a reference standard in the present assay, and thus seems a promising
lead compound for further studies. The next potent compounds were found to be 3n and 3o with fluoro
groups at positions-2,4 and -2,6 of the phenyl ring, showing toxic activity with LD50 values of
1.43 × 10−5 M and 1.34 × 10−5 M, respectively. These preliminary results indicate that structural
modifications may lead to the development of novel compounds with improved toxicity.
Table. Brine shrimp (Artemia salina) bioassay of compounds 3a-3u.
Compounds
LD50 (M)
4.49 × 10−5
>2.45 × 10−4
>2.51 × 10−4
>2.51 × 10−4
3.72 × 10−5
>2.42 × 10−4
>2.18 × 10−4
>1.98 × 10−4
1.80 × 10−4
1.70 × 10−4
1.11 × 10−5
1.81 × 10−5
>2.27 × 10−4
1.43 × 10−5
1.34 × 10−5
4.32 × 10−5
1.60 × 10−4
>2.23 × 10−4
>2.23 × 10−4
>2.34 × 10−4
>2.07 × 10−4
>3.38 × 10−4
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
2-(2-Oxo-1,2-dihydro-3H-indol-3-ylidene)-N-
phenyl-1-hydrazinecarbothioamide *
* [21,22].
3. Experimental
3.1. General
Melting points were taken on a Fisher-Johns melting point apparatus and are uncorrected. Elemental
analyses were performed on a Leco CHNS-9320 (USA) elemental analyzer. Infrared spectra (KBr
1
discs) were run on Shimadzu Prestige-21 FT-IR spectrometer. The H-NMR spectra were recorded in
DMSO-d6 on Bruker (Rhenistetten-Forchheim, Germany) AM 300 spectrometer, operating at
1
300 MHz and using TMS as an internal standard. H chemical shifts are reported in δ/ppm and
coupling constants in Hz. The electron impact mass spectra (EIMS) were determined with JEOL
MSRoute mass spectrometer. The progress of the reaction and purity of the products were checked on

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TLC plates coated with Merck silica gel 60 GF254, and the spots were visualized under ultraviolet light
at 254/366 nm and/or spraying with iodine vapours. In vitro biological evaluation of the synthesized
compounds was done at Panjwani Center for Molecular Medicine and Drug Research, H.E.J. Research
Institute of Chemistry, University of Karachi, Pakistan.
3.2. Synthesis
3.2.1. General Procedure for the Preparation of 5-Ttrifluoromethoxyisatin-thiosemicarbazones 3a-3u
To a solution of 5-trifluoromethoxyisatin (2.5 mmol) in 50% aqueous ethanol (10 mL) containing a
catalytic amount of glacial acetic acid was added the appropriate thiosemicarbazide (2.5 mmol)
dissolved in ethanol (10 mL) 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-3u in
pure form.
N-Phenyl-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3a). Yield 93% as yellow fluffy crystals, mp: 230–232 °C (229–232 °C, lit. [15]). IR: 3319, 3178,
1
3119 (NH stretching), 1695 (C=O), 1629 (C=N), 1143 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d,
J = 8.4 Hz, 1H, indole C7-H), 7.30 (t, J = 7.5 Hz, 1H, phenyl C4-H), 7.38 (dd, J = 8.4, 1.8 Hz, 1H,
indole C6-H), 7.45 (t, J = 7.5 Hz, 2H, phenyl C3-H, C5-H), 7.59 (d, J = 8.1 Hz, 2H, phenyl C2-H,
C6-H), 7.81 (d, J = 1.8 Hz, 1H, indole C4-H), 10.90 (s, 1H, CS-NH ), 11.41 (s, 1H, indole NH), 12.66
(s, 1H, N-NH). MS, m/z (rel. int.): 380 (M+, 42), 352 (100), 287 (13), 263 (10), 245 (66), 229 (15), 217
(18), 216 (20), 202 (19), 201 (22), 188 (52), 159 (16), 150 (8), 135 (71), 118 (24), 104 (6), 93 (38).
Anal calcd. for C16H11F3N4O2S: C, 50.53, H, 2.92, N, 14.73; found: C, 50.47, H, 2.92, N, 15.01.
N-(4-Ethylphenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3b). Yield 83% as yellow fluffy crystals, mp: 258 °C. IR: 3200, 3150, 3067 (NH stretching), 1701
(C=O), 1597 (C=N), 1150 (C=S) cm−1. 1H-NMR (DMSO-d6): δ 1.21 (t, J = 7.5 Hz, 3H, CH3), 2.63 (q,
J = 7.5 Hz, 2H, CH2), 7.03 (d, J = 8.4 Hz, 1H, indole C7-H), 7.27 (d, J = 8.1 Hz, 2H, phenyl C2-H,
C6-H), 7.37 (d, J = 8.4 Hz, 1H, indole C6-H), 7.48 (d, J = 8.4 Hz, 2H, phenyl C3-H, C5-H), 7.81 (s, 1H,
indole C4-H), 10.85 (s, 1H, CS-NH), 11.42 (s, 1H, indole NH), 12.63 (s, 1H, N-NH). MS, m/z (rel.
int.): 408 (M+, 34), 380 (100), 365 (5), 311 (7), 245 (11), 230 (13), 217 (7), 216 (6), 202 (10), 188 (7),
163 (11), 131 (19), 106 (24), 77 (5). Anal calcd. for C18H15F3N4O2S: C, 52.94, H, 3.70, N, 13.72;
found: C, 52.85, H, 3.69, N, 13.69.
N-(2-Fluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3c). Yield 76% as yellow powder, mp: 225 °C. IR: 3289, 3264, 3073 (NH stretching), 1703 (C=O),
1
1607 (C=N), 1155 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d, J = 8.7 Hz, 1H, indole C7-H),
7.27–7.52 (m, 5H, indole C6-H, phenyl C3-H, C4-H, C5-H, C6-H), 7.73 (s, 1H, indole C4-H), 10.81
(s, 1H, CS-NH ), 11.43 (s, 1H, indole NH), 12.71 (s, 1H, N-NH). MS, m/z (rel. int.): 398 (M+, 54), 371
(28), 370 (100), 351 (2), 301 (12), 263 (8), 245 (12), 234 (11), 216 (11), 188 (13), 154 (16), 134 (15),

Molecules 2011, 16
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111 (11), 83 (7), 69 (7), 44 (3). Anal calcd. for C16H10F4N4O2S: C, 48.24, H, 2.53, N, 14.07; found: C,
48.05, H 2.52, N 14.04.
N-(3-Fluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3d). Yield 81% as yellow powder, mp: 238 °C. IR: 3289, 3264, 3073(NH stretching), 1701 (C=O),
1
1597 (C=N), 1161 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.03 (d, J = 8.4 Hz, 1H, indole C7-H),
7.11–7.18 (m, 1H, phenyl C4-H), 7.38 (dd, J = 8.4, 1.5 Hz, 1H, indole C6-H), 7.49 (t, J = 9.9 Hz, 2H,
phenyl C6-H, C5-H), 7.61 (d, J = 10.8 Hz, 1H, phenyl C2-H), 7.80 (s, 1H, indole C4-H), 10.94 (s, 1H,
CS-NH), 11.45 (s, 1H, indole NH), 12.72 (s, 1H, N-NH). MS, m/z (rel. int.): 398 (M+, 9), 370 (27), 338
(7), 312 (3), 284 (46), 269 (3), 256 (45), 241 (27), 230 (31), 213 (29), 202 (26), 185 (39), 171 (22), 157
(25), 136 (46), 129 (65), 111 (45), 85 (36), 73 (100), 69 (36), 61 (25), 60 (73), 57 (76). Anal calcd. for
C16H10F4N4O2S: C, 48.24, H, 2.53, N, 14.07; found: C, 48.17, H, 2.53, N, 14.05.
N-(4-Fluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3e). Yield 76% as yellow fluffy crystals, mp: 224–226 °C (224–227 °C, lit. [15]). IR: 3320, 3197 (NH
1
stretching), 1692 (C=O), 1600 (C=N), 1155 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.03 (d, J = 8.7 Hz,
1H, indole C7-H), 7.26–7.32 (m, 2H, phenyl C2-H, C6-H), 7.37 (dd, J = 8.7, 2.1 Hz, 1H, indole C6-H),
7.57–7.61 (m, 2H, phenyl C3-H, C5-H), 7.78 (s, 1H, indole C4-H), 10.91 (s, 1H, CS-NH), 11.43 (s, 1H,
N-NH), 12.66 (s, 1H, indole NH). MS, m/z (rel. int.): 398 (M+, 24), 370 (100), 364 (28), 338 (12), 301
(9), 287 (7), 263 (8), 243 (63), 230 (45), 215 (39), 202 (31), 187 (30), 136 (81), 133 (31), 109 (27), 95
(15), 83 (12), 69 (27), 52 (5), 44 (25). Anal calcd. for C16H10F4N4O2S: C, 48.24, H, 2.53, N, 14.07;
found: C, 48.10, H, 2.52, N, 14.12.
N-(4-Chlorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3f). Yield 75% as shinning yellow powder, mp: 235 °C. IR: 3258, 3107, 3080 (NH stretching), 1701
(C=O), 1589 (C=N), 1150 (C=S) cm−1. 1H-NMR (DMSO-d6): δ 7.03 (d, J = 8.1 Hz, 1H, indole C7-H),
7.38 (d, J = 7.5 Hz, 1H, indole C6-H), 7.51 (d, J = 7.5 Hz, 2H, phenyl C2-H, C6-H), 7.65 (d, J = 7.2 Hz,
2H, phenyl C3-H, C5-H), 7.78 (s, 1H, indole C4-H), 10.93 (s, 1H, CS-NH), 11.44 (s, 1H, indole NH),
12.70 (s, 1H, N-NH). MS, m/z (rel. int.): 416/414 (M+, 20/53), 388/386 (39/100), 287 (32), 247/245
(8/13), 230/228 (18/15), 217/215 (8/7), 198/196 (5/5), 171 (17), 152 (12), 127 (31), 92 (4). Anal calcd.
for C16H10ClF3N4O2S: C, 46.33, H, 2.43, N, 13.51; found: C, 46.34, H, 2.43, N, 13.51.
N-(4-Bromophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3g). Yield 70% as yellow powder, mp: 240 °C. IR: 3281, 3088 (NH stretching), 1699 (C=O), 1600
1
(C=N), 1148 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d, J = 8.7 Hz, 1H, indole C7-H), 7.39 (d,
J = 7.8 Hz, 1H, indole C6-H), 7.60 (d, J = 9.0 Hz, 2H, phenyl C2-H, C6-H), 7.64 (d, J = 8.7 Hz, 2H,
phenyl C3-H, C5-H), 7.79 (s, 1H, indole C4-H), 10.92 (s, 1H, CS-NH), 11.44 (s, 1H, indole NH), 12.70
(s, 1H, N-NH). MS, m/z (rel. int.): 460/458 (M+, 5/5), 432/430 (7/5), 354 (5), 287 (45), 262 (5), 245
(36), 230/228 (5/5), 215/213 (20/20), 199/197 (39/28), 173/171 (100/96), 148 (9), 134 (4), 92 (13), 65
(19). Anal calcd. for C16H10BrF3N4O2S: C, 41.85, H, 2.19, N, 12.20; found: C, 42.00, H, 2.19,
N, 12.19.

Molecules 2011, 16
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N-(4-Iodophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-hydrazinecarbothioamide
(3h). Yield 80% as yellow powder, mp: 246 °C. IR: 3291, 3196, 3080 (NH stretching), 1703 (C=O),
1
1591 (C=N), 1146 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.03 (d, J = 8.7 Hz, 1H, indole C7-H), 7.38
(d, J = 8.1 Hz, 1H, indole C6-H), 7.45 (d, J = 8.4 Hz, 2H, phenyl C2-H, C6-H), 7.79 (d, J = 8.1 Hz, 3H,
indole C4-H , phenyl C3-H, C5-H), 10.89 (s, 1H, CS-NH ), 11.44 (s, 1H, indole NH), 12.70 (s, 1H,
N-NH). MS, m/z (rel. int.): 506 (M+, 32), 478 (69), 352 (5), 287 (100), 261 (95), 254 (30), 245 (23),
230 (56), 229 (49), 228 (49), 220 (35), 219 (100), 202 (39), 159 (7), 127 (26), 92 (72), 64 (24). Anal
calcd. for C16H10F3IN4O2S: C, 37.96, H, 1.99, N, 11.07; found: C, 37.92, H, 1.99, N, 11.04.
2-[2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-N-[2-(trifluoromethyl)phenyl]-
1-hydrazinecarbothioamide (3i). Yield 87% as yellow fluffy crystals, mp: 234 °C. IR: 3346, 3188,
1
3171(NH stretching), 1695 (C=O), 1625 (C=N), 1182 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.03 (d,
J = 8.4 Hz, 1H, indole C7-H), 7.38 (dd, J = 8.4, 1.8 Hz, 1H, indole C6-H), 7.57 (d, J = 8.1 Hz, 1H,
phenyl C4-H), 7.63 (d, J = 7.5 Hz, 1H, phenyl C6-H), 7.72 (s, 1H, indole C4-H), 7.77-7.86 (m, 2H,
phenyl C3-H, C5-H), 10.89 (s, 1H, CS-NH), 11.43 (s, 1H, indole NH), 12.71 (s, 1H, N-NH). MS, m/z
(rel. int.): 448 (M+, 3), 420 (23), 414 (38), 388 (4), 345 (9), 311 (4), 243 (63), 228 (73), 215 (58), 202
(36), 187 (76), 186 (89), 167 (33), 166 (100), 159 (38), 145 (16), 139 (13), 133 (35), 128 (21), 114
(19), 83 (46), 78 (12), 69 (36), 44 (53). Anal calcd. for C17H10F6N4O2S: C, 45.54, H, 2.25, N, 12.50;
found: C, 45.53, H, 2.24, N, 12.50.
2-[2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-N-[3-(trifluoromethyl)phenyl]-1-
hydrazinecarbothioamide (3j). Yield 79% as yellow powder, mp: 220 °C. IR: 3310, 3196, 3063 (NH
1
stretching), 1699 (C=O), 1600 (C=N), 1161 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d, J = 8.4 Hz,
1H, indole C7-H), 7.40 (dd, J = 8.7, 1.8 Hz, 1H, indole C6-H), 7.64–7.72 (m, 2H, phenyl C5-H, C6-H),
7.78 (s, 1H, indole C4-H), 7.99 (d, J = 7.2 Hz, 1H, phenyl C4-H), 8.04 (s, 1H, phenyl C2-H), 11.06
(s, 1H, CS-NH), 11.47 (s, 1H, indole NH), 12.76 (s, 1H, N-NH). MS, m/z (rel. int.): 448 (M+, 3), 420
(12), 414 (33), 395 (8), 388 (23), 372 (4), 346 (4), 345 (4), 330 (16), 277 (3), 243 (49), 230 (40), 228
(89), 216 (13), 215 (60), 203 (41), 202 (47), 188 (33), 187 (96), 186 (100), 167 (25), 161 (23), 160
(31), 159 (98), 145 (66), 139 (20), 133 (57), 109 (14), 105 (13), 95 (14), 85 (31), 83 (46), 82 (17), 80
(18), 69 (44), 63 (16), 44 (79). Anal calcd. for C17H10F6N4O2S: C, 45.54, H, 2.25, N, 12.50; found: C,
45.50, H, 2.24, N, 12.48.
2-[2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-N-[4-(trifluoromethyl)phenyl]-1-
hydrazinecarbothioamide (3k). Yield 75% as yellow powder, mp: 238–240 °C. IR: 3318, 3161, 3130
1
(NH stretching), 1701 (C=O), 1587 (C=N), 1167 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d,
J = 8.7 Hz, 1H, indole C7-H), 7.39 (dd, J = 9.9 Hz, 1H, indole C6-H), 7.82 (d, J = 9.3 Hz, 3H, indole
C4-H, phenyl C2-H, C6-H ), 7.93 (d, J = 8.4 Hz, 2H, phenyl C3-H, C5-H), 11.07 (s, 1H, CS-NH), 11.47
(s, 1H, N-NH), 12.78 (s, 1H, indole NH). MS, m/z (rel. int.): 448 (M+, 24), 421 (24), 420 (100), 414
(18), 388 (26), 372 (2), 351 (10), 331 (3), 287 (3), 264 (7), 243 (31), 230 (71), 215 (31), 202 (70), 188
(27), 187 (50), 186 (84), 167 (36), 161 (25), 160 (15), 159 (39), 145 (50), 133 (80), 120 (7), 105 (22),
95 (13), 78 (15), 69 (43), 44 (6). Anal calcd. for C17H10F6N4O2S: C, 45.54, H, 2.25, N, 12.50; found: C,
45.53, H, 2.23, N, 12.49.

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2-[2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-N-[4-(trifluoromethoxy)phenyl]-1-
hydrazinecarbothioamide (3l). Yield 72% as orange fluffy crystals, mp: 228–230 °C. IR: 3308, 3100,
1
3055 (NH stretching), 1699 (C=O), 1600 (C=N), 1159 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d,
J = 8.4 Hz, 1H, indole C7-H), 7.38 (dd, J = 8.4, 1.8 Hz, 1H, indole C6-H), 7.45 (d, J = 8.4 Hz, 2H,
phenyl C2-H, C6-H), 7.71–7.77 (m, 3H, phenyl C3-H, C5-H, indole C4-H), 7.77 (d, J = 6.3 Hz, 1H,
indole C4-H), 10.97 (s, 1H, CS-NH), 11.44 (s, 1H, indole NH), 12.71 (s, 1H, N-NH). MS, m/z (rel.
int.): 464 (M+, 12), 436 (88), 430 (28), 404 (21), 388 (2), 367 (10), 345 (4), 287 (5), 245 (18), 243 (55),
230 (56), 228 (60), 220 (13), 219 (23), 215 (46), 203 (29), 202 (100), 188 (27), 187 (44), 177 (18), 160
(12), 159 (24), 134 (15), 133 (100), 131 (11), 108 (13), 105 (32), 95 (10), 86 (7), 78 (9), 69 (53), 63
(10), 44 (33). Anal calcd. for C17H10F6N4O3S: C, 43.97, H, 2.17, N, 12.07; found: C, 44.10, H, 2.17,
N, 12.09.
N-(2,4-Dimethoxyphenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3m). Yield 89% as orange fluffy crystals, mp: 248 °C. IR: 3269, 3200, 3100
1
(NH stretching), 1705 (C=O), 1608 (C=N), 1165 (C=S) cm−1. H-NMR (DMSO-d6): δ 3.80 (s, 6H,
OCH3), 6.58 (dd, J = 8.7, 2.4 Hz, 1H, phenyl C5-H), 6.69 (d, J = 2.4 Hz, 1H, phenyl C3-H), 7.03 (d,
J = 8.7 Hz, 1H, indole C7-H), 7.30–7.38 (m, 2H, indole C6-H, phenyl C6-H), 7.76 (s, 1H, indole C4-H),
10.45 (s, 1H, CS-NH), 11.39 (s, 1H, indole NH), 12.58 (s, 1H, N-NH). MS, m/z (rel. int.): 440 (M+,
20), 426 (5), 412 (44), 381 (3), 343 (4), 287 (8), 245 (76), 228 (33), 217 (11), 195 (100), 188 (40), 180
(30), 152 (41), 138 (27), 120 (11), 95 (11), 69 (27). Anal calcd. for C18H15F3N4O4S: C, 49.09, H, 3.43,
N, 12.72; found: C, 49.13, H, 3.43, N, 12.74.
N-(2,4-Difluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3n). Yield 81% as yellow fluffy crystals, mp: 228–230 °C. IR: 3316, 3206,
1
3084 (NH stretching), 1692 (C=O), 1611 (C=N), 1155 (C=S) cm-1. H-NMR (DMSO-d6): δ 7.04 (d,
J = 8.7 Hz, 1H, indole C7-H), 7.19 (td, J = 8.4,1.2 Hz, 1H, phenyl C5-H), 7.37–7.58 (m, 3H, indole C6-H,
phenyl C3-H, C6-H), 7.71 (s, 1H, indole C4-H), 10.77 (s, 1H, CS-NH), 11.44 (s, 1H, indole NH), 12.73
(s, 1H, N-NH). MS, m/z (rel. int.): 416 (M+, 18), 389 (20), 388 (100), 382 (36), 356 (12), 337 (2), 319
(13), 297 (2), 287 (6), 263 (6), 243 (45), 230 (38), 229 (13), 228 (41), 215 (33), 202 (33), 188 (24), 187
(28), 172 (11), 159 (18), 155 (12), 154 (85), 133 (32), 129 (20), 101(16), 82 (4), 69 (24), 44 (15). Anal.
calcd. for C16H9F5N4O2S: C, 46.16, H, 2.18, N, 13.46; found: C, 46.11, H, 2.17, N, 13.43.
N-(2,6-Difluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3o). Yield 70% as yellow fluffy crystals, mp: 248–250 °C. IR: 3333, 3256,
1
3100 (NH stretching), 1694 (C=O), 1610 (C=N), 1165 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d,
J = 8.4 Hz, 1H, indole C7-H), 7.28 (t, J = 8.1 Hz, 2H, phenyl C3-H, C5-H), 7.39 (dd, J = 8.4, 1.8 Hz,
1H, indole C6-H), 7.48–7.57 (m, 1H, phenyl C4-H), 7.69 (s, 1H, indole C4-H), 10.66 (s, 1H, CS-NH),
11.44 (s, 1H, indole NH), 12.81 (s, 1H, N-NH). MS, m/z (rel. int.): 416 (M+, 13), 389 (20), 388 (99),
382 (44), 356 (7), 337 (3), 319 (10), 287 (9), 267 (7), 243 (48), 230 (39), 229 (15), 228 (44), 216 (14),
215 (39), 202 (36), 188 (24), 187 (32), 172 (11), 159 (21), 154 (100), 134 (15), 133 (38), 127 (23), 101
(13), 85 (11), 83 (16), 69 (31), 44 (4). Anal calcd. for C16H9F5N4O2S: C, 46.16, H, 2.18, N, 13.46;
found: C, 46.17, H, 2.19, N, 13.45.

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N-(3,5-Difluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3p). Yield 70% as yellow powder, mp: 236–238 °C. IR: 3308, 3262, 3102
1
(NH stretching), 1703 (C=O), 1600 (C=N), 1155 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.03 (d,
J = 8.7 Hz, 1H, indole C7-H), 7.19 (tt, J = 9.3, 2.4 Hz, 1H, phenyl C4-H), 7.39 (dd, J = 8.7, 1.8 Hz, 1H,
indole C6-H), 7.56 (dd, J = 9.0, 2.1 Hz, 2H, phenyl C2-H, C6-H), 7.78 (d, J = 1.5 Hz, 1H, indole C4-H),
10.95 (s, 1H, CS-NH), 11.47 (s, 1H, indole NH), 12.79 (s, 1H, N-NH). MS, m/z (rel. int.): 416 (M+, 7),
388 (38), 382 (19), 356 (14), 319 (5), 287 (12), 287 (12), 267 (6), 245 (14), 243 (38), 230 (58), 228
(60), 215 (32), 202 (53), 188 (24), 187 (32), 171 (14), 159 (30), 155 (18), 154 (100), 133 (60), 127
(65), 113 (20), 101 (16), 83 (37), 69 (28), 44 (11). Anal calcd. for C16H9F5N4O2S: C, 46.16, H, 2.18, N,
13.46, found: C, 46.14, H, 2.18, N, 13.42.
N-(4-Bromo-2-fluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3q). Yield 74% as yellow powder, mp: 238–240 °C. IR: 3300, 3173, 3129
1
(NH stretching), 1701 (C=O), 1580 (C=N), 1144 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d,
J = 8.4 Hz, 1H, indole C7-H), 7.39 (d, J = 8.7 Hz, 1H, indole C6-H), 7.45–7.54 (m, 2H, phenyl C5-H,
C6-H), 7.70–7.76 (m, 2H, indole C4-H, phenyl C3-H ), 10.79 (s, 1H, CS-NH), 11.44 (s, 1H, indole
NH), 12.76 (s, 1H, N-NH). MS, m/z (rel. int.): 478/476 (M+, 7/8), 450/448 (55/51), 444/442 (16/15),
418/416 (9/9), 381/379 (5/5), 363 (3), 287 (9), 245/243 (19/47), 243 (47), 233/231 (18/24), 230/228
(60/85), 217/215 (28/58), 216/214 (100/100), 203/201 (7/20), 191 (18) 189/187 (28/30), 159 (33),
135/133 (18/60), 131 (16), 115 (15), 108 (43), 83 (15), 69 (33), 44 (10). Anal calcd. for
C16H9BrF4N4O2S: C, 40.27, H, 1.90, N, 11.74, found: C, 40.26, H, 1.90, N, 11.70.
N-(2,4-Dichlorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3r). Yield 50% as shinning yellow powder, mp: 253–255 °C. IR: 3258,
3184, 3080 (NH stretching), 1701 (C=O), 1580 (C=N), 1155 (C=S) cm−1. 1H-NMR (DMSO-d6): δ 7.04
(d, J = 8.1 Hz, 1H, indole C7-H), 7.39 (d, J = 7.5 Hz, 1H phenyl C6-H), 7.55 (s, 2H, phenyl C5-H,
C6-H), 7.72 (s, 1H, phenyl C3-H), 7.82 (s, 1H, indole C4-H), 10.91 (s, 1H, CS-NH), 11.44 (s, 1H,
indole NH), 12.73 (s, 1H, N-NH). MS, m/z (rel. int.): 422 (7), 420 (10), 417 (5), 416 (17), 415 (64),
414 (30), 413 (100), 287 (7), 228 (6), 186 (13), 161 (13), 78 (5). Anal calcd. for C16H9Cl2F3N4O2S: C,
42.78, H, 2.02, N, 12.47; found: C, 42.75, H, 2.01, N, 12.44.
N-(3,4-Dichlorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3s). Yield 82% as yellow powder, mp: 245 °C. IR: 3318, 3177, 3080 (NH
1
stretching), 1697 (C=O), 1600 (C=N), 1153 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d, J = 8.4 Hz,
1H, indole C7-H), 7.39 (d, J = 8.1 Hz, 1H, indole C6-H), 7.71 (s, 2H, phenyl C5-H, C6-H) 7.77 (s, 1H,
phenyl C2-H), 8.01 (s, 1H, indole C4-H), 10.97 (s, 1H, CS-NH), 11.46 (s, 1H, indole NH), 12.76 (s,
1H, N-NH). MS, m/z (rel. int.): 450/448 (M+, 16/ 27), 422 (87), 420 (100), 351 (11), 287 (78), 263 (9),
245 (53), 230/228 (11/18), 229 (43), 206/204 (7/12), 201 (52), 188 (10), 161 (98), 133 (12), 120 (10),
99 (13), 77 (6). Anal calcd. for C16H9Cl2F3N4O2S: C, 42.78, H, 2.02, N, 12.47; found: C, 42.79, H,
2.02, N, 12.49.
N-(5-Chloro-2-methylphenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-1-
hydrazinecarbothioamide (3t). Yield 64% as orange powder, mp: 240 °C. IR: 3302, 3233, 3082 (NH

Molecules 2011, 16
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1
stretching), 1701 (C=O), 1584 (C=N), 1146 (C=S) cm−1. H-NMR (DMSO-d6): δ 2.23 (s, 3H, CH3),
7.03 (d, J = 8.7 Hz, 1H, indole C7-H), 7.37–7.39 (m, 4H, indole C6-H, phenyl C3-H, C4-H, C6-H), 7.73
(s, 1H, indole C4-H), 10.84 (s, 1H, CS-NH), 11.42 (s, 1H, indole NH), 12.66 (s, 1H, N-NH). MS, m/z
(rel. int.): 430/428 (M+, 9/22), 402 (47), 400 (100), 333/331 (4/9), 287 (4), 263 (6), 246 (6), 245 (33),
234 (7), 216 (12), 188 (20), 183 (15), 166 (7), 148 (21), 120 (7), 106 (7), 89 (8), 77 (7), 69 (18). Anal
calcd. for C17H12ClF3N4O2S: C, 47.62, H, 2.82, N, 13.07; found: C, 47.60, H, 2.83, N, 13.03.
2-[2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-N-(2,4,6-trichlorophenyl)-1-
hydrazinecarbothioamide (3u). Yield 79% as yellow powder, mp: 240 °C. IR: 3354, 3204, 3069 (NH
1
stretching), 1701 (C=O), 1564 (C=N), 1167 (C=S) cm−1. H-NMR (DMSO-d6): δ 7.04 (d, J = 8.1 Hz,
1H, indole C7-H), 7.39 (d, J = 7.2 Hz, 1H, indole C6-H), 7.69 (s, 2H, phenyl C3-H, C5-H), 7.88 (s, 1H,
indole C4-H), 10.94 (s, 1H, CS-NH), 11.44 (s, 1H, indole NH), 12.81 (s, 1H, N-NH). MS, m/z (rel.
int.): 456/454 (6/6), 451/449 (15/70), 448/446 (33/13), 447 (100), 432/430 (9/42), 402 (7), 385/383
(7/14), 287 (38), 262 (5), 245 (19), 239/237 (11/12), 199 (26), 197/195 (76/82), 69 (6). Anal calcd. for
C16H8Cl3F3N4O2S: C, 39.73, H, 1.67, N, 11.58; found: C, 39.82, H, 1.66, N, 11.56.
3.3. Bioassay of Toxic Activity
Brine shrimp (Artemia salina Leach) eggs were hatched in a shallow rectangular plastic dish
(22 × 32 cm) filled with artificial sea water, which was prepared with a commercial salt mixture
(Instant Ocean, Aquarium System, Inc., Mentor, OH, 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 two days, nauplii were collected by a pipette from the lighted side.
A sample of each test compound was prepared by dissolving 2 mg in 2 mL of methanol. From this
stock solution, 100, 10 and 1 µL 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 two
days, when shrimp larvae were ready, 1 mL of sea water and 10 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 [35,36]. Data were analyzed by a Finney computer programme to
determine the LD50 values [37].
4. Conclusions
We have demonstrated the potential of N4-aryl substituted 5-trifluoromethoxyisatin-3-
thiosemicarbazones to show toxic activity. Based on the preliminary data given in the Table and in
terms of further development and structure-activity relationship (SAR) studies, simultaneous
substitution of different substituents at position-5 of the isatin scaffold and on the phenyl ring attached
to N4 of the thiosemicarbazone moiety certainly warrants further investigation. Work in this regard
along with extended SAR studies will be reported in the near future.

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Acknowledgments
This work was supported by the Higher Education Commission (HEC), Pakistan (Project No.
20-873/R&D/07/452).
Conflict of Interest
The authors declare no conflict of interest.
References and Notes
1. da Silva, J.F.M.; Garden, S.J.; Pinto, A. da C. The chemistry of isatins: A review from 1975 to
1999. J. Braz. Chem. Soc. 2001, 12, 273-324 and references therein.
2. Pandeya, S.N.; Smitha, S.; Jyoti, M.; Sridhar, S.K. Biological activities of isatin and its
derivatives. Acta Pharm. 2005, 55, 27-46 and references therein.
3. Vine, K.L.; Matesic, L.; Locke, J.M.; Ranson, M.; Skropeta, D. Cytotoxic and anticancer
activities of isatin and its derivatives: A comprehensive review from 2000-2008. Anti-Cancer
Agents Med. Chem. 2009, 9, 397-414 and references therein.
4. Aboul-Fadl, T.; Bin-Jubair, F.A.S. Anti-tubercular activity of isatin derivatives. Int. J. Res.
Pharm. Sci. 2010, 1, 113-126 and references therein.
5. Chiyanzu, I.; Hansell, E.; Gut, J.; Rosenthal, P.J.; McKerrow, J.H.; Chibale, K. Synthesis and
evaluation of isatins and thiosemicarbazone derivatives against cruzain, falcipain-2 and rhodesain.
Bioorg. Med. Chem. Lett. 2003, 13, 3527-3530.
6. Chiyanzu, I.; Clarkson, C.; Smith, P.J.; Lehman, J.; Gut, J.; Rosenthal, P.J.; Chibale, K. Design,
synthesis and anti-plasmodial evaluation in vitro of new 4-aminoquinoline isatin derivatives.
Bioorg. Med. Chem. 2005, 13, 3249-3261.
7. Chen, L.R.; Wang, Y.C.; Lin, Y.W.; Chou, S.Y.; Chen, S.F.; Liu, L.T.; Wu, Y.T.; Kuo, C.J.;
Chen, T.S.; Juang, S.H. Synthesis and evaluation of isatin derivatives as effective SARS
coronavirus 3CL protease inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 3058-3062.
8. Bal, T.R.; Anand, B.; Yogeeswari, P.; Sriram, D. Synthesis and evaluation of anti-HIV activity of
isatin β-thiosemicarbazone derivatives. Bioorg. Med. Chem. Lett. 2005, 15, 4451-4455.
9. Pirrung, M.C.; Pansare, S.V.; Sarma, K.D.; Keith, K.A.; Kern, E.R. Combinatorial optimization of
isatin-β-thiosemicarbazones as anti-poxvirus agents. J. Med. Chem. 2005, 48, 3045-3050.
10. Patel, A.; Bari, S.; Talele, G.; Patel, J.; Sarangapani, M. Synthesis and antimicrobial activity of
some new isatin derivatives. Iran. J. Pharm. Res. 2006, 4, 249-254.
11. Beauchard, A.; Ferandin, Y.; Frere, S.; Lozach, O.; Blairvacq, M.; Meijer, L.; Thiery, V.; Besson, T.
Synthesis of novel 5-substituted indirubins as protein kinases inhibitors. Bioorg. Med. Chem.
2006, 14, 6434-6443.
12. Terzioglu, N.; Karali, N.; Gursoy, A.; Pannecouque, C.; Leysen, P.; Paeshuyse, J.; Neyts, J.;
de Clercq, E. Synthesis and primary antiviral activity evaluation of 3-hydrazono-5-nitro-2-indolinone
derivatives. ARKIVOC 2006, i, 109-118.

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13. Hyatt, J.L.; Moak, T.; Hatfield, M.J.; Tsurkan, L.; Edwards, C.C.; Wierdl, M.; Danks, M.K.;
Wadkins, R.M.; Potter, P.M. Selective inhibition of carboxylesterases by isatins, indole-2,3-diones.
J. Med. Chem. 2007, 50, 1876-1885.
14. Ravichandran, V.; Mohan, S.; Kumar, K.S. Synthesis and antimicrobial activity of Mannich bases
of isatin and its derivatives with 2-[(2,6-dichlorophenyl)amino]phenylacetic acid. ARKIVOC
2007, xiv, 51-57.
15. Guzel, O.; Karali, N.; Salman, A. Synthesis and antituberculosis activity of
5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazone derivatives. Bioorg. Med.
Chem. 2008, 16, 8976-8987.
16. Smitha, S.; Pandeya, S.N.; Stables. J.P.; Ganapathy, S. Anticonvulsant and sedative-hypnotic
activeties of N-acetyl/methyl isatin derivatives. Sci. Pharm. 2008, 76, 621-636.
17. Singh, U.K.; Pandeya, S.N.; Singh, A.; Srivastava, B.K.; Pandey, M. Synthesis and antimicrobial
activity of Schiff’s and N-Mannich bases of isatin and its derivatives with 4-amino-N-carbamimidoyl
benzene sulfonamide. Int. J. Pharm. Sci. Drug Res. 2010, 2, 151-154.
18. Banerjee, D.; Yogeeswari, P.; Bhat, P.; Thomas, A.; Srividya, M.; Sriram, D. Novel isatinyl
thiosemicarbazones derivatives as potential molecule to combat HIV-TB co-infection. Eur. J.
Med. Chem. 2011, 46, 106-121.
19. Quenelle, D.C.; Keith, K.A.; Kern, E.R. In vitro and in vivo evaluation of
isatin-β-thiosemicarbazone and marboran against vaccinia and cowpox virus infections. Antivir.
Res. 2006, 71, 24-30.
20. Hall, M.D.; Salam, N.K.; Hellawell, J.L.; Fales, H.M.; Kensler, C.B.; Ludwig, J.A.; Szakacs, G.;
Hibbs, D.E.; Gottesman, M.M. Synthesis, activity, and pharmacophore development for
isatin-β-thiosemicarbazones with selective activity toward multidrug-resistant cells. J. Med.
Chem. 2009, 52, 3191-3204.
21. Pervez, H.; Iqbal, M.S.; Tahir, M.Y.; Choudhary, M.I.; Khan, K.M. Synthesis of some
N4-substituted isatin-3-thiosemicarbazones. Nat. Prod. Res. 2007, 21, 1178-1186.
22. Pervez, H.; Iqbal, M.S.; Tahir, M.Y.; Nasim, F.H.; Choudhary, M.I.; Khan, K.M. In vitro
cytotoxic, antibacterial, antifungal and urease inhibitory activities of some N4-substituted
isatin-3-thiosemicarbazones. J. Enzyme Inhib. Med. Chem. 2008, 23, 848-854.
23. Pervez, H.; Chohan, Z.H.; Ramzan, M.; Nasim, F.H.; Khan, K.M. Synthesis and biological
evaluation of some new N4-substituted isatin-3-thiosemicarbazones. J. Enzyme Inhib. Med. Chem.
2009, 24, 437-446.
24. Pervez, H.; Manzoor, N.; Yaqub, M.; Khan, A.; Khan, K.M.; Nasim, F.H.; Choudhary, M.I.
Synthesis and urease inhibitory properties of some new N4-substituted 5-nitroisatin-3-
thiosemicarbazones. Lett. Drug. Des. Discov. 2010, 7, 102-108.
25. Pervez, H.; Ramzan, M.; Yaqub, M.; Khan, K.M. Synthesis, cytotoxic and phytotoxic effects of some
new N4-aryl substituted isatin-3-thiosemicarbazones. Lett. Drug. Des. Discov. 2011, 8, 452-458.
26. Karali, N. Synthesis and primary cytotoxicity evaluation of new 5-nitroindole-2,3-dione
derivatives. Eur. J. Med. Chem. 2002, 37, 909-918.
27. Karali, N.; Terzioglu, N.; Gursoy, A. Synthesis and primary cytotoxicity evaluation of new
5-bromo-3-substituted hydrazono-1H-2-indolinones. Arch. Pharm. 2002, 335, 374-380.

Molecules 2011, 16
6421
28. Omar, A.-M.E.; Eshba, N.H.; Salama, H.M. Synthesis of some substituted isatin-β-thio-semicarbazones
and isatin-β-hydrazonothiazoline derivatives as potential antiviral and antimicrobial agents. Arch.
Pharm. 1984, 317, 701-709.
29. Petrov, I.; Grupce, O.; Stafilov, T. The nitrogen-hydrogen stretching region of some imides and
thioimides. J. Mol. Struct. 1986, 142, 275-278.
30. Naumov, P.; Anastasova, F. Experimental and theoretical vibrational study of isatin, its 5-(NO2, F,
Cl, Br, I, CH3) analogues and the isatinato anion. Spectrochim. Acta 2001, 57A, 469-481.
31. Nizamuddin, N.; Khan, M.H.; Alauddin, S.; Haque, R. Synthesis and fungicidal activity of some
2-arylamino-1,3,4-thiadiazino[6,5-b]indoles and 2-aryl-1,3,4-oxadiazolo-[2,3-c]-1,2,4-triazino[5,6-b]
indoles. Indian J. Chem. 1999, 38B, 501-504.
32. Laatsch, H.; Thomson, R.H.; Cox, P.J. Spectroscopic properties of violacein and related
compounds: Crystal structure of tetramethyl-violacein. J. Chem. Soc. Perkin Trans. 2 1984,
1331-1339.
33. Pervez, H.; Iqbal, M.S.; Saira, N.; Yaqub, M.; Tahir, M.N. 4-(5-Chloro-2-methylphenyl)-
1-[2-oxo-5-(trifluoromethoxy)indolin-3-ylidene]thiosemicarbazide. Acta Cryst. 2010, E66,
o1169-o1170.
34. Pervez, H.; Iqbal, M.S.; Saira, N.; Yaqub, M.; Tahir, M.N. 1-[2-Oxo-5-(trifluoromethoxy)indolin-
3-ylidene]-4-[4-(trifluoromethyl)phenyl]thiosemicarbazide. Acta Cryst. 2010, E66, o1749.
35. McLaughlin, J.L.; Chang, C.J.; Smith, D.L. Bench Top Bioassays for the Discovery of Bioactive
Natural Products: An Update; In Studies in Natural Products Chemistry: Structure and Chemistry
(Part B); Rahman, A.U., Ed.; Elsevier: Amsterdam, The Netherlands, 1991; Volume 9,
pp. 383-409.
36. Meyer, B.N.; Ferrigni, N.R.; Putnam, J.E.; Jacobsen, L.B.; Nicholas, D.E.; McLaughlin, J.L.
Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med. 1982, 45,
31-34.
37. Finney, D.J. Probit Analysis, 3rd ed.; Cambridge University Press: London, UK, 1971.
Sample Availability: Samples of the compounds 3a-3u are available from the authors.
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