Synthesis and Toxicity Evaluation of Some N4-Aryl Substituted 5-Trifluoromethoxyisatin-3-thiosemicarbazones.

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 (IR, 1H-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.

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 [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. Amongst n class="Chemical">isatin derivatives, isatin-thiosemicarbazones have been found to demonstrate numerous chemotherapeutic properties such as anticancer, antimicrobial, antituberculosis, antiulcer, antiviral, antiplasmodial, cytotoxic and enzymatic inhibition [1,2,3,4,5,6,8,9,12,19,20]. Prompted by this and in continuation of our work on bioactive isatin derivatives [21,22,23,24,25], 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 [22,23,24,25]. 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 class="Chemical">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 analytical (CHN) and spectral (IR, 1H-NMR, EIMS) data. Satisfactory elemental analysis (±0.4% of 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 [26,28,29,30]. 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.

2.2. Toxicity

All the synthesized thiosemicarbazones 3a-3u were screened for toxic activity by a brine shrimp (n class="Species">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 1 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 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.

Table 1

Brine shrimp (Artemia salina) bioassay of compounds 3a-3u.

Compounds	LD50 (M)
3a	4.49 × 10−5
3b	>2.45 × 10−4
3c	>2.51 × 10−4
3d	>2.51 × 10−4
3e	3.72 × 10−5
3f	>2.42 × 10−4
3g	>2.18 × 10−4
3h	>1.98 × 10−4
3i	1.80 × 10−4
3j	1.70 × 10−4
3k	1.11 × 10−5
3l	1.81 × 10−5
3m	>2.27 × 10−4
3n	1.43 × 10−5
3o	1.34 × 10−5
3p	4.32 × 10−5
3q	1.60 × 10−4
3r	>2.23 × 10−4
3s	>2.23 × 10−4
3t	>2.34 × 10−4
3u	>2.07 × 10−4
2-(2-Oxo-1,2-dihydro-3H-indol-3-ylidene)-N-phenyl-1-hydrazinecarbothioamide *	>3.38 × 10−4

* [21,22].

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 n class="Disease">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 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.

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 discs) were run on Shimadzu Prestige-21 FT-IR spectrometer. The 1H-NMR spectra were recorded in n class="Chemical">DMSO-d6 on Bruker (Rhenistetten-Forchheim, Germany) AM 300 spectrometer, operating at 300 MHz and using TMS as an internal standard. 1H 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 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, 3119 (NH stretching), 1695 (C=O), 1629 (C=N), 1143 (C=S) cm−1. 1H-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-n class="Chemical">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-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3c). Yield 76% as yellow powder, mp: 225 °C. IR: 3289, 3264, 3073 (NH stretching), 1703 (C=O), 1607 (C=N), 1155 (C=S) cm−1. 1H-NMR (DMSO-d): 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), 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3d). Yield 81% as yellow powder, mp: 238 °C. IR: 3289, 3264, 3073(NH stretching), 1701 (C=O), 1597 (C=N), 1161 (C=S) cm−1. 1H-NMR (DMSO-d): 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-n class="Chemical">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 stretching), 1692 (C=O), 1600 (C=N), 1155 (C=S) cm−1. 1H-NMR (DMSO-d): 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-n class="Chemical">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-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3g). Yield 70% as yellow powder, mp: 240 °C. IR: 3281, 3088 (NH stretching), 1699 (C=O), 1600 (C=N), 1148 (C=S) cm−1. 1H-NMR (DMSO-d): 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.

N-(4-Iodophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3h). Yield 80% as yellow powder, mp: 246 °C. IR: 3291, 3196, 3080 (NH stretching), 1703 (C=O), 1591 (C=N), 1146 (C=S) cm−1. 1H-NMR (DMSO-d): 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-hydrazinecarbon class="Chemical">thioamide (3i). Yield 87% as yellow fluffy crystals, mp: 234 °C. IR: 3346, 3188, 3171(NH stretching), 1695 (C=O), 1625 (C=N), 1182 (C=S) cm−1. 1H-NMR (DMSO-d): 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-hydrazinecarbon class="Chemical">thioamide (3j). Yield 79% as yellow powder, mp: 220 °C. IR: 3310, 3196, 3063 (NH stretching), 1699 (C=O), 1600 (C=N), 1161 (C=S) cm−1. 1H-NMR (DMSO-d): 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-hydrazinecarbon class="Chemical">thioamide (3k). Yield 75% as yellow powder, mp: 238–240 °C. IR: 3318, 3161, 3130 (NH stretching), 1701 (C=O), 1587 (C=N), 1167 (C=S) cm−1. 1H-NMR (DMSO-d): 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.

2-[2-Oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]-N-[4-(trifluoromethoxy)phenyl]-1-hydrazinecarbon class="Chemical">thioamide (3l). Yield 72% as orange fluffy crystals, mp: 228–230 °C. IR: 3308, 3100, 3055 (NH stretching), 1699 (C=O), 1600 (C=N), 1159 (C=S) cm−1. 1H-NMR (DMSO-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3m). Yield 89% as orange fluffy crystals, mp: 248 °C. IR: 3269, 3200, 3100 (NH stretching), 1705 (C=O), 1608 (C=N), 1165 (C=S) cm−1. 1H-NMR (DMSO-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3n). Yield 81% as yellow fluffy crystals, mp: 228–230 °C. IR: 3316, 3206, 3084 (NH stretching), 1692 (C=O), 1611 (C=N), 1155 (C=S) cm-1. 1H-NMR (DMSO-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3o). Yield 70% as yellow fluffy crystals, mp: 248–250 °C. IR: 3333, 3256, 3100 (NH stretching), 1694 (C=O), 1610 (C=N), 1165 (C=S) cm−1. 1H-NMR (DMSO-d): 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.

N-(3,5-Difluorophenyl)-2-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3p). Yield 70% as yellow powder, mp: 236–238 °C. IR: 3308, 3262, 3102 (NH stretching), 1703 (C=O), 1600 (C=N), 1155 (C=S) cm−1. 1H-NMR (DMSO-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3q). Yield 74% as yellow powder, mp: 238–240 °C. IR: 3300, 3173, 3129 (NH stretching), 1701 (C=O), 1580 (C=N), 1144 (C=S) cm−1. 1H-NMR (DMSO-d): 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-n class="Chemical">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-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3s). Yield 82% as yellow powder, mp: 245 °C. IR: 3318, 3177, 3080 (NH stretching), 1697 (C=O), 1600 (C=N), 1153 (C=S) cm−1. 1H-NMR (DMSO-d): 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-n class="Chemical">3H-indol-3-ylidene]-1-hydrazinecarbothioamide (3t). Yield 64% as orange powder, mp: 240 °C. IR: 3302, 3233, 3082 (NH stretching), 1701 (C=O), 1584 (C=N), 1146 (C=S) cm−1. 1H-NMR (DMSO-d): 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-hydrazinecarbon class="Chemical">thioamide (3u). Yield 79% as yellow powder, mp: 240 °C. IR: 3354, 3204, 3069 (NH stretching), 1701 (C=O), 1564 (C=N), 1167 (C=S) cm−1. 1H-NMR (DMSO-d): 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 n class="Disease">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 1 and in terms of further development and structure-activity relationship (SAR) studies, simultaneous substitution of different substituents at position-5 of the n class="Chemical">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.