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

Abstract

A new series of thirty nine 5-trifluoromethoxy/fluoro/chloro-isatin 3-hydrazonothiazolines 5a–n, 6a–o and 7a–j were synthesized by cyclization of the corresponding intermediate N⁴-aryl-substituted isatin-3-thiosemicarbazones 3 (prepared by condensation of appropriate isatin 1 with appropriate N⁴-aryl-substituted 3-thiosemicarbazides 2) with 4-chlorophenacyl bromide 4 in absolute ethanol or ethanol–benzene mixture and screened for their cytotoxicity, phytotoxicity, antifungal and urease inhibitory potential. All the synthesized compounds were found to be almost inactive in a brine shrimp (Artemia salina) bioassay, demonstrating IC₅₀ values > 1.62 × 10⁻⁴ to 2.17 × 10⁻⁴ M. In a phytotoxicity assay, out of thirty-nine compounds tested, six i.e. 5i, 6h, 6i, 6k, 7c and 7h proved to be active, showing weak or non-significant (5–30%) activity at the highest tested concentration (500 μg mL⁻¹). Similarly, in antifungal assay, twenty-six compounds i.e. 5a, 5b, 5d–f, 5h–j, 5m, 6a, 6b, 6d, 6j, 6l–o, 7a, 7b and 7d–j were found to be active against one, two, or three selected fungal strains, exhibiting weak or non-significant inhibition (10–30%). Of these, 6d and 6o displayed a relatively better activity profile in terms of the number of organisms inhibited. On the other hand, in a urease inhibition bioassay, all the synthesized hydrazonothiazolines proved to be potent enzyme inhibitors, demonstrating inhibitory activity with IC₅₀ values ranging from 3.70 ± 0.62 to 849 ± 2.26 μM. Compounds 5c, 5g–i, 5k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o, 7a, 7e, 7i and 7j were, however, found to be relatively very potent, displaying outstanding enzymatic activity (IC₅₀ = 3.70 ± 0.62 to 20.9 ± 0.57 μM), even better than the reference inhibitor thiourea (IC₅₀ = 22.3 ± 1.12 μM), and may thus act as valid leads for further studies. Molecular docking studies of the synthesized isatin–thiazolines 5a–n, 6a–o and 7a–j were also carried out to elucidate their relationship with the binding pockets of the enzyme. This study offers the first example of exhibition of urease inhibitory potential by isatin–thiazolines and as such provides a solid basis for further research on these compounds to develop more potent antiurease compounds of medicinal/agricultural interest.

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Introduction

A literature survey indicates that isatin and its derivatives possess a wide spectrum of biological properties.^1–20 Among isatin derivatives, isatin-thiosemicarbazones are reported to possess diverse pharmacological activities, including antiulcer,¹ analgesic and anti-inflammatory,^1,7 anticancer,^{1,4,7,12,21,22} antimicrobial,^1–3,5,7,22 antituberculosis,^23–25 antiviral,^{1,2,5,7,12,25} and enzyme inhibition.^1,4,11,17 Stimulated by this and as a part of our research work aiming to synthesise isatin-based organic compounds of medicinal/agricultural interest, we recently reported the synthesis of a number of 5-(un)-N⁴-aryl-substituted isatin-3-thiosemicarbazones as antimicrobial, cytotoxic, phytotoxic and more importantly antiurease compounds.^26–33 Investigation of the structure–activity relationship (SAR) studies in the synthesized compounds revealed that in certain cases, the type and position of different substituents about the phenyl ring attached to N⁴ of the thiosemicarbazone part^26,27,29 and/or the presence of an inductively electron-withdrawing group (NO₂, F₃CO, F, or Cl) at position-5 of the isatin moiety^28,30–33 played a key role in the inducement and/or increment of different activities. Moreover, it has been reported by some other authors that certain 5-bromoisatin-3-hydrazonothiazolines, prepared by cyclization of the respective thiosemicarbazones with phenacyl bromides, exhibited high cytotoxicity at low concentrations when tested for antiviral activity against MDCK cells. The presence of more than one lipophilic functions in these compounds was considered responsible for such a display of activity.³⁴ Likewise, 5-bromo isatin-derived 3-hydrazonothiazolidinones, obtained by cyclization of the corresponding thiosemicarbazones with ethyl 2-bromopropionate, have been found to show favourable cytotoxicity.²¹ Also, certain 5-fluoroisatin-derived 3-hydrazonothiazolidinones, obtained by condensation of 5-fluoroisatin with different thiazolidin-4-ones, have been claimed to possess antimicrobial activity.³⁵ In view of these points, it was envisioned that the simultaneous presence of different lipophilic groups at position-5 of the isatin scaffold and at the N atom of thiazoline moiety in the isatin–thiazoline hybrids would result in an increase in the selected activities. Thus, the present work to synthesize three new series of thirty nine title hydrazonothiazolines (derived by cyclization of a variety of N⁴-aryl-substituted 5-trifluoromethoxy/fluoro/chloro-isatin 3-thiosemicarbazones with 4-chlorophenacyl bromide) and screen them for their cytotoxicity, phytotoxicity, antifungal and specially urease inhibitory potential was accomplished. It is pertinent to mention here that such a category of isatin derivatives has been scarcely investigated before by other researchers for their antiurease potential. The work presented herein illustrates the influences of the type of aryl groups (modified by placing one, two, or three functionalities on the phenyl ring) attached to N atom of the thiazoline moiety as well as the presence of inductively electron-attracting/lipophilic trifluoromethoxy, fluoro and chloro groups at position-5 of the isatin part on the cytotoxic, phytotoxic, antifungal and urease inhibitory properties of the synthesized isatin–thiazoline hybrids. This study furnishes some motivating and attention-grabbing results, which are reported herein.

Results and discussion

The present study describes the synthesis and in vitro evaluation of cytotoxic, phytotoxic, antifungal and urease inhibitory potential of thirty seven currently and two previously reported^36,37 isatin–hydrazonothiazolines 5a–n, 6a–d, 6f–h, 6j–o, 7a–j and 6e, 6i, respectively.

Chemistry of compounds 5a–n, 6a–o and 7a–j

The designed isatin–hydrazonothiazolines 5–7 were prepared in 73–91% yield from 5-substituted isatins 1 by first reacting them with equimolar quantities of appropriate N⁴-substituted 3-thiosemicarbazides 2 in aqueous ethanol containing a catalytic amount of glacial acetic acid, and then by treating the resultant intermediate thiosemicarbazones 3 with 4-chlorophenacyl bromide 4 in absolute ethanol or ethanol–benzene mixture at reflux temperature (Scheme 1).

Scheme 1 Synthesis of title hydrazonothiazolines 5a–n, 6a–o and 7a–j.

The structures of the synthesized isatin-3-hydrazonothiazolines 5a–n, 6a–o and 7a–j were established on the basis of elemental (CHN) and spectral (IR, ¹H NMR, EIMS) analyses. Satisfactory elemental analyses were obtained for all the compounds synthesized in this study. Also, the spectral data were in agreement with the relevant literature.^21,34,38 The IR spectra of isatin–thiazolines 5a–n, 6a–o and 7a–j showed a single band resulting from NH stretching of indole in the 3261–3008 cm⁻¹ region. The lactam C=O and azomethine C=N were observed in the 1739–1693 and 1631–1558 cm⁻¹ regions, respectively.^21,34,38 The ¹H-NMR spectra of 5a–n, 6a–o and 7a–j did not show the down field signals of thiosemicarbazone N⁴-H and N²-H, and displayed one or two separate singlets at δ 10.39–10.75 and δ 10.35–10.77, respectively, for indole NH. The thiazoline ring C₅–H resonated either as a singlet at δ 7.01–7.22 or two separate singlets at δ 6.90–7.12 or a multiplet at δ 6.95–7.13.^21,34,38 Furthermore, the ¹H-NMR spectra of compounds 6c, 6d, 6f, 6o, 7a, 7c, 7d and 7f–j exhibited duplicate signals, confirming the presence of two isomers. It is suggested that in these compounds, the restricted rotation about the azomethine (C=N) linkage and the partial double bond character of the lactam C–N bond, induced by delocalization of the nitrogen lone pair of electrons onto the carbonyl oxygen, led to the formation of E and Z isomers.^21,38 The EI mass spectra of 5a–n, 6a–o and 7a–j showed molecular ions of different intensity peculiar to the isatin and thiazoline moieties. The key fragmentation pathways involved the N–N, N–C, and N–CO bonds fission.³⁸ The proposed fragmentation patterns of compounds 5c, 6g and 7f, representing each series of isatin–thiazoline hybrids, are illustrated in Fig. 1–3, respectively. X-ray structures of two representative examples 6e and 6i were determined in order to prove the allocated structures and to substantiate conformations of the synthesized isatin–hydrazonothiazolines. The related crystallographic data and refinement details of 6e and 6i have been reported somewhere else.^36,37

Fig. 1 The proposed fragmentation pattern of compound 5c.

Fig. 2 The proposed fragmentation pattern of compound 6g.

Fig. 3 The proposed fragmentation pattern of compound 7f.

Biology

Cytotoxicity (in vitro) of compounds 5a–n, 6a–o and 7a–j. The synthesized isatin-3-hydrazonothiazolines 5a–n, 6a–o and 7a–j were screened for their cytotoxic effects by a brine shrimp (Artemia salina) lethality bioassay using etoposide, a standard anticancer drug as a reference point for comparison to the trial compounds. All the compounds of this series gave LD₅₀ values > 1.62 × 10⁻⁴ to 2.17 × 10⁻⁴ M in the present assay when tested at 100, 10 and 1 μg mL⁻¹ concentrations and thus can be considered to be almost inactive.

Phytotoxicity (in vitro) of compounds 5a–n, 6a–o and 7a–j. The synthetic compounds 5a–n, 6a–o and 7a–j were further screened for their phytotoxicity potential at 500, 50 and 5 μg mL⁻¹ concentrations. Out of thirty nine compounds tested, only six i.e. 5i, 6h, 6i, 6k, 7c and 7h appeared to be active, exhibiting weak or non-significant (5–30%) plant growth inhibition at the highest tested concentration (500 μg mL⁻¹) compared to the standard drug, paraquat, which displayed 100% inhibition at 0.015 μg mL⁻¹ concentration (Table 1).

Table 1 Growth inhibition of Lemna aequinoctialis by compound 5a–n, 6a–o and 7a–j^a,b at different concentrations

Compound	R	R1	% growth inhibition
			500 μg mL^−1	50 μg mL^−1	5 μg mL^−1
a The reference compound, paraquat, shows 100% growth inhibition at 0.015 μg mL^−1.b All others inactive.
5i	OCF3	4-F	05	00	00
6h	F	3-F	05	00	00
6i	F	4-F	05	00	00
6k	F	2,6-(F)2	15	00	00
7c	Cl	4-CH3	18	00	00
7h	Cl	3-F	30	00	00

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Antifungal activity (in vitro) of compounds 5a–n, 6a–o and 7a–j. Antifungal activity of the synthesized isatin–hydrazonothiazolines 5a–n, 6a–o and 7a–j was determined against five fungal cultures i.e. Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata at 200 μg mL⁻¹ in DMSO (Table 2). Of the thirty nine compounds tested, twenty six i.e. 5a, 5b, 5d–f, 5h–j, 5m, 6a, 6b, 6d, 6j, 6l–o, 7a, 7b and 7d–j were found to be active against one, two, or three selected pathogens, displaying varied but low or non-significant inhibition (10–30%). Among these, 6d and 6o bearing fluoro group at position-5 of the isatin scaffold and methoxy and chloro functions, respectively, at positions-2 (ortho) and -2,4,6 (ortho, para, ortho) of the phenyl ring attached to N atom of the thiazoline moiety exhibited relatively better activity profile in terms of the number of organisms affected.

Table 2 Antifungal activity (in vitro) of compounds 5a–n, 6a–o and 7a–j^a,b (% inhibition)

Compound	R	R1	Microbial species
			C. albicans	A. flavus	M. canis	F. solani	C. glabrata
a Concentration used 200 μg mL^−1.b Standard drugs (MIC in μg mL^−1): A = miconazole (110.8 μg mL^−1); B = amphotericin B (20 μg mL^−1); C = miconazole (98.4 μg mL^−1); D = miconazole (73.25 μg mL^−1); E = miconazole (110.8 μg mL^−1); 00: absence of measurable inhibitory action.
5a	OCF3	2-CH3	00	00	20	00	00
5b	OCF3	3-CH3	00	00	20	25	00
5d	OCF3	2-OCH3	00	00	00	10	00
5e	OCF3	3-OCH3	00	00	20	00	00
5f	OCF3	4-OCH3	00	00	00	10	00
5h	OCF3	3-F	00	00	25	00	00
5i	OCF3	4-F	00	00	00	30	00
5j	OCF3	2,4-(Cl)2	00	00	00	25	00
5m	OCF3	3,4-(Cl)2	00	00	00	10	00
6a	F	2-CH3	00	00	00	25	00
6b	F	3-CH3	00	00	20	00	00
6d	F	2-OCH3	00	20	20	20	00
6j	F	2,4-(F)2	00	00	10	00	00
6l	F	2,4-(Cl)2	00	20	30	00	00
6m	F	2,5-(Cl)2	00	00	10	00	00
6n	F	2,6-(Cl)2	00	00	30	00	00
6o	F	2,4,6-(Cl)3	00	10	20	25	00
7a	Cl	2-CH3	00	00	20	00	00
7b	Cl	3-CH3	00	00	20	00	00
7d	Cl	2-OCH3	00	20	00	00	00
7e	Cl	3-OCH3	00	00	20	00	00
7f	Cl	4-OCH3	00	00	25	00	00
7g	Cl	2-F	00	00	25	00	00
7h	Cl	3-F	00	00	20	00	00
7i	Cl	4-F	00	00	25	00	00
7j	Cl	2,4-(F)2	00	00	00	20	00
Standard Drug^b			A	B	C	D	E

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Urease inhibition (in vitro) of compounds 5a–n, 6a–o and 7a–j. The synthesized 5-trifluoromethoxy/fluoro/chloro-isatin 3-hydrazonothiazolines 5a–n, 6a–o and 7a–j, respectively, were tested for their antiurease activity against Jack bean urease. Thiourea served as reference standard in this assay. All the compounds proved to be potent inhibitors of the enzyme, exhibiting inhibitory activity with IC₅₀ values of 3.70 ± 0.62 to 849 ± 2.26 μM. Compounds 5c, 5g–i, 5k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o, 7a, 7e, 7i and 7j were, however, found to be relatively very much potent, displaying excellent activity (IC₅₀ values 3.70 ± 0.62 to 20.9 ± 0.57 μM) even better than the reference inhibitor, thiourea (IC₅₀ = 22.3 ± 1.12 μM) and may thus act as compelling leads for further studies. The remaining compounds i.e. 5a, 5b, 5d–f, 5j, 5l, 5m, 6a, 6d–h, 6j, 6m, 7b–d and 7f–h showed a varying degree of activity with IC₅₀ values of 24.6 ± 2.14 to 849 ± 2.26 μM (Table 3).

Table 3 Inhibition of Jack been urease by compounds 5a–n, 6a–o and 7a–j

Compound	R	R1	IC50 ± SEM (μM)	Compound	R	R1	IC50 ± SEM (μM)
a Reference inhibitor of urease enzyme.
5a	OCF3	2-CH3	336 ± 5.45	6g	F	2-F	26.1 ± 1.11
5b	OCF3	3-CH3	427 ± 2.89	6h	F	3-F	32.6 ± 3.42
5c	OCF3	4-CH3	10.3 ± 0.51	6i	F	4-F	8.20 ± 0.01
5d	OCF3	2-OCH3	42.9 ± 4.67	6j	F	2,4-(F)2	66.6 ± 5.40
5e	OCF3	3-OCH3	42.6 ± 1.40	6k	F	2,6-(F)2	11.0 ± 0.86
5f	OCF3	4-OCH3	102 ± 5.82	6l	F	2,4-(Cl)2	8.40 ± 0.23
5g	OCF3	2-F	7.27 ± 0.33	6m	F	2,5-(Cl)2	33.1 ± 2.88
5h	OCF3	3-F	12.0 ± 0.83	6n	F	2,6-(Cl)2	12.7 ± 0.62
5i	OCF3	4-F	11.7 ± 0.07	6o	F	2,4,6-(Cl)3	14.3 ± 0.12
5j	OCF3	2,4-(Cl)2	44.5 ± 3.15	7a	Cl	2-CH3	9.20 ± 0.77
5k	OCF3	2,5-(Cl)2	3.70 ± 0.62	7b	Cl	3-CH3	24.6 ± 2.14
5l	OCF3	2,6-(Cl)2	129 ± 5.91	7c	Cl	4-CH3	25.3 ± 3.20
5m	OCF3	3,4-(Cl)2	134 ± 2.21	7d	Cl	2-OCH3	38.6 ± 1.50
5n	OCF3	2,4,6-(Cl)3	14.1 ± 1.31	7e	Cl	3-OCH3	10.3 ± 0.38
6a	F	2-CH3	275 ± 4.99	7f	Cl	4-OCH3	81.3 ± 7.99
6b	F	3-CH3	15.0 ± 1.92	7g	Cl	2-F	25.6 ± 2.91
6c	F	4-CH3	11.4 ± 1.51	7h	Cl	3-F	32.0 ± 0.69
6d	F	2-OCH3	40.1 ± 5.32	7i	Cl	4-F	11.3 ± 0.32
6e	F	3-OCH3	29.3 ± 1.17	7j	Cl	2,4-(F)2	20.9 ± 0.57
6f	F	4-OCH3	849 ± 2.26	Thiourea^a			22.3 ± 1.12

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The structure–activity relationship (SAR) studies in the case of 5-trifluoromethoxyisatin derivatives 5a–n revealed that compound 5k having chloro substituents at positions-2 and -5 of the phenyl ring attached to N atom of the thiazoline moiety was the most potent urease inhibitor of the series, demonstrating several fold more activity (IC₅₀ = 3.70 ± 0.62 μM) than the reference inhibitor, thiourea (IC₅₀ = 22.3 ± 1.12 μM). Comparison of the urease inhibitory potential of compound 5k with that of closely related dichloro-substituted compounds 5j, 5l and 5m indicated that chloro substitution at positions-2,5 (ortho, meta) of the phenyl ring was more favourable than at positions-2,4 (ortho, para), -2,6 (ortho, ortho) and -3,4 (meta, para), respectively. The dichloro-substituted derivatives 5j, 5l and 5m displayed relatively much lower activity (IC₅₀ = 44.5 ± 3.15, 129 ± 5.91 and 134 ± 2.21 μM, respectively) in the present assay. This clearly showed that compound 5k compared to 5j, 5l and 5m interfered with the enzyme in a different fashion. Interestingly, the only trichloro-substituted compound tested in this assay i.e. 5n bearing the substituents at positions-2,4,6 (ortho, para, ortho) of the phenyl ring demonstrated much higher activity in contrast to the dichloro-substituted compounds 5j and 5l with the substituents at positions-2,4 (ortho, para) and -2,6 (ortho, ortho) of the phenyl ring, respectively (IC₅₀ value 14.1 ± 1.31 vs. 44.5 ± 3.15 and 129 ± 5.91 μM). Chlorine is both electron-donating by mesomeric effect (+M) and electron-withdrawing by inductive effect (−I). Much higher enzyme inhibitory activity presented by compound 5n in the present assay clearly indicated that the ultimate or overall electron-withdrawing effect of the three chloro functions increased its inhibitory potential, though not exclusively. The next most potent urease inhibitor was compound 5g having a fluoro group at position-2 of the phenyl ring, showing inhibitory activity with IC₅₀ value of 7.27 ± 0.33 μM. This compound was found to be three fold more active than the reference inhibitor, thiourea, but two fold less active than the most potent urease inhibitor 5k. The other highly active monofluoro-substituted derivatives 5h and 5i, possessing the substituent at positions-3 and -4 of the phenyl ring exhibited enzymatic inhibition with IC₅₀ values of 12.0 ± 0.83 and 11.7 ± 0.07 μM, respectively. This clearly indicated that compound 5g in comparison to 5h and 5i meddled with the enzyme differently and more competently. The remaining relatively very much potent urease inhibitor was compound 5c with a methyl substituent at position-4 of the phenyl ring attached to N atom of the thiazoline moiety. This compound was found to be two fold more active than the reference inhibitor (thiourea) but three times less active than the most potent urease inhibitor of the series i.e. compound 5k (IC₅₀ value 10.3 ± 0.51 vs. 22.3 ± 1.12 and 3.70 ± 0.62 μM, respectively). The other methyl-substituted compounds 5a and 5b bearing the substituent at positions-2 and -3 of the phenyl ring displayed relatively much lower inhibitory activity (IC₅₀ values 336 ± 5.45 and 427 ± 2.89 μM, respectively). These results showed that steric hindrance played a significant role in reducing the inhibitory potential of the compounds.

Among 5-fluoroisatin derivatives 6a–o, compounds 6i and 6l having fluoro and chloro substituents at positions-4 (para) and -2,4 (ortho, para) of the phenyl ring attached to N atom of the thiazoline moieties were the most potent urease inhibitors of the series, presenting excellent and almost the same inhibitory activity (IC₅₀ values 8.20 ± 0.01 and 8.40 ± 0.23 μM, respectively). Comparison of the urease inhibitory potential of compound 6i with that of closely related fluoro-substituted derivatives 6g and 6h showed that fluoro substitution at position-4 of the phenyl ring was more favourable than at positions-2 and -3. The fluoro-substituted compounds 6g and 6h exhibited relatively much lower activity (IC₅₀ values 26.1 ± 1.11 and 32.6 ± 3.42 μM, respectively) in the present assay. Also, comparison of the antiurease potential of compound 6l with that of 6m and 6n indicated that chloro substitution at positions-2,4 (ortho, para) was more favourable than at -2,5 (ortho, meta) and -2,6 (ortho, ortho). The dichloro-substituted derivative 6n compared to 6l displayed lower but still exciting activity (IC₅₀ value 12.7 ± 0.62 vs. 8.40 ± 0.23 μM), while 6m demonstrated relatively much lower activity with IC₅₀ value of 33.1 ± 2.88 μM. Similarly, the trichloro-substituted compound 6o having the chloro substituents at positions-2,4,6 (ortho, para, ortho) of the phenyl ring exhibited lower but still stimulating inhibitory activity (IC₅₀ = 14.3 ± 0.12 μM) in comparison to the corresponding dichloro-substituted compounds 6l and 6n, showing enzyme inhibition with IC₅₀ values of 8.40 ± 0.23 and 12.7 ± 0.62 μM, respectively. In case of difluoro-substituted compounds tested in this assay, compound 6k bearing the fluoro substituents at positions-2,6 (ortho, ortho) of the phenyl ring was found to show much higher inhibitory potential compared to compound 6j having the substituents at positions-2,4 (ortho, para) (IC₅₀ value 11.0 ± 0.86 vs. 66.6 ± 5.40 μM). This indicated that compound 6k in contrast to 6j intermingled with the enzyme in a different manner, resulting into much marked enhancement in enzyme inhibitory potential. Amongst methyl-substituted compounds 6a–6c, compound 6c possessing the substituent at positions-4 of the phenyl ring was found to be the most active one, displaying enzyme inhibition with IC₅₀ value of 11.4 ± 1.51 μM. The next most active derivative was 6b with the substituent at position-3 of the ring, which showed inhibitory potential with IC₅₀ value of 15.0 ± 1.92 μM. The remainder derivative 6a having the substituent at position-2 of the phenyl ring exhibited much lower inhibitory activity with IC₅₀ value of 275 ± 4.99 μM. This indicated that in case of 6a, steric hindrance played a pivotal role in decreasing the inhibitory potential of the compound. The above results demonstrated that compounds 6g and 6h in contrast to 6i, and 6m in comparison to 6l, 6n and 6o, interacted with the enzyme differently, resulting into reduction in the inhibitory potential to a smaller or greater extent.

The results given in the table revealed that compared to compounds 5a–n having trifluoromethoxy substituent at position-5 of the isatin part, substitution of fluoro group at the same position in the case of 6a–o caused either a decrement or an increment in the enzymatic activity in certain cases. For example, compound 6c having a methyl substituent at position-4 of the phenyl ring attached to N atom of the thiazoline moiety showed inhibition of the enzyme with IC₅₀ value of 11.4 ± 1.51 μM, whereas the respective compound 5c bearing trifluoromethoxy function at position-5 of the isatin moiety displayed more inhibitory activity (IC₅₀ = 10.3 ± 0.51 μM). Similarly, compounds 6f–h possessing methoxy and fluoro substituents at positions-4 and -2, -3 of the phenyl ring, respectively, were found to demonstrate reduced activity (IC₅₀ values 849 ± 2.26, 26.1 ± 1.11 and 32.6 ± 3.42 μM) in comparison to the corresponding 5-trifluoromethoxyisatin derivatives 5f–h, which exhibited inhibitory activity with IC₅₀ values of 102 ± 5.82, 7.27 ± 0.33 and 12.0 ± 0.83 μM, respectively. Also, compound 6m possessing chloro functions at positions-2 and -5 of the phenyl ring was found to show decreased activity (IC₅₀ = 33.1 ± 2.88 μM) when compared with the respective compound 5k having trifluoromethoxy substituent at position-5 of the isatin moiety, which displayed enzyme inhibition with IC₅₀ value 3.70 ± 0.62 μM. Much marked decrement in enzyme inhibitory activity was found to occur in case of compounds 6g, 6h and 6m, respectively, when compared with the corresponding 5-trifuoromethoxyisatin-derived hydrazonothiazolines 5g, 5h and 5k (IC₅₀ values 7.27 ± 0.33 → 26.1 ± 1.11 μM, 12.0 ± 0.83 → 32.6 ± 3.42 μM and 3.70 ± 0.62 → 33.1 ± 2.88 μM, respectively). On the contrary, compounds 6a and 6b bearing methyl function at positions-2 and -3 of the phenyl ring, respectively, displayed increased inhibitory activity (IC₅₀ values 275 ± 4.99 and 15.0 ± 1.92 μM, respectively) in contrast to the respective compounds 5a and 5b, showing enzyme inhibition with IC₅₀ values of 336 ± 5.45 and 427 ± 2.89 μM. Similarly, compounds 6d and 6e having methoxy substituent at positions-2 and -3 of the phenyl ring displayed increased enzyme inhibition with IC₅₀ values 40.1 ± 5.32 and 29.3 ± 1.17 μM, respectively, when compared with the corresponding compounds 5d and 5e bearing trifluoromethoxy group at position-5 of the isatin scaffold, demonstrating inhibition of the enzyme with IC₅₀ values of 42.9 ± 4.67 and 42.6 ± 1.40 μM. Furthermore, the monofluoro-substituted derivative 6i possessing the substituent at position-4 of the phenyl ring showed enhanced enzyme inhibition with IC₅₀ value of 8.20 ± 0.01 μM in comparison to the corresponding 5-trifluoromethoxyisatin-derived hydrazonothiazoline 5i, which displayed inhibitory activity with IC₅₀ value of 11.7 ± 0.07 μM. Also, the dichloro-substituted compounds 6l and 6n having the substituents at positions-2,4 (ortho, para) and -2,6 (ortho, ortho) exhibited enhanced enzyme inhibition (IC₅₀ values 8.40 ± 0.23 and 12.7 ± 0.62 μM, respectively) in contrast to the respective compounds 5j and 5l bearing trifluoromethoxy residue at position-5 of the isatin moiety, demonstrating inhibitory activity with IC₅₀ values of 44.5 ± 3.15 and 129 ± 5.91 μM. Relatively, marked increment in enzyme inhibitory activity (IC₅₀ values 427 ± 2.89 → 15.0 ± 1.92 μM, 44.5 ± 3.15 → 8.40 ± 0.23 μM and 129 ± 5.91 → 12.7 ± 0.62 μM, respectively) occurred in case of compounds 6b, 6l and 6n. Among the remaining compounds of the series i.e. 6j and 6k, compound 6j having fluoro substituents at positions-2 and -4 of the phenyl ring exhibited inhibitory activity with IC₅₀ value of 66.6 ± 5.40 μM. In contrast, 6k possessing the same substituents at positions-2 and -6 of the phenyl ring showed much enhanced activity (IC₅₀ = 11.0 ± 0.86 μM). These results showed that the presence of fluoro group (exercising both −ve inductive and +ve mesomeric effects) at position-5 of the isatin scaffold as well as the nature, number and position of different functions present in the phenyl ring caused the molecules to meddle with the enzyme in a different fashion.

In case of 5-chloroisatin-derived hydrazonothiazolines 7a–j, 7a bearing methyl group at position-2 of the phenyl ring attached to N atom of the thiazoline moiety was found to be the most potent antiurease compound, showing enzymatic activity (IC₅₀ = 9.20 ± 0.77 μM) much higher than the reference inhibitor, thiourea (IC₅₀ = 22.3 ± 1.12 μM). Comparison of the antiurease activity of compound 7a with that of closely related methyl-substituted compounds 7b and 7c having the substituent at positions-3 and -4 of the phenyl ring, respectively, revealed that methyl substitution at position-2 of the phenyl ring was more favourable than at -3 and -4. Compound 7b was found to be >two fold less active than 7a but slightly more active than 7c (IC₅₀ value 24.6 ± 2.14 vs. 9.20 ± 0.77 and 25.3 ± 3.20 μM, respectively). This showed that compounds 7b and 7c compared to 7a interfered with the enzyme much less efficiently, resulting in a decrement in their inhibitory potential. The next most potent antiurease compound was 7e possessing methoxy substituent at position-3 of the phenyl ring. This compound exhibited slightly less inhibitory activity (IC₅₀ = 10.3 ± 0.38 μM) than the most potent inhibitor 7a (IC₅₀ = 9.20 ± 0.77 μM) but much more than the reference inhibitor, thiourea (IC₅₀ = 22.3 ± 1.12 μM). The other potent methoxy-substituted compounds 7d and 7f having the substituent at position-2 and -4 of the phenyl ring attached to N atom of the thiazoline moiety, however, displayed relatively much lower activity with IC₅₀ values of 38.6 ± 1.50 μM and 81.3 ± 7.99 μM, respectively. This clearly demonstrated that compound 7e intermingled with the enzyme differently and much more competently. The remaining relatively much more potent urease inhibitor was compound 7i bearing a fluoro substituent at position-4 of the phenyl ring. This compound was found to be slightly less active than the most potent urease inhibitor of the series i.e. compound 7a but two fold more active than the reference inhibitor, thiourea (IC₅₀ value 11.3 ± 0.32 vs. 9.20 ± 0.77 and 22.3 ± 1.12 μM, respectively). The other fluoro-substituted compound 7g possessing the substituent at position-2 of the phenyl ring showed inhibitory activity with IC₅₀ value of 25.6 ± 2.91 μM. On the contrary, compound 7h with the fluoro substituent at position-3 of the phenyl ring displayed reduced enzymatic activity with IC₅₀ value of 32.0 ± 0.69 μM. Finally, the only difluoro-substituted compound tested in the present assay i.e. 7j having fluoro substituents at positions-2,4 (ortho, para) of the phenyl ring, though found to be markedly less active than the monofluoro-substituted compound 7i (20.9 ± 0.57 vs. 11.3 ± 0.32 μM), but was still more active than the monofluoro-substituted compound 7g (IC₅₀ = 25.6 ± 2.91 μM) as well as the reference inhibitor, thiourea (IC₅₀ = 22.3 ± 1.12 μM). Fluorine is both strongly electron-attracting by inductive effect (−I) and electron-donating by mesomeric effect (+M). Noticeably higher inhibitory activity shown by compounds 7g, 7i and 7j compared to 7h in the present assay is attributed, though not exclusively, to the overall or ultimate electron-donating influences of the fluoro functions present at position-2 (ortho) and -4 (para) of the phenyl ring attached to N atom of the thiazoline moiety.

The results given in the table further revealed that compared with the 5-fluoroisatin-derived thiazolines 6a–o, thiazolines of this series exhibited either increased or decreased enzyme inhibition in certain cases. For example, compound 7a possessing a methyl group at position-2 of the phenyl ring attached to N atom of the thiazoline moiety showed inhibitory activity with IC₅₀ value 9.20 ± 0.77 μM, whereas the respective compound 6a displayed enzymatic inhibition with IC₅₀ value of 275 ± 4.99 μM. Similarly, compounds 7d–f bearing methoxy functions at position-2, -3 and -4 of the phenyl ring, respectively, showed enhanced activity (IC₅₀ values 38.6 ± 1.50, 10.3 ± 0.38 and 81.3 ± 7.99 μM) compared to the corresponding compounds 6d–f, demonstrating enzyme inhibition with IC₅₀ values of 40.1 ± 5.32, 29.3 ± 1.17 and 849 ± 2.26 μM, respectively. Also, the monofluoro-substituted compounds 7g and 7h having the substituent at position-2 and -3 of the phenyl ring, respectively, displayed enhanced activity (IC₅₀ values 25.6 ± 2.91 and 32.0 ± 0.69 μM) in comparison to the respective compounds 6g and 6h, exhibiting enzyme inhibitory activity with IC₅₀ values of 26.1 ± 1.11 and 32.6 ± 3.42 μM, respectively. Furthermore, the difluoro-substituted compound 7j with the substituents at positions-2 and -4 of the phenyl ring showed increased activity (IC₅₀ = 20.9 ± 0.57 μM) in contrast to the corresponding compound 6j, displaying inhibition of the enzyme with IC₅₀ value of 66.6 ± 5.40 μM. Marked increment was found to occur in case of compounds 7a, 7e, 7f and 7j. To the contrary, compounds 7b and 7c possessing the methyl substituent at position-3 and -4 of the phenyl ring, respectively, exhibited decreased enzyme inhibitory activity (IC₅₀ values 24.6 ± 2.14 and 25.3 ± 3.20 μM) compared to the corresponding compounds 6b and 6c, showing activity with IC₅₀ values of 15.0 ± 1.92 and 11.4 ± 1.51 μM. Also, compound 7i bearing the fluoro function at position-4 of the phenyl ring showed reduced inhibitory activity (IC₅₀ value 11.3 ± 0.32 μM) in contrast to the respective compound 6i, exhibiting enzyme inhibition with IC₅₀ value of 8.20 ± 0.01 μM. Relatively, pronounced reduction in the enzymatic activity was observed in the case of 7c.

The above results showed that the simultaneous presence of varied inductively electron-withdrawing groups at position-5 of the isatin scaffold and the variously substituted aryl functions at N atom of the thiazoline moiety caused the isatin–thiazoline hybrids to meddle with the enzyme differently and sometimes relatively much more competently.

Molecular docking studies of compounds 5a–n, 6a–o and 7a–j. Molecular docking studies of isatin–hydrazonothiazolines 5a–n, 6a–o and 7a–j were performed using LeadIT to investigate the binding modes of inhibitors as well as interactions in the active site. The structures were docked to the crystallographic structure of Jack bean urease PDB ID: 3LA4 having bi-nickel center in its active site. After reproducing the co-crystallized reference ligand into the active site of receptor, all the derivatives were docked inside the active site. Most of the compounds were found interacting deep inside with the active site residues and showed coordinated interactions with the nickel ions in the active site of the receptor. The active site of the receptor contains the amino acids residues His409, Ala436, Arg439, Ala440, His492, Asp494, His519, His593, His594, Arg609, Ala636 and Met637.

Overall, the structures of the compounds are too large to be accommodated in the active pocket of the enzyme; therefore, some part lies within the bottom, while the rest interacts with the mid gorge area. The putative binding modes of the most active compounds among the three series i.e. 5a–n, 6a–o and 7a–j are given below:

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

Fig. 4 The putative binding mode of compound 5k bound to the active site of Jack bean urease. Carbon atoms of the compound are golden coloured and that of protein are blue. Dark green spheres are two nickel ions.

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

Fig. 5 The putative binding mode of compound 6i bound to the active site of Jack bean urease. Carbon atoms of the compound are magenta coloured and that of protein are blue. Dark green spheres are two nickel ions.

Binding mode analysis of compounds 7a–j. Docking studies supported the experimental results that potent inhibitors inhibit the catalytic site of the enzyme. The docking studies revealed the stable hydrogen bonding interactions between the oxygen and hydrogen atoms of the inhibitors and the amino acid residues Arg439, His593 and Arg609. Fig. 6 reports the prioritized binding mode of the most potent inhibitor 7a; the pose was selected after Hyde assessment and visualization of 30 lowest energy docked conformations. The 5-chloroisatin part of the molecule was oriented towards amino acids His409, Ala436, Arg439, Ala440 and His593 where π–π interactions were observed with His593 in the active pocket; the 4-chlorophenyl and 2-methylphenyl substituents attached to C and N atoms of the thiazoline moiety, respectively, were inclined towards Asp494.

Fig. 6 The putative binding mode of compound 7a bound to the active site of Jack bean urease. Carbon atoms of the compound are light green coloured and that of protein are blue. Dark green spheres are two nickel ions.

Conclusions

Conclusively, three new series of thirty nine isatin-3-hydrazonothiazolines have been synthesized in this study and evaluated for their in vitro cytotoxic, phytotoxic, antifungal and urease inhibitory effects. All the compounds proved to be almost inactive in the brine shrimp (Artemia salina) lethality bioassay. However, in phytotoxicity bioassay, six compounds were found to be active, exhibiting weak or non-significant (5–30%) activity at the highest tested concentration (500 μg mL⁻¹). Similarly, in antifungal assay, out of thirty nine compounds tested, twenty six proved to be active, showing weak or non-significant activity (10–30%) against one, two, or three fungal species. On the other hand, the urease inhibition testing results have demonstrated that all the thirty nine compounds tested appeared as potent urease inhibitors; seventeen of these i.e. 5c, 5g–i, 5k, 5n, 6b, 6c, 6i, 6k, 6l, 6n, 6o, 7a, 7e, 7i and 7j proved to be relatively highly potent ones and may act as convincing leads for further studies. These compounds, being non toxic, could be potent candidates for orally effective therapeutic agents used for the treatment of certain clinical conditions induced by microbial ureases. Also, by demonstrating weak or non-significant phytotoxicity at the highest tested concentration, they invite much attention to their utility as potent inhibitors of soil ureases, as they could be mixed with fertilizers in small quantities to enhance the overall effectiveness of nitrogen utilization. Generally, urease inhibitory potential of the synthesized isatin–thiazolines was found to be dependent upon electronic effects of the substituents at position-5 of the isatin scaffold and on the phenyl ring attached to N atom of the thiazoline moiety. In order to understand the binding mechanism, molecular docking studies were performed for compounds 5a–n, 6a–o and 7a–j, which elucidated their relationship with the binding pockets of urease. Based on the data presented in Table 3 and in terms of further development and SAR studies, simultaneous substitution of diverse inductively electron-withdrawing groups at position-5 of the isatin scaffold and the aryl substituents (modified by placing one or more lipophilic functions about the phenyl ring) at N as well as C-4 atoms of the thiazoline moiety in the hybrid molecules certainly warrants investigations.

Experimental

General procedure

Chemical materials and solvents were obtained from Merck-Schuchardt, Fluka and Sigma-Aldrich. Melting points were determined on a Fisher–Johns melting point apparatus and are not corrected. Elemental analyses were performed by using a Leco CHNS-9320 (USA) elemental analyzer. The data obtained from all the synthesized compounds were found to be satisfactory. FT-IR spectra (KBr) were run on a Shimadzu 8400 or Thermo Scientific Nicolet 6700 FTIR spectrophotometer using ATR facility. ¹H-NMR spectra were recorded in C₂D₆SO on Bruker (Rheinstetten-Forchheim, Germany) AM 300, Bruker Avance AV300 and Bruker Spectrospin 300 spectrometers, operating at 300 MHz and using Si(CH₃)₄ as an internal standard. Mass spectra were obtained on MAT-312 and JEOL JMS-600H mass spectrometers.

Synthesis

General procedure for the synthesis of isatin-3-thiosemicarbazones 3. An appropriate 5-substituted isatin 1 (0.01 mol), N⁴-substituted thiosemicarbazide 2 (0.01 mol) and 50% aqueous ethanol (40 mL) containing a catalytic amount of acetic acid were mixed and heated under reflux for 2 h. The solid obtained in each case during heating was filtered and washed thoroughly with hot 50% aqueous ethanol to give the desired thiosemicarbazones 3, which were used as such in the next step without further purification.^{30,32,33,39,40}

General procedure for the synthesis of isatin-3-hydrazonothiazolines 5–7. An appropriate isatin-3-thiosemicarbazone 3 (0.005 mol) was mixed with 4-chlorophenacyl bromide 4 (0.005 mol) in absolute ethanol (25 mL) or in a mixture of ethanol–benzene and heated under reflux for 12–15 h. The refluxate was concentrated on a rotary evaporator and the precipitate thus formed in each case was filtered. Thorough washing with warm diethyl ether or n-hexane followed by crystallization from ethanol–water furnished the target compounds 5–7 in pure form.

The different compounds are characterized as under:

(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5a. Yield 82% as orange brown crystals; mp 248–250 °C; IR (KBr, cm⁻¹): 3122 (NH stretching), 1717 (C=O), 1618 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.08 (s, 3H, CH₃), 6.84 (d, J = 9.1 Hz, 1H, indole C₇–H), 7.13–7.16 (m, 2H, N-phenyl C₄–H, C₅–H), 7.17 (s, 1H, thiazoline =CH), 7.27 (d, J = 9.1 Hz, 2H, phenyl C₂–H, C₆–H) 7.29 (dd, J = 9.1 Hz, 1H, indole C₆–H), 7.31–7.38 (m, 4H, N-phenyl C₆–H, phenyl C₃–H, C₅–H, indole C₄–H), 7.42 (d, J = 7.4 Hz, 1H, N-phenyl C₃–H), 10.66 (s, 1H, indole NH); EIMS (70 eV): m/z (%): 530 (M⁺ + 2, 41), 529 (M⁺ + 1, 31), 528 (M⁺, 100), 499 (5), 301 (7), 300 (19), 299 (39), 298 (40), 297 (77), 288 (8), 287 (12), 286 (48), 285 (23), 284 (81), 240 (14), 228 (7), 187 (6), 174 (6), 170 (5), 168 (12), 162 (19), 139 (5), 134 (6), 133 (7), 91 (14), 77 (5); anal. calcd for C₂₅H₁₆ClF₃N₄O₂S (528): C 56.76, H 3.03, N 10.06; found: C 56.77, H 2.98, N 10.11.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5b. Yield 88% as orange yellow fluffy crystals; mp 268–270 °C; IR (KBr, cm⁻¹): 3111 (NH stretching), 1709 (C=O), 1622 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.28 (s, 3H, CH₃), 6.83 (d, J = 8.2 Hz, 1H, indole C₇–H), 7.05 (s, 1H, N-phenyl C₂–H), 7.09 (s, 1H, thiazoline =CH), 7.13–7.37 (m, 9H, N-phenyl C₄–H, C₅–H, C₆–H, phenyl C₂–H, C₃–H, C₅–H, C₆–H, indole C₄–H, C₆–H), 10.59 (s, 1H, indole NH); EIMS (70 eV): m/z (%): 530 (M⁺ + 2, 47), 529 (M⁺ + 1, 34), 528 (M⁺, 100), 501 (6), 500 (16), 500 (7), 300 (6), 299 (11), 298 (6), 288 (10), 287 (7), 286 (32), 285 (6), 284 (30), 228 (6), 187 (4), 170 (5), 168 (12), 149 (13), 133 (4), 91 (9), 69 (9); anal. calcd for C₂₅H₁₆ClF₃N₄O₂S (528): C 56.76, H 3.03, N 10.06; found: C 56.70, H 3.00, N 10.00.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5c. Yield 92% as orange yellow fluffy crystals; mp 276–278 °C; IR (KBr, cm⁻¹): 3072 (NH stretching), 1701 (C=O), 1622 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.31 (s, 3H, OCH₃), 6.83 (d, J = 8.4 Hz, 1H, indole C₇–H), 7.11 (s, 1H, thiazoline =CH), 7.16 (dd, J = 8.3, 1.4 Hz, 1H, indole C₆–H), 7.23 (d, J = 8.4 Hz, 2H, N-phenyl C₂–H, C₆–H), 7.28 (d, J = 8.4 Hz, 2H, N-phenyl C₃–H, C₅–H), 7.30 (d, J = 8.4 Hz, 2H, phenyl C₂–H, C₆–H), 7.38 (d, J = 8.5 Hz, 2H, phenyl C₃–H, C₅–H), 7.39 (d, J = 2.0 Hz, 1H, indole C₄–H), 10.66 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 530 (M⁺ + 2, 76), 529 (M⁺ + 1, 81), 528 (M⁺, 100), 502 (11), 501 (13), 500 (37), 499 (13), 468 (8), 301 (8), 300 (10), 299 (18), 298 (14), 288 (22), 287 (16), 286 (95), 285 (19), 284 (69), 228 (12), 215 (5), 187 (7), 174 (11), 169 (6), 168 (28), 149 (40), 133 (8), 91 (18), 69 (12); anal. calcd for C₂₅H₁₆ClF₃N₄O₂S (528): C 56.76, H 3.03, N 10.06; found: C 56.72, H 3.05, N 10.11.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5d. Yield 88% as orange fluffy crystals; mp 250–252 °C; IR (KBr, cm⁻¹): 3217 (NH stretching), 1720 (C=O), 1607 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.37 (s, 3H, OCH₃), 6.84 (d, J = 8.4 Hz, 1H, indole C₇–H), 6.99 (dd, J = 7.7, 0.9 Hz, 1H, indole C₆–H), 7.03 (s, 1H, thiazoline =CH), 7.08 (d, J = 8.4 Hz, 2H, phenyl C₂–H, C₆–H), 7.2 (td, J = 8.6, 1.8 Hz, 2H, N-phenyl C₄–H, C₅–H), 7.34 (d, J = 8.5 Hz, 3H, phenyl C₃–H, C₅–H, N-phenyl C₆–H), 7.40 (dd, J = 8.3, 1.5 Hz, 1H, N-phenyl C₃–H), 7.45 (d, J = 1.6 Hz, 1H, indole C₄–H), 10.57 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 546 (M⁺ + 2, 40), 545 (M⁺ + 1, 31), 544 (M⁺, 100), 516 (6), 485 (11), 302 (39), 300 (74), 287 (8), 285 (11), 229 (5), 228 (8), 215 (3), 201 (4), 187 (4), 168 (6), 150 (3), 136 (4), 132 (5), 122 (4), 92 (4), 77 (5); anal. calcd for C₂₅H₁₆ClF₃N₄O₃S (544): C 55.10, H 2.94, N 10.28; found: C 55.07, H 2.92, N 10.20.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5e. Yield 91% as orange yellow fluffy crystals; mp 259–261 °C; IR (KBr, cm⁻¹): 3261 (NH stretching), 1719 (C=O), 1605 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.77 (s, 3H, OCH₃), 6.73 (dd, J = 7.7, 1.1 Hz, 1H, N-phenyl C₄–H), 6.86 (d, J = 8.5 Hz, 1H, indole C₇–H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H, indole C₆–H), 7.07 (s, 1H, thiazoline =CH), 7.17 (t, J = 2.1 Hz, 1H, N-phenyl C₂–H), 7.22–7.26 (m, 4H, N-phenyl C₅–H, C₆–H, phenyl C₂–H, C₆–H), 7.36 (d, J = 8.6 Hz, 2H, phenyl C₃–H, C₅–H), 7.38 (d, J = 1.8 Hz, 1H, indole C₄–H), 10.63 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 546 (M⁺ + 2, 46), 545 (M⁺ + 1, 36), 544 (M⁺, 100), 517 (6), 516 (13), 515 (7), 483 (3), 317 (6), 315 (16), 302 (43), 300 (24), 287 (3), 244 (5), 228 (3), 215 (3), 201 (4), 187 (4), 170 (4), 168 (11), 165 (14), 136 (4), 133 (5), 92 (6), 77 (5); anal. calcd for C₂₅H₁₆ClF₃N₄O₃S (544): C 55.10, H 2.94, N 10.28; found: C 54.97, H 2.90, N 10.28.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone 5f. Yield 91% as orange yellow fluffy crystals; mp 256–258 °C; IR (KBr, cm⁻¹): 3210 (NH stretching), 1719 (C=O), 1618 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.76 (s, 3H, OCH₃), 6.85 (d, J = 8.4 Hz, 1H, indole C₇–H), 6.92 (d, J = 8.9 Hz, 2H, N-phenyl C₂–H, C₆–H), 7.05 (s, 1H, thiazoline =CH), 7.22 (d, J = 8.6 Hz, 3H, phenyl C₂–H, C₆–H, indole C₆–H), 7.27 (d, J = 8.9 Hz, 2H, N-phenyl C₃–H, C₅–H), 7.36 (d, J = 8.6 Hz, 2H, phenyl C₃–H, C₅–H), 7.37 (d, J = 2.0 Hz, 1H, indole C₄–H), 10.59 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 546 (M⁺ + 2, 42), 545 (M⁺ + 1, 30), 544 (M⁺, 100), 518 (15), 517 (112), 516 (34), 515 (6), 503 (5), 501 (12), 316 (7), 315 (9), 314 (6), 302 (27), 301 (17), 300 (14), 244 (8), 201 (5), 187 (4), 174 (7), 168 (21), 165 (14), 150 (8), 133 (7), 92 (5), 77 (6); anal. calcd for C₂₅H₁₆ClF₃N₄O₃S (544): C 55.10, H 2.94, N 10.28; found: C 55.00, H 2.94, N 10.23.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5g. Yield 84% as yellow crystals; mp 256–258 °C; IR (KBr, cm⁻¹): 3221 (NH stretching), 1724 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.84 (d, J = 8.4 Hz, 1H, indole C₇–H), 7.16 (s, 1H, thiazoline =CH), 7.19–7.22 (m, 2H, N-phenyl C₅–H, C₆–H), 7.30 (d, J = 8.5 Hz, 3H, phenyl C₂–H, C₆–H, indole C₆–H), 7.35–7.42 (m, 3H, phenyl C₃–H, C₅–H, indole C₄–H), 7.50–7.57 (m, 1H, N-phenyl C₄–H), 7.69 (t, J = 7.2 Hz, 1H, N-phenyl C₃–H), 10.71 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 534 (M⁺ + 2, 84), 533 (M⁺ + 1, 64), 532 (M⁺, 100), 504 (21), 503 (7), 292 (32), 291 (17), 290 (80), 266 (7), 241 (8), 232 (16), 215 (8), 187 (9), 170 (8), 168 (19), 153 (35), 136 (12), 133 (6), 95 (5), 69 (12); anal. calcd for C₂₄H₁₃ClF₄N₄O₂S (532): C 54.08, H 2.44, N 10.52; found: C 54.04, H 2.46, N 10.49.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5h. Yield 86% as orange yellow crystals; mp 248–250 °C; IR (KBr, cm⁻¹): 3219 (NH stretching), 1720 (C=O), 1606 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.84 (d, J = 8.4 Hz, 1H, indole C₇–H), 7.13 (s, 1H, thiazoline =CH), 7.18 (dd, J = 8.4, 1.7 Hz, 1H, indole C₆–H), 7.22 (dd, J = 7.9, 0.9 Hz, 1H, N-phenyl C₆–H), 7.25–7.29 (m, 1H, N-phenyl C₅–H), 7.33 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.36 (d, J = 1.7 Hz, 1H, indole C₄–H), 7.40 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 7.44–7.49 (m, 1H, N-phenyl C₄–H), 7.59 (dt, J = 9.5, 2.1 Hz, 1H, N-phenyl C₄–H), 10.69 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 534 (M⁺ + 2, 93), 533 (M⁺ + 1, 77), 532 (M⁺, 100), 506 (10), 505 (11), 504 (25), 503 (12), 304 (7), 303 (13), 302 (6), 293 (7), 292 (44), 291 (23), 290 (90), 289 (8), 288 (30), 241 (11), 232 (16), 215 (15), 187 (14), 170 (8), 168 (20), 153 (23), 136 (19), 133 (7), 95 (13), 69 (13); anal. calcd for C₂₄H₁₃ClF₄N₄O₂S (532): C 54.08, H 2.44, N 10.52; found: C 54.06, H 2.49, N 10.54.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5i. Yield 88% as orange yellow crystals; mp 274–276 °C; IR (KBr, cm⁻¹): 3221 (NH stretching), 1724 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.84 (d, J = 8.5 Hz, 1H, indole C₇–H), 7.11 (s, 1H, thiazoline =CH), 7.16 (dd, J = 8.5, 1.7 Hz, 1H, indole C₆–H), 7.27 (d, J = 8.6 Hz, 2H, phenyl C₂–H, C₆–H), 7.35 (d, J = 8.6 Hz, 2H, phenyl C₃–H, C₅–H), 7.41 (dd, J = 8.5, 1.9 Hz, 2H, N-phenyl C₂–H, C₆–H), 7.51 (d, J = 1.5 Hz, 1H, indole C₄–H), 7.54 (t, J = 8.7 Hz, 2H, N-phenyl C₃–H, C₅–H), 10.67 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 534 (M⁺ + 2, 32), 533 (M⁺ + 1, 30), 532 (M⁺, 100), 504 (11), 503 (7), 303 (5), 292 (11), 291 (8), 290 (33), 288 (10), 232 (7), 215 (5), 187 (6), 168 (9), 153 (15), 136 (4), 133 (4), 95 (6), 69 (9); anal. calcd for C₂₄H₁₃ClF₄N₄O₂S (532): C 54.08, H 2.44, N 10.52; found: C 54.06, H 2.46, N 10.55.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,4-dichlorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5j. Yield 78% as orange brown crystals; mp 258–260 °C; IR (KBr, cm⁻¹): 3130 (NH stretching), 1715 (C=O), 1618 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.85 (d, J = 9.0 Hz, 1H, indole C₇–H), 7.16 (s, 1H, thiazoline =CH), 7.16–7.20 (m, 1H, indole C₆–H), 7.31 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.44 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 7.55 (d, J = 2.0 Hz, 1H, indole C₄–H), 7.62 (dd, J = 8.6, 2.3 Hz, 1H, N-phenyl C₅–H), 7.84 (d, J = 2.3 Hz, 1H, N-phenyl C₃–H), 7.89 (d, J = 8.6 Hz, 1H, N-phenyl C₆–H), 10.71 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 584 (M⁺ + 1, 94), 583 (M⁺, 26), 582 (M⁺ − 1, 100), 555 (13), 553 (11), 549 (9), 547 (12), 521 (23), 519 (30), 376 (16), 374 (13), 344 (16), 342 (49), 341 (11), 319 (10), 318 (8), 292 (7), 291 (8), 245 (14), 215 (12), 205 (22), 203 (30), 188 (9), 187 (12), 170 (9), 168 (24), 136 (9), 133 (7), 69 (13); anal. calcd for C₂₄H₁₂Cl₃F₃N₄O₂S (583): C 49.36, H 2.06, N 9.60; found: C 49.36, H 2.03, N 9.57.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,5-dichlorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5k. Yield 86% as orange brown crystals; mp 266–268 °C; IR (KBr, cm⁻¹): 3121 (NH stretching), 1717 (C=O), 1620 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.86 (d, J = 8.3 Hz, 1H, indole C₇–H), 7.16 (s, 1H, thiazoline =CH), 7.18–7.33 (m, 1H, indole C₆–H), 7.35 (d, J = 8.6 Hz, 2H, phenyl C₂–H, C₆–H), 7.45 (d, J = 8.6 Hz, 2H, phenyl C₃–H, C₅–H), 7.60 (d, J = 2.5 Hz, 1H, indole C₄–H), 7.67 (d, J = 8.7 Hz, 2H, N-phenyl C₃–H, C₄–H), 8.16 (d, J = 2.4 Hz, N-phenyl C₆–H), 10.72 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 584 (M⁺ + 1, 99), 583 (M⁺, 32), 582 (M⁺ − 1, 100), 549 (20), 547 (29), 521 (19), 519 (24), 484 (24), 344 (15), 342 (39), 340 (41), 319 (12), 318 (11), 205 (10), 203 (14), 168 (14), 136 (12), 69 (11); anal. calcd for C₂₄H₁₂Cl₃F₃N₄O₂S (583): C 49.36, H 2.06, N 9.60; found: C 49.33, H 2.03, N 9.59.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,6-dichlorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5l. Yield 83% as orange yellow fluffy crystals; mp 262–264 °C; IR (KBr, cm⁻¹): 3225 (NH stretching), 1717 (C=O), 1605 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.86 (d, J = 8.4 Hz, 1H, indole C₇–H), 7.13–7.18 (m, 2H, indole C₄–H, C₆–H), 7.20 (s, 1H, thiazoline =CH), 7.30 (d, J = 8.6 Hz, 2H, phenyl C₂–H, C₆–H), 7.45 (d, J = 8.6 Hz, 2H, phenyl C₃–H, C₅–H), 7.57 (t, J = 9.0 Hz, 1H, N-phenyl C₄–H), 7.70 (dd, J = 8.7, 1.1 Hz, 2H, N-phenyl C₃–H, C₅–H), 10.73 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 586 (M⁺ + 3, 61), 584 (M⁺ + 1, 100), 583 (M⁺, 40), 582 (M⁺ − 1, 100), 521 (27), 520 (11), 519 (46), 484 (7), 344 (21), 343 (10), 342 (67), 341 (12), 340 (40), 319 (7), 293 (8), 292 (7), 291 (9), 284 (7), 215 (8), 205 (18), 203 (27), 187 (9), 168 (22), 136 (16), 69 (11); anal. calcd for C₂₄H₁₂Cl₃F₃N₄O₂S (583): C 49.36, H 2.06, N 9.60; found: C 49.34, H 2.01, N 9.54.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3,4-dichlorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5m. Yield 84% as orange yellow crystals; mp 276–278 °C; IR (KBr, cm⁻¹): 3163 (NH stretching), 1699 (C=O), 1622 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.87 (d, J = 8.5 Hz, 1H, indole C₇–H), 7.01 (s, 1H, thiazoline =CH), 7.24–7.29 (m, 4H, N-phenyl C₆–H, phenyl C₂–H, C₆–H, indole C₆–H), 7.36 (d, J = 1.6 Hz, 1H, indole C₄–H), 7.42 (d, J = 8.6 Hz, 2H, phenyl C₃–H, C₅–H), 7.64 (d, J = 8.6 Hz, 1H, N-phenyl C₅–H), 7.91 (d, J = 2.4 Hz, IH, N-phenyl C₂–H), 10.62 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 584 (M⁺ + 1, 92), 583 (M⁺, 32), 582 (M⁺ − 1, 100), 556 (14), 554 (15), 532 (8), 344 (14), 342 (48), 341 (10), 340 (47), 284 (7), 215 (12), 203 (10), 187 (11), 168 (12), 136 (7), 69 (11); anal. calcd for C₂₄H₁₂Cl₃F₃N₄O₂S (583): C 49.36, H 2.06, N 9.60; found: C 49.30, H 2.03, N 9.66.
(3Z)-5-(Trifluoromethoxy)-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,4,6-trichlorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 5n. Yield 81% as orange yellow crystals; mp 280–282 °C; IR (KBr, cm⁻¹): 3167 (NH stretching), 1711 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.87 (d, J = 8.5 Hz, 1H, indole C₇–H), 7.16–7.24 (m, 3H, indole C₄–H, indole C₆–H, thiazoline =CH), 7.30 (d, J = 8.6 Hz, 2H, phenyl C₂–H, C₆–H), 7.50 (d, J = 8.6 Hz, 2H, phenyl C₃–H, C₅–H), 7.98 (s, 2H, N-phenyl C₃–H, C₅–H), 10.75 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 620 (M⁺ + 2, 17), 618 (M⁺, 32), 584 (94), 582 (100), 555 (13), 553 (11), 549 (9), 547 (12), 521 (23), 519 (30), 376 (16), 374 (13), 344 (16), 342 (49), 341 (11), 319 (10), 318 (8), 292 (7), 291 (8), 245 (14), 215 (12), 205 (22), 203 (30), 188 (9), 187 (12), 170 (9), 168 (24), 136 (9), 133 (7), 69 (13); anal. calcd for C₂₄H₁₁Cl₄F₃N₄O₂S (618): C 46.60, H 1.78, N 9.06; found: C 46.60, H 1.77, N 9.02.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6a. Yield 79% as bright orange crystals; mp 276–278 °C; IR (KBr, cm⁻¹): 3179 (NH stretching), 1705 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.08 (s, 3H, CH₃), 6.72 (dd, J = 8.7, 4.5 Hz, 1H, indole C₇–H), 6.89 (dd, J = 9.3, 2.4 Hz, 1H, N-phenyl C₆–H), 6.99 (dd, J = 8.7, 2.7 Hz, 1H, N-phenyl C₃–H), 7.15 (s, 1H, thiazoline =CH), 7.28 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.33–7.46 (m, 6H, N-phenyl C₄–H, C₅–H, phenyl C₃–H, C₅–H, indole C₄–H, indole C₆–H), 10.45 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 464 (M⁺ + 2, 39), 463 (M⁺ + 1, 28), 462 (M⁺, 100), 433 (6), 300 (17), 299 (43), 298 (38), 297 (82), 288 (13), 287 (16), 286 (64), 285 (24), 284 (100), 240 (16), 225 (8), 168 (18), 162 (25), 136 (11), 134 (11), 121 (17), 108 (15), 91 (19), 89 (10), 65 (17); anal. calcd for C₂₄H₁₆ClFN₄OS (462): C 62.27, H 3.46, N 12.11; found: C 62.25, H 3.43, N 12.10.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6b. Yield 87% as orange fluffy crystals; mp 278–280 °C; IR (KBr, cm⁻¹): 3217 (NH stretching), 1720 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.27 (s, 3H, CH₃), 6.75 (dd, J = 8.4, 4.2 Hz, 1H, indole C₇–H), 7.02–7.08 (m, 3H, thiazoline =CH, N-phenyl C₄–H, C₆–H), 7.13–7.28 (m, 6H, N-phenyl C₂–H, C₅–H, phenyl C₂–H, C₆–H, indole C₄–H, C₆–H), 7.35 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H) 10.39 (s, 1H, indole NH); EIMS (70 eV): m/z (%): 464 (M⁺ + 2, 37), 463 (M⁺ + 1, 29), 462 (M⁺, 100), 434 (13), 299 (15), 288 (13), 286 (44), 284 (25), 228 (7), 168 (16), 149 (21), 136 (7), 121 (8), 91 (13), 65 (8); anal. calcd for C₂₄H₁₆ClFN₄OS (462): C 62.27, H 3.46, N 12.11; found: C 62.24, H 3.43, N 12.13.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6c. Yield 89% as bright orange yellow fluffy crystals; mp 278–280 °C; IR (KBr, cm⁻¹): 3217 (NH stretching), 1717 (C=O), 1606 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.31, 2.34 (2s, 3H, CH₃), 6.75, 6.79 (2d, J = 8.4, 4.2 Hz, 1H, indole C₇–H), 6.95–7.13 (m, 2H, indole C₆–H, thiazoline =CH), 7.18–7.49 (m, 9H, N-phenyl C₂–H, C₃–H, C₅–H, C₆–H, phenyl C₂–H, C₃–H, C₅–H, C₆–H, indole C₄–H), 10.48, 10.52 (2s, 1H, indole NH); EIMS (70 eV): m/z (%): 464 (M⁺ + 2, 96), 463 (M⁺ + 1, 82), 462 (M⁺, 100), 461 (13), 436 (13), 435 (15), 434 (34), 433 (20), 402 (9), 301 (112), 300 (16), 299 (28), 298 (16), 288 (37), 287 (25), 286 (91), 285 (21), 284 (58), 240 (9), 228 (15), 170 (12), 168 (31), 149 (37), 136 (8), 133 (8), 121 (9), 91 (24), 65 (17); anal. calcd for C₂₄H₁₆ClFN₄OS (462): C 62.27, H 3.46, N 12.11; found: C 62.23, H 3.44, N 12.15.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6d. Yield 88% as orange crystals; mp 274–276 °C; IR (KBr, cm⁻¹): 3209 (NH stretching), 1719 (C=O), 1606 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.58, 3.61 (2s, 3H, OCH₃), 6.73 (dd, J = 8.4, 4.2 Hz, 1H, indole C₇–H), 6.95–7.07 (m, 4H, thiazoline =CH, N-phenyl C₃–H, C₄–H, C₅–H), 7.17 (dd, J = 8.7 Hz, phenyl C₂–H, C₆–H), 7.26 (d, J = 8.7 Hz, 1H, N-phenyl C₆–H), 7.32 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 7.36–7.52 (m, 2H, indole C₄–H, C₆–H), 10.35, 10.44 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 480 (M⁺ + 2, 44), 479 (M⁺ + 1, 24), 478 (M⁺, 100), 450 (6), 419 (12), 304 (9), 303 (10), 302 (36), 301 (14), 300 (58), 287 (8), 286 (10), 285 (13), 272 (12), 270 (16), 168 (9), 136 (7), 121 (7), 108 (9), 77 (6); anal. calcd for C₂₄H₁₆ClFN₄O₂S (478): C 60.19, H 3.37, N 11.70; found: C 60.17, H 3.33, N 11.73.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6e. Yield 88% as orange brown crystals; mp 276–278 °C; IR (KBr, cm⁻¹): 3171 (NH stretching), 1693 (C=O), 1607 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.73 (s, 3H, OCH₃), 6.76 (dd, J = 8.7, 4.5 Hz, 1H, indole C₇–H), 6.91 (dd, J = 7.8, 1.2 Hz, 1H, N-phenyl C₆–H), 6.97–7.05 (m, 2H, N-phenyl C₄–H, C₅–H), 7.11 (s, 1H, thiazoline =CH), 7.19 (t, J = 2.1 Hz, 1H, N-phenyl C₂–H), 7.27 (dd, J = 9.0, 2.7 Hz, 1H, indole C₆–H), 7.32 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.36 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 7.41 (dd, J = 8.7, 2.1 Hz, 1H, indole C₄–H), 10.51 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 480 (M⁺ + 2, 81), 479 (M⁺ + 1, 67), 478 (M⁺, 100), 451 (10), 450 (21), 449 (11), 317 (11), 316 (11), 315 (25), 304 (29), 303 (18), 302 (92), 301 (12), 300 (42), 287 (6), 244 (9), 168 (15), 165 (19), 136 (8), 121 (6), 108 (5), 92 (7), 77 (7); anal. calcd for C₂₄H₁₆ClFN₄O₂S (478): C 60.19, H 3.37, N 11.70; found: C 60.10, H 3.36, N 11.71.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6f. Yield 90% as orange crystals; mp 270–272 °C; IR (KBr, cm⁻¹): 3177 (NH stretching), 1693 (C=O), 1607 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.75, 3.77 (2s, 3H, OCH₃), 6.76 (dd, J = 8.7, 4.5 Hz, 1H, indole C₇–H), 6.90, 6.93 (2s, 1H, thiazoline =CH), 6.98–7.01 (m, 3H, N-phenyl C₂–H, C₆–H, indole C₆–H), 7.13–7.29 (m, 4H, phenyl C₂–H, C₆–H, N-phenyl C₃–H, C₅–H), 7.32–7.41 (m, 3H, phenyl C₃–H, C₅–H, indole C₄–H), 10.38, 10.45 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 480 (M⁺ + 2, 44), 479 (M⁺ + 1, 35), 478 (M⁺, 100), 452 (14), 451 (14), 450 (29), 449 (9), 435 (10), 316 (10), 315 (16), 304 (10), 303 (12), 302 (33), 301 (19), 300 (18), 286 (11), 284 (8), 244 (13), 168 (21), 165 (14), 150 (11), 136 (10), 135 (9), 121 (8), 108 (12), 77 (7); anal. calcd for C₂₄H₁₆ClFN₄O₂S (478): C 60.19, H 3.37, N 11.70; found: C 60.17, H 3.33, N 11.73.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6g. Yield 67% as bright orange crystals; mp 272–274 °C; IR (KBr, cm⁻¹): 3146 (NH stretching), 1715 (C=O), 1600 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.74 (dd, J = 8.7, 4.5 Hz, 1H, indole C₇–H), 7.06–7.09 (m, 3H, thiazoline =CH, N-phenyl C₄–H, C₅–H), 7.15–7.23 (m, 2H, N-phenyl C₃–H, C₆–H), 7.25 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.38 (d, J = 8.7 Hz, 3H, phenyl C₃–H, C₅–H, indole C₆–H), 7.46 (d, J = 9.9 Hz, 1H, indole C₄–H), 10.41 (s, 1H, indole NH); EIMS (70 eV): m/z (%): 468 (M⁺ + 2, 43), 467 (M⁺ + 1, 23), 466 (M⁺, 100), 438 (11), 437 (7), 305 (7), 304 (10), 303 (18), 292 (29), 291 (16), 290 (80), 288 (11), 232 (16), 170 (8), 168 (19), 153 (14), 149 (14), 136 (19), 135 (8), 134 (8), 133 (8), 121 (14), 108 (12), 95 (17), 75 (10); anal. calcd for C₂₃H₁₃ClF₂N₄OS (466): C 59.16, H 2.79, N 12.00; found: C 59.19, H 2.77, N 11.98.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6h. Yield 87% as orange crystals, mp 278–280 °C; IR (KBr, cm⁻¹): 3192 (NH stretching), 1707 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76 (dd, J = 9.3, 4.2 Hz, 1H, indole C₇–H), 7.03 (dd, J = 6.9, 2.1 Hz, 2H, N-phenyl C₅–H, C₆–H), 7.15 (s, 1H, thiazoline =CH), 7.31 (d, J = 8.4 Hz, 2H, phenyl C₂–H, C₆–H), 7.35–7.47 (m, 4H, phenyl C₃–H, C₅–H, indole C₄–H, C₆–H), 7.55–7.66 (m, 1H, N-phenyl C₄–H), 7.70 (td, J = 7.5, 1.5 Hz, 1H, N-phenyl C₂–H), 10.53 (s, 1H, indole NH); EIMS (70 eV): m/z (%): 468 (M⁺ + 2, 87), 467 (M⁺ + 1, 72), 466 (M⁺, 100), 440 (8), 439 (8), 438 (20), 437 (8), 304 (7), 292 (49), 291 (24), 290 (89), 289 (6), 241 (11), 234 (12), 233 (12), 232 (25), 170 (10), 168 (26), 153 (41), 149 (13), 136 (17), 121 (14), 108 (12), 95 (9), 75 (9); anal. calcd for C₂₃H₁₃ClF₂N₄OS (466): C 59.16, H 2.79, N 12.00; found: C 59.17, H 2.79, N 12.01.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6i. Yield 88% as orange yellow crystals; mp 298–300 °C; IR (KBr, cm⁻¹): 3177 (NH stretching), 1705 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.75 (dd, J = 8.7, 4.5 Hz, 1H, indole C₇–H), 7.03 (td, J = 9.3, 2.7 Hz, 1H, indole C₆–H), 7.09 (dd, J = 9.0, 2.7 Hz, 1H, indole C₄–H), 7.11 (s, 1H, thiazoline =CH), 7.29 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.37 (t, J = 8.7 Hz, 2H, N-phenyl C₂–H, C₆–H), 7.41 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 7.55 (dd, J = 8.7, 4.5 Hz, 2H, N-phenyl C₃–H, C₅–H), 10.50 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 468 (M⁺ + 2, 75), 467 (M⁺ + 1, 55), 466 (M⁺, 100), 440 (8), 439 (9), 438 (18), 437 (11), 305 (6), 304 (8), 303 (12), 292 (35), 291 (19), 290 (82), 288 (22), 234 (7), 232 (16), 170 (8), 168 (19), 153 (26), 149 (13), 136 (9), 134 (7), 133 (6), 121 (12), 108 (6), 95 (14), 75 (7); anal. calcd for C₂₃H₁₃ClF₂N₄OS (466): C 59.16, H 2.79, N 12.00; found: C 59.16, H 2.77, N 11.99.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,4-difluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6j. Yield 91% as orange yellow crystals; mp 298–300 °C; IR (KBr, cm⁻¹): 3184 (NH stretching), 1707 (C=O), 1606 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.75 (dd, J = 8.4, 4.2 Hz, 1H, indole C₇–H), 7.01 (d, J = 9.3 Hz, 2H, indole C₄–H, C₆–H), 7.13 (s, 1H, thiazoline =CH), 7.32 (d, J = 8.4, 3H, phenyl C₂–H, C₆–H, N-phenyl C₆–H), 7.44 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H), 7.55 (td, J = 8.4, 2.4 Hz, 1H, N-phenyl C₅–H), 7.82–7.86 (m, 1H, N-phenyl C₃–H), 10.50 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 486 (M⁺ + 2, 56), 485 (M⁺ + 1, 40), 484 (M⁺, 100), 456 (12), 310 (28), 308 (75), 259 (6), 250 (14), 171 (19), 168 (14), 149 (12), 136 (10), 135 (8), 121 (11), 111 (11); anal. calcd for C₂₃H₁₂ClF₃N₄OS (484): C 56.97, H 2.48, N 11.56; found: C 56.99, H 2.44, N 11.57.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,6-difluoro-phenyl)1,3-thiazol-2(3H)-ylidene]hydrazone} 6k. Yield 78% as orange brown crystals; mp 278–280 °C; IR (KBr, cm⁻¹): 3185 (NH stretching), 1722 (C=O), 1622 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76 (dd, J = 8.4, 4.2 Hz, 1H, indole C₇–H), 6.96–7.07 (m, 2H, indole C₄–H, C₆–H), 7.18 (s, 1H, thiazoline =CH), 7.29 (d, J = 8.4, 2H, phenyl C₂–H, C₆–H), 7.39 (t, J = 8.7 Hz, 2H, N-phenyl C₃–H, C₅–H), 7.45 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H), 7.62–7.74 (m, 1H, N-phenyl C₄–H), 10.54 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 486 (M⁺ + 2, 43), 485 (M⁺ + 1, 31), 484 (M⁺, 94), 456 (11), 455 (18), 453 (20), 344 (23), 343 (14), 342 (64), 341 (13), 340 (63), 310 (41), 309 (19), 308 (97), 294 (13), 284 (14), 282 (14), 259 (11), 258 (10), 252 (11), 250 (38), 205 (27), 203 (45), 177 (16), 171 (61), 170 (44), 168 (98), 162 (18), 156 (18), 150 (19), 149 (65), 145 (18), 140 (15), 139 (32), 138 (41), 137 (15), 136 (100), 135 (38), 134 (41), 133 (40), 125 (15), 123 (16), 122 (22), 121 (98), 113 (32), 111 (83), 108 (70), 101 (26), 100 (14), 94 (56), 89 (57); anal. calcd for C₂₃H₁₂ClF₃N₄OS (484): C 56.97, H 2.48, N 11.56; found: C 56.96, H 2.49, N 11.58.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,4-dichlorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6l. Yield 86% as orange crystals; mp 270–272 °C; IR (KBr, cm⁻¹): 3171 (NH stretching), 1708 (C=O), 1624 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76 (dd, J = 8.4, 4.2 Hz, 1H, indole C₇–H), 6.89 (dd, J = 9.0, 2.7 Hz, 1H, indole C₄–H), 7.05–7.11 (m, 1H, indole C₆–H), 7.15 (s, 1H, thiazoline =CH), 7.33 (d, J = 8.4 Hz, 2H, phenyl C₂–H, C₆–H), 7.44 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H), 7.68 (dd, J = 8.4, 2.4 Hz, 1H, N-phenyl C₅–H), 7.91 (d, J = 7.5 Hz, 1H, N-phenyl C₆–H), 7.89 (d, J = 2.1 Hz, 1H, N-phenyl C₃–H), 10.54 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 518 (M⁺ + 1, 88), 517 (M⁺, 22), 516 (M⁺ − 1, 87), 455 (20), 553 (23), 344 (26), 342 (86), 340 (83), 319 (27), 284 (20), 282 (22), 205 (39), 203 (63), 170 (30), 169 (16), 168 (95), 162 (34), 149 (58), 147 (16), 145 (31), 139 (24), 138 (27), 136 (76), 135 (34), 134 (55), 133 (38), 121 (100), 111 (64), 109 (54), 108 (61), 94 (47); anal. calcd for C₂₃H₁₂Cl₃FN₄OS (517): C 53.33, H 2.32, N 10.82; found: C 53.34, H 2.34, N 10.84.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,5-dichlorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6m. Yield 87% as orange brown crystals; mp 274–276 °C; IR (KBr, cm⁻¹): 3225 (NH stretching), 1697 (C=O), 1590 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76 (dd, J = 8.4, 4.2 Hz, 1H, indole C₇–H), 6.98 (dd, J = 8.7, 3.0 Hz, 1H, indole C₄–H), 7.03 (td, J = 9.3, 2.7 Hz, 1H, indole C₆–H), 7.20 (s, 1H, thiazoline =CH), 7.31 (d, J = 8.4, 2H, phenyl C₂–H, C₆–H), 7.34–7.48 (m, 4H, N-phenyl C₃–H, C₆–H, phenyl C₃–H, C₅–H), 7.59–7.74 (m, 1H, N-phenyl C₄–H), 10.58 (s, 1H, indole NH); EIMS (70 eV): m/z (%): 518 (M⁺ + 1, 45), 517 (M⁺, 34), 516 (M⁺ − 1, 34), 455 (6), 343 (37), 342 (38), 341 (40), 340 (42), 319 (20), 284 (10), 282 (12), 205 (38), 203 (54), 187 (14), 180 (17), 179 (35), 177 (9), 170 (15), 168 (47), 163 (12), 162 (30), 150 (17), 149 (43), 147 (25), 145 (40), 141 (24), 139 (71), 137 (29), 136 (100), 135 (35), 134 (50), 133 (34), 122 (41), 121 (84), 111 (89), 109 (80), 108 (96), 107 (23), 101 (34), 100 (24), 95 (52), 75 (67); anal. calcd for C₂₃H₁₂Cl₃FN₄OS (517): C 53.33, H 2.32, N 10.82; found: C 53.32, H 2.30, N 10.85.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,6-dichlorophenyl)1,3-thiazol-2(3H)-ylidene]hydrazone} 6n. Yield 88% as reddish brown crystals; mp 272–274 °C; IR (KBr, cm⁻¹): 3177 (NH stretching), 1703 (C=O), 1620 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76 (dd, J = 8.7, 4.5 Hz, 1H, indole C₇–H), 6.91 (dd, J = 8.7, 3.0 Hz, 1H, indole C₄–H), 7.03 (td, J = 9.3, 2.7 Hz, 1H, indole C₆–H), 7.13 (s, 1H, thiazoline =CH), 7.31 (d, J = 8.4, 2H, phenyl C₂–H, C₆–H), 7.45 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 7.63 (t, J = 7.2 Hz, 1H, N-phenyl C₄–H), 7.76 (d, J = 7.5 Hz, 2H, N-phenyl C₃–H, C₅–H), 10.41 (s, 1H, indole NH); EIMS (70 eV): m/z (%): 518 (M⁺ + 1, 11), 516 (M⁺ − 1, 13), 340 (12), 319 (13), 284 (7), 282 (9), 205 (8), 203 (10), 180 (21), 168 (28), 164 (8), 162 (31), 149 (20), 141 (28), 139 (100), 138 (14), 136 (46), 134 (28), 133 (11), 121 (32), 113 (18), 111 (70), 109 (35), 108 (23), 107 (12), 94 (16), 75 (59); anal. calcd for C₂₃H₁₂Cl₃FN₄OS (517): C 53.33, H 2.32, N 10.82; found: C 53.33, H 2.34, N 10.82.
(3Z)-5-Fluoro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,4,6-trichlorophe-nyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 6o. Yield 85% as brown crystals; mp 258–260 °C; IR (KBr, cm⁻¹): 3195 (NH stretching), 1701 (C=O), 1631 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76 (dd, J = 8.7, 4.5 Hz, 1H, indole C₇–H), 6.86 (dd, 1H, J = 8.7, 3.0 Hz indole C₄–H), 7.05 (td, J = 8.7, 2.7 Hz, 1H, indole C₆–H), 7.22 (s, 1H, thiazoline =CH), 7.32 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.49 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 8.06 (s, 2H, N-phenyl C₃–H, C₅–H), 10.43, 10.57 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 552 (M⁺, 3), 550 (M⁺ − 2, 3), 518 (76), 516 (53), 344 (23), 342 (57), 340 (56), 319 (24), 284 (19), 282 (19), 205 (44), 203 (70), 179 (29), 170 (25), 169 (17), 168 (85), 162 (43), 149 (66), 147 (23), 145 (36), 141 (23), 139 (90), 138 (26), 136 (71), 135 (38), 134 (55), 133 (37), 124 (20), 122 (39), 121 (88), 113 (29), 111 (100), 109 (53), 108 (71), 94 (55), 89 (50), 75 (73); anal. calcd for C₂₃H₁₁Cl₄FN₄OS (552): C 50.00, H 1.99, N 10.14; found: C 50.03, H 1.99, N 10.16.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-methylphenyl)1,3-thiazol-2(3H)-ylidene]hydrazone} 7a. Yield 85% as orange yellow fluffy crystals; mp 288–290 °C; IR (KBr, cm⁻¹): 3215 (NH stretching), 1719 (C=O), 1615 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.49 (s, 3H, CH₃), 6.76 (2d, J = 8.4, 8.1 Hz, 1H, indole C₇–H), 6.94–7.03 (m, 1H, N-phenyl C₄–H), 7.07 (s, 1H, thiazoline =CH), 7.18 (d, J = 8.4 Hz, 2H, N-phenyl C₃–H, C₆–H), 7.25 (td, J = 8.4, 2.1 Hz, 1H, N-phenyl C₅–H), 7.32 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.35 (d, J = 8.7 Hz, 2H, phenyl C₃–H, C₅–H), 7.42 (d, J = 9.0 Hz, 1H, indole C₆–H), 7.47, 7.50 (2s, 1H, indole C₄–H), 10.46, 10.54 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 480 (M⁺ + 2, 78), 478 (M⁺, 100), 301 (19), 300 (29), 299 (54), 288 (25), 287 (18), 286 (100), 284 (58), 228 (32), 180 (17), 178 (27), 170 (17), 168 (50), 152 (18), 151 (13), 150 (25), 149 (57), 139 (35), 137 (17), 136 (21), 133 (19), 111 (21), 91 (98), 89 (25); anal. calcd for C₂₄H₁₆Cl₂N₄OS (478): C 60.13, H 3.34, N 11.69; found: C 60.15, H 3.34, N 11.70.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7b. Yield 86% as orange yellow fluffy crystals; mp 290–292 °C; IR (KBr, cm⁻¹): 3224 (NH stretching), 1719 (C=O), 1619 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.27 (s, 3H, CH₃), 6.76 (d, J = 8.1 Hz, 1H, indole C₇–H), 7.04–7.08 (m, 2H, N-phenyl C₄–H, thiazoline =CH), 7.15–7.27 (m, 6H, N-phenyl C₂–H, C₄–H, C₆–H, phenyl C₂–H, C₆–H, indole C₆–H), 7.35 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H), 7.40 (d, J = 2.1 Hz, 1H, indole C₄–H), 10.52 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 479 (M⁺ + 1, 88), 478 (M⁺, 78), 301 (16), 300 (19), 299 (41), 288 (23), 287 (15), 286 (80), 284 (37), 228 (27), 180 (19), 178 (25), 170 (17), 169 (13), 168 (42), 165 (13), 152 (24), 151 (15), 150 (15), 149 (54), 139 (34), 138 (17), 137 (21), 136 (16), 133 (23), 124 (15), 111 (17), 102 (14), 91 (100), 89 (28); anal. calcd for C₂₄H₁₆Cl₂N₄OS (478): C 60.13, H 3.34, N 11.69; found: C 60.14, H 3.36, N 11.66.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-methylphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7c. Yield 87% as orange yellow fluffy crystals; mp 284–286 °C; IR (KBr, cm⁻¹): 3218 (NH stretching), 1720 (C=O), 1625 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 2.31, 2.34 (2s, 3H, CH₃), 6.76, 6.80 (2d, J = 8.4, 8.1 Hz, 1H, indole C₇–H), 7.11 (s, 1H, thiazoline =CH), 7.17–7.26 (m, 3H, N-phenyl C₂–H, C₆–H, indole C₆–H), 7.29–7.42 (m, 7H, N-phenyl C₃–H, C₅–H, phenyl C₂–H, C₃–H, C₅–H, C₆–H, indole C₄–H), 10.59, 10.60 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 479 (M⁺ + 1, 100), 477 (93), 301 (14), 300 (15), 299 (33), 288 (16), 286 (70), 284 (32), 228 (22), 178 (15), 168 (35), 149 (38), 91 (68), 89 (16); anal. calcd for C₂₄H₁₆Cl₂N₄OS (478): C 60.13, H 3.34, N 11.69; found: C 60.13, H 3.33, N 11.71.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7d. Yield 91% as bright orange crystals; mp 250–252 °C; IR (KBr, cm⁻¹): 3096 (NH stretching), 1721 (C=O), 1619 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.60, 3.63 (2s, 3H, OCH₃), 6.77 (d, J = 8.4 Hz, 1H, N-phenyl C₃–H), 6.83 (d, J = 8.4 Hz, 1H, indole C₇–H), 7.00–7.12 (m, 2H, N-phenyl C₆–H, thiazoline =CH), 7.19 (t, J = 7.5 Hz, 2H, N-phenyl C₄–H, C₅–H), 7.25 (d, J = 8.1 Hz, phenyl C₂–H, C₆–H), 7.35 (d, J = 8.4 Hz, 1H, indole C₆–H), 7.46 (d, J = 8.1 Hz, 2H, phenyl C₃–H, C₅–H), 7.50 (s, 1H, indole C₄–H), 10.59, 10.73 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 496 (M⁺ + 2, 38), 494 (M⁺, 59), 462 (16), 303 (13), 302 (60), 301 (19), 300 (100), 287 (15), 286 (30), 285 (25), 284 (13), 272 (21), 270 (38), 229 (12), 180 (12), 178 (29), 170 (13), 168 (39), 165 (29), 152 (17), 151 (18), 150 (31), 149 (17), 139 (19), 138 (21), 137 (25), 136 (24), 134 (15), 133 (23), 132 (23), 124 (23), 123 (20), 122 (31), 108 (27), 102 (17), 92 (40), 91 (31), 89 (31); anal. calcd for C₂₄H₁₆Cl₂N₄O₂S (494): C 58.18, H 3.23, N 11.31; found: C 58.17, H 3.24, N 11.30.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-methoxyphenyl) 1,3-thiazol-2(3H)-ylidene]hydrazone} 7e. Yield 85% as orange crystals; mp 296–298 °C; IR (KBr, cm⁻¹): 3195 (NH stretching), 1720 (C=O), 1614 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.72 (s, 3H, OCH₃), 6.77, 6.80 (2d, J = 8.4, 8.1 Hz, 1H, indole C₇–H), 6.93 (td, J = 8.4, 2.4 Hz, 1H, N-phenyl C₅–H), 7.03 (dd, J = 8.4, 2.4 Hz, 1H, N-phenyl C₆–H), 7.10, 7.12 (2s, 1H, thiazoline =CH), 7.14–7.19 (m, 1H, N-phenyl C₄–H), 7.21 (t, J = 2.0 Hz, 1H, N-phenyl C₂–H), 7.25 (dd, J = 8.1, 2.4 Hz, 1H, indole C₆–H), 7.34 (d, J = 8.4 Hz, 2H, phenyl C₂–H, C₆–H), 7.40 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H), 7.51 (d, J = 2.1 Hz, 1H, indole C₄–H), 10.58 (s, 1H, indole NH); EIMS (70 eV) m/z (%): 496 (M⁺ + 2, 37), 494 (M⁺, 52), 315 (20), 304 (16), 302 (52), 300 (28), 244 (11), 178 (12), 170 (11), 168 (33), 165 (45), 152 (13), 151 (11), 137 (17), 136 (16), 133 (12), 124 (12), 107 (10), 92 (41), 89 (16), 77 (100); anal. calcd for C₂₄H₁₆Cl₂N₄O₂S (494): C 58.18, H 3.23, N 11.31; found: C 58.16, H 3.22, N 11.33.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-methoxyphenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7f. Yield 86% as orange crystals; mp 294–296 °C; IR (KBr, cm⁻¹): 3224 (NH stretching), 1718 (C=O), 1614 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 3.76 (s, 3H, OCH₃), 6.77, 6.84 (2d, J = 8.4, 8.1 Hz, 1H, indole C₇–H), 7.02 (d, J = 9.0 Hz, 2H, N-phenyl C₂–H, C₆–H), 7.10 (s, 1H, thiazoline =CH), 7.20–7.25 (m, 2H, phenyl C₂–H, C₆–H), 7.31 (dd, J = 8.4, 2.1 Hz, 1H, indole C₆–H), 7.34 (d, J = 9.0 Hz, 2H, N-phenyl C₃–H, C₅–H), 7.38 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H), 7.44, 7.46 (2d, J = 2.1, 1.8 Hz, 1H, indole C₄–H), 10.60, 10.77 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 496 (M⁺ + 2, 76), 494 (M⁺, 100), 468 (18), 466 (30), 317 (16), 316 (25), 315 (34), 304 (25), 303 (23), 302 (72), 301 (35), 300 (30), 258 (12), 257 (16), 246 (11), 244 (42), 214 (12), 180 (17), 179 (14), 177 (26), 170 (28), 169 (19), 168 (75), 165 (66), 152 (27), 151 (23), 150 (52), 147 (12), 139 (32), 138 (13), 137 (20), 136 (12), 134 (20), 133 (25), 125 (11), 124 (22), 122 (13), 115 (12), 111 (16), 92 (40), 89 (21), 77 (61); anal. calcd for C₂₄H₁₆Cl₂N₄O₂S (494): C 58.18, H 3.23, N 11.31; found: C 58.19, H 3.23, N 11.35.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7g. Yield 86% as yellowish brown crystals; mp 290–292 °C; IR (KBr, cm⁻¹): 3125 (NH stretching), 1720 (C=O), 1599 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76 (dd, J = 8.4, Hz, 1H, indole C₇–H), 7.14 (s, 1H, thiazoline =CH), 7.21 (td, J = 8.4, 2.1 Hz, 2H, N-phenyl C₄–H, C₅–H), 7.26–7.31 (m, 3H, phenyl C₂–H, C₆–H, indole C₆–H), 7.36 (d, J = 2.4 Hz, 1H, indole C₄–H), 7.41 (d, J = 8.4 Hz, phenyl C₃–H, C₅–H), 7.58 (m, 1H, N-phenyl C₆–H), 7.67 (t, J = 7.2 Hz, 1H, N-phenyl C₃–H), 10.49, 10.59 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 484 (M⁺ + 2, 63), 483 (M⁺ + 1, 28), 482 (M⁺, 92), 456 (6), 454 (6), 419 (6), 304 (9), 292 (36), 291 (18), 290 (100), 241 (21), 234 (10), 232 (37), 180 (10), 178 (16), 170 (24), 169 (15), 168 (73), 167 (10), 165 (27), 154 (9), 153 (87), 152 (13), 150 (11), 139 (33), 138 (27), 137 (37), 136 (48), 134 (19), 133 (33), 124 (14), 122 (8), 113 (9), 111 (26), 110 (9), 102 (12), 95 (28), 89 (21), 75 (33); anal. calcd for C₂₃H₁₃Cl₂FN₄OS (482): C 57.14, H 2.69, N 11.59; found: C 57.13, H 2.66, N 11.60.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(3-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7h. Yield 88% as orange crystals; mp 258–260 °C; IR (KBr, cm⁻¹): 3008 (NH stretching), 1718 (C=O), 1584 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76, 6.79 (2d, J = 8.4, 8.1 Hz, 1H, indole C₇–H), 7.07–7.11 (m, 2H, N-phenyl C₆–H, thiazoline =CH), 7.18–7.32 (m, 4H, N-phenyl C₂–H, C₄–H, C₅–H, indole C₆–H), 7.34 (d, J = 8.4 Hz, 2H, phenyl C₂–H, C₆–H), 7.40 (d, J = 8.4 Hz, 2H, phenyl C₃–H, C₅–H), 7.42 (s, 1H, indole C₄–H), 10.52, 10.58 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 484 (M⁺ + 2, 17), 483 (M⁺ + 1, 7), 482 (M⁺, 22), 292 (11), 290 (34), 232 (21), 195 (15), 178 (8), 170 (8), 168 (21), 167 (10), 165 (14), 153 (16), 152 (8), 141 (52), 140 (18), 139 (100), 138 (25), 136 (20), 125 (13), 124 (13), 113 (30), 112 (10), 111 (84), 110 (11), 102 (14), 95 (41), 89 (17), 75 (70); anal. calcd for C₂₃H₁₃Cl₂FN₄OS (482): C 57.14, H 2.69, N 11.59; found: C 57.10, H 2.67, N 11.64.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(4-fluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7i. Yield 87% as brown crystals; mp 249–251 °C; IR (KBr, cm⁻¹): 3020 (NH stretching), 1739 (C=O), 1558 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.76, 6.78 (2d, J = 8.4, 8.1 Hz, 1H, indole C₇–H), 7.07, 7.11 (2s, 1H, thiazoline =CH), 7.21 (d, J = 8.7 Hz, 2H, phenyl C₂–H, C₆–H), 7.25 (dd, J = 8.4, 2.1 Hz, 2H, N-phenyl C₂–H, C₆–H), 7.31–7.41 (m, 5H, N-phenyl C₃–H, C₅–H, phenyl C₃–H, C₅–H, indole C₆–H), 7.54 (m, 1H, indole C₄–H), 10.51, 10.57 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 484 (M⁺ + 2, 66), 483 (M⁺ + 1, 65), 467 (17), 454 (6), 305 (11), 304 (17), 303 (25), 292 (33), 291 (13), 290 (99), 288 (18), 241 (11), 234 (15), 232 (41), 178 (19), 170 (14), 169 (9), 168 (45), 167 (10), 165 (21), 154 (14), 153 (73), 152 (9), 150 (11), 141 (19), 139 (87), 138 (25), 137 (26), 136 (35), 134 (118), 133 (17), 125 (13), 124 (14), 121 (12), 115 (12) 111 (52), 102 (13), 95 (100), 89 (24), 75 (68); anal. calcd for C₂₃H₁₃Cl₂FN₄OS (482): C 57.14, H 2.69, N 11.59; found: C 57.14, H 2.66, N 11.58.
(3Z)-5-Chloro-1H-indole-2,3-dione-3-{[(2Z)-4-(4-chlorophenyl)-3-(2,4-difluorophenyl)-1,3-thiazol-2(3H)-ylidene]hydrazone} 7j. Yield 73% as orange crystals; mp 238–240 °C; IR (KBr, cm⁻¹): 3100 (NH stretching), 1703 (C=O), 1614 (C=N); ¹H-NMR (DMSO-d₆, δ, ppm): 6.78 (d, J = 8.1 Hz, 1H, indole C₇–H), 7.08 (s, 1H, thiazoline =CH), 7.13–7.19 (m, 2H, N-phenyl C₅–H, C₆–H), 7.25 (d, J = 8.4 Hz, 2H, phenyl C₂–H, C₆–H), 7.30 (dd, J = 8.4, 2.4 Hz, 1H, indole C₆–H), 7.39–7.45 (m, 3H, phenyl C₃–H, C₅–H, indole C₄–H), 7.65 (m, 1H, N-phenyl C₃–H), 10.50, 10.60 (2s, 1H, indole NH); EIMS (70 eV) m/z (%): 502 (M⁺ + 2, 46), 501 (M⁺ + 1, 14), 500 (M⁺, 62), 310 (31), 309 (14), 308 (100), 303 (11), 259 (10), 252 (10), 250 (49), 180 (12), 178 (25), 171 (70), 170 (17), 169 (17), 168 (62), 167 (14), 165 (31), 152 (14), 150 (13), 140 (13), 139 (41), 138 (29), 137 (43), 136 (41), 134 (18), 133 (20), 125 (11), 124 (17), 115 (10), 113 (49), 111 (68), 102 (18), 101 (17), 100 (10), 89 (42), 75 (52); anal. calcd for C₂₃H₁₂Cl₂F₂N₄OS (500): C 55.09, H 2.40, N 11.18; found: C 55.07, H 2.42, N 11.20.

Biological assays

Cytotoxicity (in vitro). Cytotoxic activity was determined by a brine shrimp (Artemia salina) lethality bioassay described in our published research paper.³⁰ Etoposide, a potent anticancer drug, was used as a reference standard.

Phytotoxicity (in vitro). Phytotoxicity potential was determined in accordance with the literature protocol given in our published research article.³³

Antifungal activity (in vitro). Antifungal studies were carried out according to the literature protocol provided in our published research paper.³¹

Urease inhibition (in vitro). Urease inhibitory activity was determined in accordance with the literature protocol given in our published research article.⁴¹

Molecular docking studies

Structure selection and preparation. Molecular docking studies were conducted to investigate the putative interactions of compounds 5a–n, 6a–o and 7a–j in complex with the urease enzyme. In order to perform efficient docking studies, the crystallographic structure of Jack bean urease (PDB ID: 3LA4) was obtained from the RCSB PDB database.⁴² Prior to molecular docking studies, the structures of the enzyme and the compounds were prepared as follows. The enzyme structure was protonated with the Protonate 3D⁴³ algorithm implemented in the molecular modeling tool MOE.⁴⁴ The structure was energy minimized using Amber99 force field, including all the crystallographic solvent molecules. The backbone atoms were restrained with a small force in order to avoid collapse of the binding pockets during energy minimization calculations. After minimization, the water molecules and co-crystallized bound compounds were removed.

Compounds preparation. The 3D structural coordinates were generated for all the compounds using MOE followed by assignment of protonation and ionization states in physiological pH range by using the “wash” module. Afterwards, the compounds' structures were energy minimized with the MMFF94x force field for docking studies.⁴⁴

Docking studies. Docking studies of all the compounds were performed using LeadIT from BioSolveIT, GmbH Germany.⁴⁵ Receptor was loaded by Load or Prepare Receptor utility of the LeadIT software, while selecting the metal ions as part of the receptor. The binding site for Jack bean urease was defined in 8.0 Å spacing of the amino acid residues surrounding the bound phosphate. By FlexX utility of LeadIT, docking of the compounds was performed. Default docking parameters were not modified and top 30 highest scoring docked positions were kept for further analysis. The Discovery studio visualizer v4 was used for visualizing the results.⁴⁶ Using HYDE visual affinity⁴⁷ program of LeadIT, the binding mode analysis of the docked poses was evaluated. Binding free energy i.e. ΔG was determined for each pose. Poses with lowest ΔG values were considered as the most stable ones with highest affinity for interaction with the receptor.

Acknowledgements

We acknowledge partial funding of this research work and the award of indigenous PhD scholarship to MA by Higher Education Commission (HEC), Government of Pakistan. J. Iqbal is thankful to the Organization for the Prohibition of Chemical Weapons (OPCW), The Hague, The Netherlands and the Higher Education Commission of Pakistan for the financial support through Project No. 20-3733/NRPU/R&D/14/520.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10043k

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