Discovery and Anticancer Activity of Novel 1,3,4-Thiadiazole- and Aziridine-Based Indolin-2-ones via In Silico Design Followed by Supramolecular Green Synthesis.

Three crucial anticancer scaffolds, namely indolin-2-one, 1,3,4-thiadiazole, and aziridine, are explored to synthesize virtually screened target molecules based on the c-KIT kinase protein. The stem cell factor receptor c-KIT was selected as target because most U.S. FDA-approved receptor tyrosine kinase inhibitors bearing the indolin-2-one scaffold profoundly inhibit c-KIT. Molecular hybrids of indolin-2-one with 1,3,4-thiadiazole (IIIa-m) and aziridine (VIa and VIc) were afforded through a modified Schiff base green synthesis using β-cyclodextrin-SO3H in water as a recyclable proton-donor catalyst. A computational study found that indolin-2,3-dione forms a supramolecular inclusion complex with β-cyclodextrin-SO3H through noncovalent interactions. A molecular docking study of all the synthesized compounds was executed on the c-KIT kinase domain, and most compounds displayed binding affinities similar to that of Sunitinib. On the basis of the pharmacokinetic significance of the aryl thioether linkage in small molecules, 1,3,4-thiadiazole hybrids (IIIa-m) were extended to a new series of 3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IVa-m) via thioetherification using bis(triphenylphosphine)palladium(II)dichloride as the catalyst for C-S bond formation. Target compounds were tested against NCI-60 human cancer cell lines for a single-dose concentration. Among all three series of indolin-2-ones, the majority of compounds demonstrated broad-spectrum activity toward various cancer cell lines. Compounds IVc and VIc were further evaluated for a five-dose anticancer study. Compound IVc showed a potent activity of IC50 = 1.47 μM against a panel of breast cancer cell lines, whereas compound VIc exhibited the highest inhibition for a panel of colon cancer cell lines at IC50 = 1.40 μM. In silico ADME property descriptors of all the target molecules are in an acceptable range. Machine learning algorithms were used to examine the metabolites and phase I and II regioselectivities of compounds IVc and VIc, and the results suggested that these two compounds could be potential leads for the treatment of cancer.

Introduction

Regardless of human development, cancer is a prominent cause of mortality and a significant impediment to increasing life expectancy in every country.[1] The World Health Organization (WHO) forecast an additional 29.5 million-cancer cases by 2040.[2] According to the WHO, only 12 countries will be able to reduce cancer deaths by more than 30% by 2030.[3] Cancer chemotherapy is the most common method of cancer treatment and involves the use of cytotoxic molecules to induce cell apoptosis.[4] Many strategies attempt to battle cancer metamorphosis by targeting receptor tyrosine kinases (RTKs), which are vital units of cell signaling pathways.[5]

There are 20 subfamilies of human RTKs, one of which is the stem cell growth factor receptor often known as c-KIT.[6] c-KIT is a type III receptor tyrosine kinase that has been linked to the initiation and spread of cancer.[7] Because c-KIT is important in cellular proliferation, the use of c-KIT inhibitors has offered potential insights into cancer therapy.[8] Furthermore, because c-KIT is expressed in several normal human tissues, including the retina, the breast epithelium, the vascular endothelium, and sweat glands, not every c-KIT mutation is a cancer risk factor. As a result, it appears that targeting c-KIT when it is the tumor’s “controller or trigger” is the most viable cancer treatment strategy. Although a few RTK inhibitors, such as Imatinib, Sunitinib, and similar analogues, have been demonstrated to be effective against c-KIT, treatment with these inhibitors frequently results in c-KIT mutations that lead to resistance.[9] In addition, incidences of heart failure have been documented during Imatinib treatment, and other multikinase-including c-KIT inhibitors such as Sunitinib, Dasatinib, Sorafenib, and Bevacizumab have been linked to serious cardiac side effects.[10,11]

The development of novel antitumor agents is becoming increasingly important as a means of combating drug resistance and the cardiotoxicity associated with it. Indolin-2-one is a promising cancer chemotherapeutic scaffold. Several new chemical entities containing the indolin-2-one moiety have been discovered, and the majority of them have been approved by the U.S. Food and Drug Administration (FDA) as RTKIs for the treatment of various malignancies. In our recent review article, we reported different heterocyclic, heteroacyclic, and heteroaryl anticancer hybrids of indolin-2-ones that were explored by medicinal chemists using a molecular or pharmacophoric hybridization approach.[12]

We have focused our research on stem cell factor receptor c-KIT as a target because indolin-2-ones are effective against mast cell tumors (MCT) that involve c-KIT overexpression. Toceranib is a c-KIT inhibitor widely used as an anti-MCT agent.[13] Nintedanib inhibits human fibroblast-mediated mast cell survival via blocking SCF-stimulated c-KIT phosphorylation.[14] In a human myeloid leukemia cell line (MO7E), Orantinib (SU-6668) and Semaxanib (SU-5416) inhibit the tyrosine autophosphorylation of the receptor that is induced by c-KIT in a dose-dependent manner.[15] Famitinib and Tafetinib also act against c-KIT in patients with advanced solid cancer.[16] Vorolanib was also found to inhibit c-KIT with an excellent IC50 value.[17]

Compounds bearing the 1,3,4-thiadiazole framework with excellent in vitro activities against a range of tumor cells with diverse modes of action, including angiogenesis, tumor infiltration, and cell-cycle events, have been reported in the literature.[18] The thiadiazole’s heteroatoms can establish interactions, such as hydrogen bonding, with important kinases involved in cancer.[19] CA IX, a carbonic anhydrase enzyme that is usually overexpressed in cancer cells and causes hypoxia, is a prospective target for a spectrum of malignancies. Renowned CA inhibitors (Figure ), namely sulfonamide 1,3,4-thiadiazoles, are used in cancer treatment.[20]

Figure 1

Design strategy for the pharmacophore hybridization approach in this study.

As a DNA-alkylating agent, the aziridine ring is a widely used heterocyclic system in cancer chemotherapy. The first member of the aziridine group, thiotepa, is still in use for the treatment of bladder cancer, but it was labeled as an “orphan drug” by the U.S. FDA in 2007 because of its myelosuppression toxicity. The “Apaziquone” anticancer candidate bearing a single aziridine ring is well tolerated, with a lower toxicity profile and a higher efficacy than both Diaziquone and Triaziquone, which have two and three aziridine rings, respectively.[21]

Many bioactive natural and synthetic entities have a thioether linkage; in particular, the diaryl thioether connection has been found in naturally occurring bioactive molecules, pesticides, and small-molecule anticancer drugs, including Axitinib and Thymitaq.[22] When Doxorubicin is conjugated with the BR96-monoclonal antibody via the thioether linkage, its anticancer activity increases.[23] Although the thioether linkage is thought to increase drug likeness by improving pharmacokinetic features, thioetherification (C–S bond formation) has received less attention and resources than C–C, C–N, and C–O bond formation.[24,25]

Hence, inspired by the intrinsic anticancer potential of indolin-2-one, 1,3,4-thiadiazole, and aziridine and the pharmacokinetic importance of thioether linkage, we successfully developed three novel series of virtually screened indolin-2-ones hybrids and evaluated them against human cancer cell lines (Figure ). In the present work, Schiff bases of indolin-2-ones with 1,3,4-thiadiazole and aziridine were synthesized using a novel greener approach. Various synthetic procedures for the Schiff base formation of indolin-2-one are well-documented by conventional methods,[26−29] but the majority are time-consuming, low yielding, and not very ecologically friendly. 2-Amino-5-mercapto-1,3,4-thiadiazole has also been explored as a starting material for imine group synthesis with various aromatic aldehydes in ethanol under reflux conditions utilizing conc. H2SO4 as a catalyst.[30]

To eliminate the use of mineral acids and harmful organic solvents, the concept of green chemistry has arisen as a major approach. Various methodologies and routes have been developed for this purpose.[31] There are few reports published on green synthesis of isatin Schiff’s bases using microwave (MW), ultrasound (US) techniques,[32] and the on-water condensation of aryl amine with substituted aromatic aldehydes.[33]

In the present work, we report the exploration of supramolecular green chemistry approach toward the synthesis of indolin-2-one Schiff base hybrids using β-cyclodextrin-SO3H (β-CDSO3H) as a recyclable catalyst in water. Supramolecular chemistry involves the formation of host–guest complexes through reversible noncovalent intermolecular interactions. Cyclodextrins are promising supramolecular hosts for a range of guests, including aromatic compounds and nonpolar solutes.[34] β-Cyclodextrin possesses a hydrophilic outer surface due to a secondary hydroxyl group and a hydrophobic inner cavity.[35] The cavity size and the inner hydrophobicity are suitable for encapsulating a variety of guest molecules. Chemical modifications of endogenous cyclodextrins have been shown to accelerate the complex formation process.[36] We investigated this green catalyst in the hopes of finding a noncovalent association with indolin-2-ones via inclusion complex formation.[37]

Results and Discussion

Virtual Screening Using a Prevalidated 3D QSAR Model

Virtual screening of a library of indolin-2-ones molecules was performed through the “Virtual Screening Workflow” package implemented in Schrödinger Maestro[38] using a previously reported atom-based three-dimensional QSAR model (ADHRR.24) of anticancer activity toward c-KIT inhibition.[39]

Our earlier reported model ADHRR.24 was validated with external databases and proven to be significantly robust (R2 = 0.9378, Q2 = 0.7832). A small-molecule library comprising compounds with the indolin-2-one framework was prepared and processed for in silico virtual screening using the LigPrep module. Initially, the ADHRR.24 hypothesis was used as a filter for “search for matches” in the PHASE program for all the molecules from the library. Retrieved hits were ranked based on the obtained fitness score (≥2.3). Further, the top-ranked retrieved hits were filtered by ADME property descriptors using the QikProp module (ADME property filtration suite) and the SwissADME[40] module. Finally, 28 molecules were obtained as new hits after structure-based and ligand-based virtual screening (Table S1). None of the compounds among the final 28 hits were found to violate Lipinski’s “Rule of Five”.[41]

Pharmacophore Alignment of Synthesized Compounds (IIIa–m, IVa–m, VIa, and VIc)

In an attempt to estimate the fitness of the synthesized molecules on the basis of the prevalidated pharmacophore, the hypothesis ADHRR.24 was searched for matches within the indolin-2-one molecular library. All the molecules were well aligned on the pharmacophore model. The fitness values and predicted activity (pIC50) values toward the c-KIT protein are mentioned in the Supporting Information (Table S1). Compound IVc displayed a high fitness value (3.03) and a predicted activity (pIC50) of −0.48 against c-KIT. The common pharmacophore (ADHRR.24) was superimposed with five features of compound IVc, namely a H-bond acceptor (A) group from —C=O, a donor (D) group from −NH of indolin-2-one, a hydrophobic (H) group of −F, and an aromatic characteristic (R) from indolin-2-one and 1,3,4-thiadiazole (Figure a). Compound VIc also displayed a high fitness score (2.96) and a predicted pIC50 of −0.48 for c-KIT (Table S1). The required aromatic characteristic of the ADHRR.24 model aligned well with an aziridine ring and the indolin-2-one moeity, and other features were well matched in the same manner as compound IVc (Figure b).

Figure 2

(a) Pharmacophore alignment of compound IVc in the c-KIT 3D QSAR model “ADHRR.24”. (b) Pharmacophore alignment of compound VIc in the c-KIT 3D QSAR model “ADHRR.24”.

Chemistry

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IIIa–m) Using a Greener Approach (Schemes and 2)

The synthesis of substituted indoline-2,3-dione (Ia–m) (Scheme ) was carried out as per the reported protocol. The compounds were identified by uncorrected melting points comparable to that reported in the literature. The compounds (Ia–m) were further subjected to Schiff base formation via coupling with the 1,3,4-thiadiazole moiety (II) (Scheme ). Initially, the synthesis of 3-((5-mercapto-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IIIa–m) was attempted by the conventional Schiff base formation approach,[12],[26−29] where primary amine of 5-amino-1,3,4-thiadiazole-2-thiol was clubbed with indolin-2,3-diones using a catalytic amount of glacial AcOH, a few drops of concentrated HCl, and a suitable solvent at reflux (Scheme a).

Scheme 1

Synthesis of Substituted Isatins (Ia–m)

Scheme 2

Synthesis of Indolin-2-one Hybrids (IIIa–m) Using (a) the Conventional Approach and (b) the Greener Approach

Unfortunately, the yields obtained by this method were not promising, and most reactions were not successful, especially with the indolin-2,3-diones bearing electron-releasing substituents in the fifth position (Table ). Alternatively, to eliminate the use of acids and to follow the principles of green chemistry, the Schiff base formation was effectively performed utilizing β-cyclodextrin-SO3H in H2O as a reusable catalyst (Scheme b). Scheme b shows the formation of the titled compounds (IIIa–m) using a recyclable green catalyst in water as benign solvent.

Table 1

Melting Points and Yields of Indolin-2-one Hybrids (IIIa–m)

entry	R	mp (°C)	yielda(%)	yieldb(%)
IIIa	5-H	176	82	58
IIIb	5-Cl	184	90	36
IIIc	5-F	178	88	62
IIId	5-Br	180	86	52
IIIe	4-Cl	210	84	NA
IIIf	5-Me		74	NA
IIIg	5-NO2	186	96	66
IIIh	5-COOH	204	92	64
IIIi	5-OMe, 6-OMe		72	34
IIIj	7-COOMe		68	NA
IIIk	5-OMe		76	NA
IIIl	7-COOH	210	94	NA
IIIm	5-Me, 6-Me		78	32

Yields obtained by the green method.

Yields obtained by the conventional method.

The reaction of β-cyclodextrin and chlorosulfonic acid in CH2Cl2 at 0 °C for 3 h yielded a sulfonated derivative of β-cyclodextrin. The FT-IR spectra of β-cyclodextrin and its sulfated derivative were compared to identify the sulfate group. In the FT-IR spectra of β-CDSO3H, the characteristic −SOO– symmetric stretching vibrations and −SOO– asymmetric stretching vibrations were observed at 986 and 1224 cm–1, respectively. The −SO3H content was also measured by the titrimetric method. In order to afford the Schiff bases of compounds Ia–m, the performance of β-CDSO3H as a proton donor catalyst was explored. This catalyst is water-soluble at warm temperatures and is able to efficiently solubilize the starting materials.

The compounds (Ia–m) in methanol (1 equiv) were added to a warmed stirred solution of β-CDSO3H (1.2 equiv) in water, followed by addition of 5-amino-1,3,4-thiadiazole-2-thiol (II) (1 equiv), and the mixture was refluxed for 5–20 min. As the reflux started, the solution became clear. Reaction was monitored using precoated TLC with ethyl acetate (EA)/hexanes (HEX) (3:7) as the mobile phase. A new nonpolar spot was observed at the completion of the reaction, and the reaction mixture was then cooled to room temperature. Being insoluble in water at room temperature, β-CDSO3H was reprecipitated and used for three runs. To obtain the final solid or semisolid compounds (IIIa–m), the crude products were extracted three times with dichloromethane (DCM) or ether, then dried over anhydrous sodium sulfate and evaporated under vacuum. A triple cycle of this green catalyst enabled the expedited synthesis of the desired compounds.

Compounds IIIg, IIIh, and IIIl were obtained with highest yields; it can be noted that the electron-withdrawing substituent on fifth or seventh position of the indolin-2-one moiety affects the elctrophilicity at the C3 position, which enhances the yields. In contrast, compounds IIIf, IIIk, and IIIm possessing an electron-releasing substituent on the fifth or sixth position afforded with comparatively lower yields (Table ).

The formation of the —C=N bond was confirmed by stretching vibrations in the range from 1620 to 1600 cm–1 in the FT-IR spectra of compounds IIIa–m. Values of the KBr FT-IR vibrations (cm–1) are provided in Table S2. The representative structure of the compound IIIa was confirmed by mass spectrometry, where the molecular ion peaks at m/z 262.3 in the LC-MS spectra and m/z 262.07 in the MS spectra represent the molecular weight. Due to thiol group tautomerism (Figure ), the free mercapto proton appears as a thioamide proton (O=S—NH) with a chemical shift value of δ 13 ppm in all 1H NMR spectra.

Figure 3

Proposed mechanism for the synthesis of 1,3,4-thiadiazole-linked indolin-2-one hybrids (IIIa–m) using β-CDSO3H.

The −NH proton of indolin-2-one was observed at chemical shift values around δ 10 ppm, but −NH and O=S—NH protons were not recorded at the desired chemical shift values in the D2O exchangeable 1H NMR spectra due to exchange with deuterated water. All aromatic protons were recorded at chemical shift values of δ 8–7 ppm. The sum of number of protons in every structure matched the integral values obtained in the 1H NMR spectra. The structures were also characterized by chemical shift values (δ, ppm) obtained in the 13C NMR spectra, wherein aromatic carbons were identified at the range from δ 180 to 120 ppm. CHNS analyses provided elemental detection data that supported the structural elucidations.

Proposed Mechanism for the Synthesis of Indolin-2-one Hybrids (IIIa–m)

The proposed mechanism for the present synthetic method was deduced such that the role of β-CDSO3H as a proton donor was clearly understood (Figure ).

β-CDSO3H is the solubilizing agent that forms an inclusion complex with the isatin substrates (Ia–m) through H-bond interactions. This hypothesis was supported by an in silico inclusion complex formation study. In the initial step, the β-CDSO3H catalyst acts as a proton donor in a manner similar to those of GAA and HCl in conventional Schiff base syntheses. In the latter steps, β-CDSO3– acts as a proton acceptor to give final imine products. β-CDSO3H was almost completely recovered through extraction after the workup.

Synthesis of 3-((5-(Phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IVa–m) (Scheme )

A series of 3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IVa–m) was synthesized by the thioetherification of 1,3,4-thiadiazole-coupled indolin-2-ones (IIIa–m) and aryl halide (bromobenzene) in an alkaline medium (NaOEt) in the presence of toluene as the solvent (Scheme ).

Scheme 3

Synthesis of 3-((5-(Phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IVa–m)

The C–S bond formation was successfully achieved using bis(triphenylphosphine)palladium(II)dichloride (0.1 mol %) as a catalyst. In the 1H NMR spectra, the absence of a thioamide proton peak around δ 13 ppm confirmed the formation of the thioether linkage. The structures were also characterized by chemical shifts values (δ, ppm) obtained from the 13C NMR spectra, wherein aromatic carbons were detected near δ 180–120 ppm. Melting points and yields of the derivatives (IVa–m) are provided in Table .

Table 2

Melting Points and Yields of the Derivatives (IVa–m)

entry	R	mp (°C)	yield (%)
IVa	5-H	184–186	80
IVb	5-Cl	192–194	90
IVc	5-F	186–188	84
IVd	5-Br	188–190	82
IVe	4-Cl	218–220	82
IVf	5-Me	178–280	70
IVg	5-NO2	194–196	92
IVh	5-COOH	212–214	90
IVi	5-OMe, 6-OMe	156–158	72
IVj	7-COOMe	oily	crude
IVk	5-OMe	174–176	74
IVl	7-COOH	218–220	90
IVm	5-Me, 6-Me	180–182	80

We have proposed a mechanism for the synthesis of 3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (Scheme ) in which the concerted oxidative nucleophilic addition of bromobenzene and a mercapto group to dicoordinated palladium results in the formation of a tetracoordinated palladium complex. In the final step, reductive elimination yields the C–S-coupled product (Figure ).

Figure 4

Proposed mechanism for aryl thioetherification via C–S bond formation

In presence of the bis(triphenylphosphine)palladium(II)dichloride catalyst, the possibility of starting material (IIIa–m) forming a dimer through the S–S disulfide linkage could be overruled by the study of the reaction kinetics and transition state optimization (Figure ).

Figure 5

Energy profile diagram of transition state intermediates drawn using the GAMESS interface (PerkinElmer Inc.).

Reaction Kinetics Study of Thioetherification

Although palladium metal has gained popularity for C–S bond formation, selection of a properly coordinated palladium catalyst is essential to avoid the generation of byproducts during the reaction. For that purpose, in order to estimate the performance of the selected bis(triphenylphosphine)palladium(II)dichloride as a catalyst for C–S bond formation, it was necessary to investigate the free energies of possible transition states and products. Free energies of the reactants and possible products were obtained by performing in silico energy minimization. Product 1, the desired product, has the lowest energy (−21 kcal/mol), while the undesired product 2 has a higher energy (32 kcal/mol) than 1. The high-energy product 2 could be predicted by high-energy transition state intermediate T. S. 2. S–S bond formation in transition state 2 (T. S. 2) needs a higher energy (217 kcal/mol) than C–S bond formation in T. S. 1 (149 kcal/mol) (Figure ). Hence, C–S (thioetherification) bond formation is favored over S–S bond formation in the reaction.

Figure 6

3D visualization of (a) the T. S. 1 intermediate (147 kcal/mol energy) and (b) the T. S. 2 intermediate (217 kcal/mol energy).

Synthesis of Aziridine-indolin-2-one Hybrids (VIa and VIc) (Scheme and 5)

Synthesis of Methyl 1-Acetylaziridine-2-carboxylate (Scheme )

To afford methyl 1-acetylaziridine-2-carboxylate (Scheme ), the methyl esterification of N-acetyl-dl-serine was performed using MeOH and thionyl chloride at reflux for 3 h, followed by overnight stirring.[43] Colorless crystals of methyl 2-acetamido-3-hydroxypropanoate were obtained after the evaporation of the reaction mixture under reduced pressure. The final product was washed with cold water to remove thionyl chloride. The methyl ester of N-acetyl-dl-serine was cyclized to form an aziridine ring through a reaction with sulphuryl chloride, triethyl amine, and toluene at −10 °C for 2–3 h.[44]

Scheme 4

Synthesis of the Aziridine Scaffold (V)

The formation of methyl-1-acetylaziridine-2-carboxylate was confirmed by the 1H NMR spectrum; the doublet of doublets splitting pattern of aziridine protons clearly indicated the formation of the cyclized ring. Other protons at desired chemical shift values revealed the complete structure.

Aziridine-clubbed indolin-2-one hybrids (VIa and VIc) (Scheme ) were synthesized using the greener approach in a protocol similar to that for the synthesis of the thiadiazole conjugates. Hydrazone derivatives of isatin (Vx and Vy) and methyl 1-acetylaziridine-2-carboxylate (V) are the key starting materials required to obtain the target compounds (VIa and VIc). Hydrazones of Isatin (Vx and Vy) were afforded by adapting the reported procedure such that the greener approach was followed by microwave irradiation using ethylene glycol as the solvent.[42]

Scheme 5

Synthesis of Methyl 1-(−1-(-(2-Oxoindolin-3-ylidene)hydrazono)ethyl)aziridine-2-carboxylates (VIa and VIc)

Docking Studies on c-KIT

Several reports on the molecular docking of indolin-2-ones toward the stem cell factor receptor c-KIT are available;[39,45,46] hence, in order to investigate the anticancer potential of the molecule designed to target c-KIT, we envisaged the molecular docking pattern of the synthesized indolin-2-ones.

All the synthesized indolin-2-ones were included in an SP mode docking study performed on the c-KIT protein (PDB ID 3G0E) using the GLIDE program. The amino acid residues CYS673, TYR672, ALA621, GLU671, ALA621, VAL654, LYS623, VAL603, CYS809, PHE811, ALA814, ASP810, 674, LEU799, LEU595, and ASN680 were found to be associated in the formation of the c-KIT kinase domain’s ATP binding site. Most of the ligands in the energy-minimized states had the same binding pattern as the cocrystallized inhibitor Sunitinib and interacted with CYS673 and GLU671 via H-bond interactions. These two key amino acid residues present within the ATP-pocket of c-KIT domain are required for c-KIT inhibition.[47,48]

Docking interactions of the aziridine-indolin-2-one compound VIc with c-KIT showed the H-bond network, attractive charges, and π-interactions with the active-site residues (Figure a). In the molecular docking simulation study of all the ligands under investigation, compound VIc had the highest docking score (−10.195) and glide energy (−58.53) (Table ). Compound VIc revealed three important hydrogen bonds at the ATP binding site’s hinge region: a H-bond with a distance of 1.72 Å between the carbonyl functionality of 2-oxoindoline and an amide −NH of CYS673, a H-bond with a distance of 2.27 Å between the adjacent −NH and >C=O of GLU671, and a H-bond with a distance of 1.94 Å between the methyl ester group of the aziridine ring and LYS593 (Figure b).

Figure 7

(a) 2D representation of the complex between the ligand (VIc) and the protein (c-KIT). (b) Compound VIc docked in the hinge region of the ATP binding site of c-KIT (PDB ID 3g0e).

Table 3

Docking Results for Target Molecules

entry	compd	docking score	glide energy	entry	compd	docking score	glide energy
1	VIc	–10.195	–58.53	15	IIIf	–8.152	–50.19
2	IVc	–9.662	–57.25	16	IIIb	–7.87	–48.66
3	IVh	–9.42	–53.28	17	IVd	–7.87	–49.87
4	IVi	–9.353	–53.9	18	IIIc	–7.57	–48.36
5	VIa	–9.321	–54.23	19	IVe	–6.681	–49.49
6	IVa	–9.282	–53.77	20	IIIa	–5.31	–45.06
7	IIIk	–9.27	–51.82	21	IVf	–4.85	–41.11
8	IVb	–9.25	–53.13	22	IVg	–4.85	–44.09
9	IIIm	–9.141	–55.18	23	IIIg	–4.53	–43.81
10	IIIl	–9.11	–55.73	24	IIIe	–4.481	–44.42
11	IIIh	–8.97	–52.87	25	IIId	–3.422	–39.46
12	IVl	–8.5	–49.78	26	IVj	–3.38	–36.29
13	IIIi	–8.321	–50.99	27	IVk	–3.26	–32.65
14	IIIj	–8.23	–46.98	28	IVm	–3.123	–30.78

Compound IVc from the series of thiadiazole-indolin-2-one conjugates also demonstrated a better binding affinity, with a docking score of −9.662 kcal/mol and a glide energy −57.25 (Table ). The 2-oxoindoline moiety of compound IVc displayed H-bonds with the amide −NH of CYS673 and the carbonyl of GLU671 with distances of 1.86 and 2.14 Å, respectively (Figure ). Among the series of 1,3,4-thiadiazole and aziridine hybrids, compounds IVh, IVI, VIa, IVa, IIIk, IVb, IIIl, and IIIm exhibited good binding affinities, with docking scores from −9.11 to −9.42 kcal/mol (Table ).

Figure 8

(a) 2D representation of the complex between the ligand (IVc) and the protein (c-KIT) at the binding site. (b) Docking pose of compound IVc in the catalytic domain of c-KIT (PDB ID 3g0e).

Inclusion Complex Study of indoline-2,3-diones and β-Cyclodextrin-SO3H

An inclusion complex study of β-cyclodextrin-SO3H was carried out wherein the docking pattern and H-bonding interaction were evaluated. The representative reaction starting materials indoline-2,3-diones were observed to be docked at the outer rim of β-CDSO3H in the cavity (Figure ). Based on an SP docking score of ⩽−9.78 kcal/mol obtained for all the substituted indoline-2,3-diones, the binding interactions were determined to be favorable for the formation of inclusion complexes.

Figure 9

(a) 2D structure of β-CDSO3H, (b) inclusion complex of indoline-2,3-dione at the outer rim of β-CDSO3H, and (c) H-binding interaction of indoline-2,3-dione with β-CDSO3H.

ADME and Synthetic Accessibility Studies

None of the target molecules were found to violate Lipinski’s rule; all the target molecules were also investigated through measurements of in silico ADME property descriptors (Table ). The partition coefficient (log Po/w), the solubility (log S) parameter, and MDCK cell permeability parameters are in the acceptable ranges for all the molecules. The percent of human oral absorption is high for majority of the molecules. The synthetic accessibility was measured using SwissADME,[40] which measures synthetic accessibility via fragmental contributions (FP2) regulated by size and complexity penalties; measurements were performed on molecules from a diverse data set and tested on a small number of external molecules (R2 = 0.94). The synthetic accessibility for all the target molecules was in the range from 2.67 to 3.38 on 10-point scale (Table ).

Table 4

Combined Results of ADME Parameters Measured Using QikProp and SwissADME

entry	compd	molecular weight (g/mol)	consensus log Pao/w	log Sb (ESOL)	MDCKc	% human oral absorptiond	synthetic accessibilitye
1	IIIa	262.31	1.81	–3.03	808.596	73.027	2.68
2	IIIb	296.76	2.36	–3.60	1598.358	81.406	2.67
3	IIIc	280.30	2.12	–3.17	2215.361	79.578	2.73
4	IIId	341.21	2.46	–3.92	618.551	83.172	2.77
5	IIIe	296.76	2.29	–3.60	947.256	90.923	2.74
6	IIIf	276.34	2.15	–3.31	601.704	78.513	2.77
7	IIIg	307.31	1.05	–3.06	1428.755	76.685	2.81
8	IIIh	306.32	1.34	–2.86	2045.758	87.433	2.79
9	IIIi	322.36	1.79	–3.14	448.948	75.916	2.98
10	IIIj	320.35	1.80	–3.07	777.653	83.667	2.93
11	IIIk	292.34	1.82	–3.08	815.769	69.313	2.79
12	IIIl	306.32	1.42	–2.86	1642.82	67.485	2.82
13	IIIm	290.36	2.48	–3.61	2259.823	78.233	2.88
14	IVa	338.41	3.35	–4.64	663.013	91.031	3.05
15	IVb	372.85	3.81	–5.22	991.718	79.514	3.04
16	IVc	356.40	3.55	–4.79	771.307	87.265	3.10
17	IVd	417.30	3.94	–5.54	1635.647	74.855	3.12
18	IVe	372.85	3.85	–5.22	2252.65	85.769	3.10
19	IVf	352.43	3.68	–4.93	655.84	83.941	3.15
20	IVg	383.40	2.50	–4.68	984.545	94.689	3.18
21	IVh	382.42	2.91	–4.48	638.993	90.326	3.16
22	IVi	398.46	3.30	–4.76	1466.044	78.809	3.36
23	IVj	396.44	3.32	–4.70	2083.047	85.068	3.31
24	IVk	368.43	3.37	–4.70	486.237	92.819	3.17
25	IVl	382.42	2.94	–4.48	814.942	80.409	3.19
26	IVm	366.46	4.01	–5.23	853.058	85.97	3.27
27	VIa	286.29	1.03	–2.00	1680.109	93.721	3.38
28	VIc	304.28	1.29	–2.18	1208.459	70.158	3.36

Predicted octanol/water partition coefficient (log p = −2.0 to 6.5).

Predicted aqueous solubility (S in the range of −6.5 to 0.5 mol/L)

Predicted apparent MDCK cell permeability (nm/s).

Human oral absorption percentage (25% is poor and 80% is high).

Synthetic accessibility score range from 1 (very easy) to 10 (very difficult).

In Vitro NCI 60 Cell Line Assay (Single High Dose of 10–5 M)

Individual cell lines from nine panels revealed a distinct pattern of selectivity for the compounds examined. In terms of the percentage of growth inhibition (GI percentage = 100 – GI; GI, growth inhibition), many compounds from all three series of indolin-2-ones showed good bioactivities against the tested cell lines. Compound IVc (NSC no. D810738/1) showed the highest growth inhibitions of 77.83% and 91.00% against cell lines MCF7 and MDA-MB-468, respectively, in the breast cancer panel. The fluoro substitution renders this molecule active against breast cancer cell lines. Compounds IVf (NSC no. D810747/1) and IVa (NSC no. D810737/1) showed higher growth inhibition against the K-562 cell line in the leukemia panel at 66.14% and 59.03%, respectively (Table ). The presence of an electron-releasing substituent (methyl group) at the fifth position of indolin-2-one leads to better activity against leukemia.

Table 5

Screening Results of Representative Compounds against Human Cancer Cell Lines

Compound IIIh (NSC no. D810756/1) showed the highest growth inhibition against the CCRF-CEM cell line (47.52%), the SR (51.81%) cell line, and the HL-60(TB) cell line (51.83%) in the leukemia panel. Compound IIIk (NSC no. D810749/1) showed the highest growth inhibition against the MCF7 and MDA-MB-468 cell lines at 74.93% and 83.94%, respectively. In the absence of the thioether linkage, a carboxylic acid group or a methoxy substituent at the fifth position leads to better activity against leukemia. Compound IIIl (NSC no. D810753/1) showed the highest growth inhibition against the NCI-H522 cell line in the non-small-cell lung cancer panelat 51.03%. The carboxylic acid substituent at the seventh position enhances the activity against non-small-cell lung cancer. Compound IIIm (NSC no. D810757/1) showed the highest growth inhibition against the MALME-3 M cell line in the melanoma panel at 67.19%. Electron-releasing substituents (methyl groups) on fourth and fifth positions of the indolinone moiety lead to better activities against melanoma. Compound VIc (NSC no. D810742/1) showed the highest growth inhibition against the KM12 cell line of the colon cancer panel at 72.06%. The aziridine ring leads to better activity against colon cancer. Compound VIa (NSC no. D810746/1) showed the highest growth inhibition against the K-562 cell line in the leukemia panel at 44.10% and the UACC-62 cell line in the melanoma panel at 44.88% (Table ).

Structure–Activity Relationship

Results obtained for the single-dose anticancer study reveal that the fifth position on the indolin-2-one ring plays an important role in the anticancer properties of synthesized molecules. The absence of an arylthioether linkage and a carboxyl group at the fifth position enhances the activity against leaukemia cell lines. Structure–activity relationships of both thiadiazole and aziridine hybrids are depicted in Figure .

Figure 10

Common structure–activity relationships of indolin-2-ones.

Anticancer Activity at Five-Dose Concentration

Out of 20 compounds, IVc (NSC no. D810738/1) and VIc (NSC no. D810742/1) successfully cleared the primary cell line study and were evaluated in a five-dose screening test toward 60 human cancer cell lines. The five-dose screening test revealed that compound IVc (NSC no. D810738/1) inhibited breast cancer cell growth the most, with a subpanel average activity of 1.47 μM (Table ). The unique thioether linkage in this molecule facilitates the molecule’s cell permeability. The inhibitory concentrations against MDA-MB-231/ATCC and MCF7 cell lines were 0.71 and 1.04 μM, respectively. Compound IVc bearing thiadiazole- and fluoro-substituted indolin-2-one motifs exhibited a broad spectrum of inhibitory activity for all subpanels of cancer cell lines in the range of 1.47–3.03 μM, indicating that the molecule could be potential candidate for further cancer chemotherapeutic development.

Table 6

Five-Dose Screening Results of Compound IVc

		GI50
cancer panel	cancer cell line	concentration (μM)	subpanel MIDb	selectivity ratioa (MIDa:MIDb)	TGI	LC50
leukemia			2.01	2.46
	CCRF-CEM	1.47			>100	>100
	HL-60(TB)	2.62			58.2	>100
	K-562	1.16			>100	>100
	MOLT-4	2.34			>100	>100
	RPMI-8226	1.781			>100	>100
	SR	2.69			36.4	>100
non-small-cell lung cancer			2.40	2.06
	A549/ATCC	2.34			49.1	>100
	HOP-62	2.67			>100	>100
	HOP- 92	1.44			27	>100
	NCI-H226	2.96			>100	>100
	NCI-H460	2.86			28.2	>100
	NCI-H522	2.148			26.6	>100
colon cancer			2.54	1.95
	COLO 205	2.59			>100	>100
	HCC-2998	2.64			38	>100
	HCT-116	3.51			>100	>100
	HCT-15	2.45			>100	>100
	HT 29	2.18			>100	>100
	KM 12	1.853			46.7	>100
	SW-620	2.05			>100	>100
CNS Cancer			2.91	1.70
	SF-268	2.03			>100	>100
	SF-295	3.14			87.4	>100
	SF-539	3.45			>100	>100
	SNB-19	3.24			56.8	>100
	SNB-75	2.68			>100	>100
melanoma			1.87	2.64
	LOX IMVI	1.58			>100	>100
	MALME-3M	2.39			74.8	>100
	M14	2.12			49.1	>100
	MDA-MB-435	1.84			>100	>100
	SK-MEL-2	2.56			59.3	>100
	SK-MEL-28	2.46			>100	>100
	SK-MEL-5	1.14			50.7	13.9
	UACC-257	1.56			>100	63.3
	UACC-62	1.16			>100	76.4
ovarian cancer			3.03	1.63
	IGROV1	3.68			>100	>100
	OVCAR-3	1.41			86.5	>100
	OVCAR-4	3.12			26.2	>100
	OVCAR-5	3.47			>100	>100
	OVCAR-8	3.56			25.3	>100
	NCI/ADR-RES	3.18			>100	>100
	SK-OV-3	2.78			>100
renal cancer			2.46	2.01
	786–0	2.03				>100
	A498	5.18			57.6	>100
	ACHN	2.75			>100	>100
	CAKI-1	2.01			>100	>100
	RXF 393	1.24			62.4	>100
	SN12C	1.83			>100	>100
	TK-10	1.005			48.4	>100
	UO-31	3.66			38.9	>100
prostate cancer			2.06	2.40
	PC-3	2.17			>100	>100
	DU-145	1.95			88.6	>100
breast cancer			1.47	3.36
	MCF 7	1.04			>100	>100
	MDA- MB-231/ATCC	0.71			84.25	>100
	HS 578T	1.70			46.32	>100
	BT-549	1.63			>100	>100
	T-47D	1.98			>100	>100
	MDA-MB-468	1.77			78.20	>100
MIDa		4.94

MIDa is the average growth-inhibitory activity (μM) for all cell lines and MIDb is the average growth-inhibitory activity (μM) for the subpanel.

Another compound (VIc; NSC no. D810742/1) that was tested in the five-dose screening demonstrated the highest growth inhibition against colon cancer, with a subpanel average activity of 1.40 μM (Table ). This compound showed 0.733 and 0.93 μM activities against HCT-116 and KM-12 cell lines, respectively. In the majority of cell lines from all nine panels, compound VIc was potent in the range of 1.40–3.48 μM; thus, the aziridine–isatin hybrid molecule could be a potential lead for cancer chemotherapy, particularly colon cancer. According to the findings of the lethality tests (LC50), both compounds IVc and VIc were confirmed to be nontoxic (Tables and 7).

Table 7

Five-Dose Screening Results of Compound VIc

		GI50
panel	cell line	concentration per cell line (μM)	subpanel MIDb	selectivity ratioa (MIDa:MIDb)	TGI	LC50
leukemia			2.55	2.06
	CCRF-CEM	2.88			>100	>100
	HL-60(TB)	1.7			>100	>100
	K-562	3.16			45.60	>100
	MOLT-4	3.23			>100	>100
	RPMI-8226	2.321			72.23	>100
	SR	2.01			>100	>100
non-small cell lung cancer			2.94	1.79
	A549/ATCC	2.88			>100	>100
	HOP-62	3.21			>100	>100
	HOP- 92	1.98			>100	>100
	NCI-H226	3.5			89.6	>100
	NCI-H460	3.4			64.3	>100
	NCI-H522	2.69			22.8	>100
colon cancer			1.40	3.76
	COLO 205	2.39			>100	>100
	HCC-2998	1.52			48	>100
	HCT-116	0.733			56.7	>100
	HCT-15	1.47			23.5	>100
	HT 29	1.33			>100	>100
	KM 12	0.93			66.8	>100
	SW-620	1.06			>100	>100
CNS cancer			3.48	1.51
	SF-268	3.41			>100	>100
	SF-295	4.20			>100	>100
	SF-539	3.62			72.9	>100
	SNB-19	3.31			36.7	>100
	SNB-75	2.85			>100	>100
melanoma			2.54	2.07
	LOX IMVI	3.81			>100	>100
	MALME-3M	2.62			74.8	>100
	M14	2.35			>100	>100
	MDA-MB-435	2.07			51.6	>100
	SK-MEL-2	3.39			>100	>100
	SK-MEL-28	2.69			>100	>100
	SK-MEL-5	1.37			82.4	13.9
	UACC-257	1.79			>100	63.3
	UACC-62	2.79			>100	76.4
ovarian cancer			2.42	2.17
	IGROV1	2.67			>100	>100
	OVCAR-3	1.52			76.1	>100
	OVCAR-4	1.89			>100	>100
	OVCAR-5	2.58			>100	>100
	OVCAR-8	3.79			12.3	>100
	NCI/ADR-RES	2.29			>100	>100
	SK-OV-3	2.23			>100
renal cancer			2.87	1.83
	786–0	4.71			>100	>100
	A498	2.56			69.5	>100
	ACHN	4.19			>100	>100
	CAKI-1	2.36			>100	>100
	RXF 393	1.77			66.5	>100
	SN12C	2.54			>100	>100
	TK-10	1.54			83.6	>100
	UO-31	3.28			55.7	>100
prostate cancer			2.74	1.92
	PC-3	2.85			>100	>100
	DU-145	2.63			>100	>100
breast cancer			2.66	1.98
	MCF 7	2.48			>100	>100
	MDA- MB-231/ATCC	2.62			>100	>100
	HS 578T	2.55			88.3	>100
	BT-549	2.89			>100	>100
	T-47D	3.83			>100	>100
	MDA-MB-468	1.56			37.6	>100
MIDa		5.26

MIDa is the average growth-inhibitory activity (μM) for all cell lines and MIDb is the average growth-inhibitory activity (μM) for the subpanel.

Metabolites and Regioselectivity Predictions

Using the GLORY, GLORYx, and FAME3 tools, the potential metabolites of compounds VIc and IVc and their regioselectivity were examined for the CYP 450 and phase I and II pathways. The FAME3 tool predicted that an ester carbon attached to the aziridine ring of compound VIc provides the site of metabolism (SoM) for aliphatic hydroxylation. The highest probability score (0.776) suggests that the ester carbon of compound VIc is the top priority site for phase I and II metabolism. Similarly, SoM predictions were performed for compound IVc, and it was discovered that the sulfur atom is the top-priority site (probability score of 0.624) for the phase I and phase II pathways via S-oxidation (Figure ). Figure shows the potential structures of the metabolites predicted by GLORY and GLORYx, which are ranked according to their probability scores. Multiple metabolites were predicted for both the compounds (IVc and VIc) based on the various metabolic reactions involving aliphatic hydroxylation, aromatic hydroxylation, and S-oxidation type.

Figure 11

Metabolites and sites of metabolism (SoMs) for compounds VIc and IVc predicted using the GLORY, GLORYx, and FAME3 tools.

Experimental Section

Computational Methodology

Virtual Screening Using a Prevalidated 3D QSAR Model

The virtual screening of indolin-2-one molecules was performed using Schrödinger Maestro[38] on the c-KIT protein pharmacophore model ADHRR.24.[39] ADME studies were performed using the QikProp module implemented in Schrödinger Maestro and the SwissADME module. Transition state optimization and free energy measurements for C–S bond formation was performed on the GAMESS interface (PerkinElmer Inc.).[49]

Molecular Docking Study of Synthesized Molecules (IIIa–m, IVa–m, VIa, and VIc)

Protein Preprocess and Receptor Grid Generation

The molecular docking study of all the synthesized compounds was executed on the c-KIT kinase domain (PDB ID 3G0E; 1.6 Å resolution) with Sunitinib as a cocrystallized ligand. The simulation operations were executed using Glide (Schrödinger, LLC);[50,51] as incorporated in the Maestro program (Schrödinger, LLC). Initially, the c-KIT protein was analyzed and preprocessed in the Maestro Panel “Protein Preparation Wizard”. The Glide program was used to generate the Grid for docking. All bond orders were allocated, −H atoms were incorporated, and metals, if any, were treated. The force field OPLS-2005 achieved a global minimum for c-KIT at RMSD = 0.30 Å. The van der Waals radius was set at a scaling factor of 1, and the partial charge was set to 0.25. A cocrystallized ligand Sunitinib in c-KIT was selected, and the 20 Å area around the ligands was designated as the “grid box”. This grid was further used to dock the target molecules.

Structures of all the ligands were drawn in the Maestro workspace and processed using the LigPrep module. Molecular docking was performed using the “Ligand Docking” panel. Previously prepared grids and 3D conformers of the ligands were selected to perform standard precision (SP) docking. The ligand was docked flexibly; nonplanar conformations of bonds (if any) were penalized. In the flexible mode of docking, 5000 poses were allowed, and only poses in the top 1% were kept for energy minimization. A total of 100 minimization steps were performed during the docking run.[39]

Structure Preparation and Inclusion Complex Study of β-CDSO3H

The structure of β-cyclodextrin was obtained from the crystal structure of the β-cyclodextrin complex (PDB ID 3CGT) and further built to β-CD-SO3H using the Builder module in Maestro.[52]

A global minimum of β-CD-SO3H is achieved at RMSD = 0.30 Å using the force field OPLS-2005. The grid for the inclusion complex study was generated using Glide. The van der Waals radius was selected at a scaling factor of 1, and the partial charge was limited to 0.25. 3D structures of all the starting material (indoline-2,3-diones) as ligands were built on Schrodinger Maestro workspace. The LigPrep protocol was used to prepare these ligands. The β-CD-SO3H grid was selected in the “Ligand Docking” module of Glide. The ligands (indoline-2,3-diones) were docked flexibly in the similar manner as the synthesized molecules. One-hundred minimization steps were performed. The docking score and the docking pattern were interpreted.

Phase I and Phase II Metabolism Predictions

Cytochrome P450, phase I and II metabolites, and the regioselectivity of metabolism for compounds IVc and VIc were predicted using machine learning tools GLORY, GLORYx, and FAME3, which are freely accessible (https://nerdd.univie.ac.at/) on a noncommercial basis. The Glory tool was used to anticipate metabolites that could be produced by the cytochrome P450 (CYP) enzyme family in humans.[53,54] The phase I- and phase II-mediated metabolites of compounds IVc and VIc were predicted using GLORYx.[55] The FAME3 program estimated the regioselectivity of phase I and II metabolism based on the submitted chemical structures.[56]

Experimental Synthesis of Target Molecules

All the required chemicals, commercial-grade reagents, and solvents were procured from Sigma-Aldrich, Rankem Chemicals, Avra Chemicals, S. D. Fine, and E-Merck. Precoated 0.2 mm thick aluminum TLC sheets (Merck) with GF254 silica gel and various mobile phase systems were used to monitor the reactions. TLC plates were visualized using a UV light, KMnO4 dip, and an iodine chamber. Silica gel (100–200 mesh) was used for column chromatography. The reaction kinetics study was performed on the GAMESS interface (PerkinElmer Inc.). The melting point of the synthesized compounds was uncorrected and determined using an Analab scientific melting point apparatus.

The FT-IR spectrum was recorded using the KBr pellet technique on a Shimadzu FTIR-8400S spectrometer. 1H NMR and 13C NMR spectra were recorded on Bruker Avance II 400 and 100 MHz NMR spectrometers, respectively, using CDCl3 and DMSO-d6 as solvents and TMS as internal standard. Mass spectra of the target molecules were recorded on Q-TOF Micromass (ESI-MS) MAT 120 spectrometer.

Synthesis of Substituted 2-(Hydroxyimino)-N-phenylacetamide (Scheme )

The synthesis of substituted 2-(hydroxyimino)-N-phenylacetamide was carried out using various anilines, chloral hydrate, and hydroxylamine hydrochloride following a reported protocol.[57] Crystalline solid products were obtained in 80–90% yields and further utilized for the synthesis of the desired indolin-2,3-diones (Ia–m).

General Procedure for the Synthesis of Indolin-2,3-diones (Ia–m)[58]

Concentrated H2SO4 (20 g) was added to a round-bottom flask (RBF) and stirred at 50 °C. Then, 0.015 mol dry 2-(hydroxyimino)-N-phenylacetamide was added in portions at a temperature ≤70 °C. After the complete addition of the respective 2-(hydroxyimino)-N-phenylacetamide, the reaction continued at 80 °C for 10 min. The reaction mixture was cooled to rt and poured onto ice. After 1 h of cooling, indolin-2,3-diones were obtained with 70–78% yields.

5-Fluoroisatin (Ic) and 5-Nitroisatin (Ig) were purchased from Sigma-Aldrich, while 5,6-dimethylisatin (Im) was synthesized using above protocol; however, the final crude mixture of 5,6-dimethylisatin and 4,5-dimethylisatin was separated by adapting the literature procedure.[59]

Procedure for the Synthesis of β-CDSO3H[60]

β-Cyclodextrin (10.0 g, 4.5 mmol) was added to a RBF. To the RBF was then added CH2Cl2 (50 mL), and the mixture was stirred. To this stirring mixture was added chlorosulfonic acid (2.00 g, 10 mmoL) slowly at 0 °C over 3 h. As the reaction was stirred, HCl gas evolution was observed and monitored using pH paper. The reaction was continued for next 2 h until the evolution of HCl ceased. The resulting mixture was then filtered and washed with 60 mL of MeOH. The product was dried to afford β-CDSO3H as a colorless powder (10.56 g). The −SO3H amount was found to be 0.52 mequiv/g using the titrimetric method. FT-IR spectra confirmed the structure of β-CD-SO3H due to the characteristic -SOO– symmetric stretching vibrations at 986 cm–1 and -SOO– asymmetric stretching vibrations at 1224 cm–1.

General Protocol for the Schiff Base Synthesis of Isatin-Linked Thiadiazoles Using the Greener Approach[60,61]

The warmed solution of β-CD-SO3H (1.3 equiv) in water was prepared in a RBF, then to the solution were added compound Ia–m and 2-amino-5-mercapto-1,3,4-thiadiazolein methanol. The mixture was allowed to reflux for 20 min to 1 h, and the progress of reaction was checked using TLC. After the completion of the reaction, the mixture was cooled at rt, and products were extracted using nonpolar or semipolar solvents (ether or dichloromethane, respectively) and water. Extracts in organic solvents were dried over anhydrous sodium sulfate and evaporated under vacuum to afford crude solid or semisolid derivatives. The resulting crude products were further purified by column chromatography.

Yield, Melting Point, and Spectral Characterization Data of Compounds IIIa–m

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IIIa)

Yield 82.0%, mp 174–178 °C. 1H NMR (400 MHz, DMSO): δ 12.72 (s, 1H, O=S—NH), 10.96 (s, 1H, NH), 7.43 (m, 1H, ArH), 7.06 (m, 2H, ArH), 6.94 (m, 1H, J = 6.0 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 184.84, 159.99, 147.08, 140.04, 131.79, 122.07, 121.34, 117.48. FT-IR (KBr, cm–1): 3219 (−NH), 3014 (−CH aromatic), 1737(O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 262.3 (M + H+). Anal. Calcd for C10H6N4OS2: C, 45.79; H, 2.31; N, 21.36; O, 6.10; S, 24.44. Found: C, 45.81; H, 2.29; N, 21.34; O, 6.10; S, 24.46.

Synthesis of 5-cChloro-3-((5-mercapto-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IIIb)

Yield 90.0%, mp 182–186 °C. 1H NMR (400 MHz, DMSO): δ 13.10 (s, 1H, O=S—NH), 10.08 (s, 1H, NH), 8.04 (s, 1H, ArH), 7.69 (d, 1H, ArH), 7.58 (d, 1H, J = 6.0 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 184.04, 162.34, 149.06, 138.16, 131.48, 130.19, 128.07, 125.34. FT-IR (KBr, cm–1): 3192 (−NH), 2926 (−CH aromatic), 1734(O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 296.5 (M + H+). Anal. Calcd for C10H5ClN4OS2: C, 40.47; H, 1.70; Cl, 11.95; N, 18.88; O, 5.39; S, 21.61. Found: C, 40.37; H, 1.80; Cl, 11.90; N, 18.93; O, 5.35; S, 21.66.

Synthesis of 5-Fluoro-3-((5-mercapto-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IIIc)

Yield 88.0%, mp 176–180 °C. 1H NMR (400 MHz, DMSO): δ 13.26 (s, 1H, O=S—NH), 10.14 (s, 1H, NH), 7.94 (s, 1H, ArH), 7.70 (d, 1H, ArH), 7.42 (d, 1H, J = 5.2 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 184.02, 158.89, 163.22, 153.17, 150.17, 118.48, 116.07, 110.74. FT-IR (KBr, cm–1): 3190 (−NH), 2922 (−CH aromatic), 1728 (O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 280.3 (M + H+). Anal. Calcd for C10H5FN4OS2: C, 42.85; H, 1.80; F, 6.78; N, 19.99; O, 5.71; S, 22.88. Found: C, 42.76; H, 1.80; F, 6.69; N, 19.99; O, 5.71; S, 22.88.

Synthesis of 5-Bromo-3-((5-mercapto-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IIId)

Yield 84.0%, mp 208–212 °C. 1H NMR (400 MHz, DMSO): δ 13.26 (s, 1H, O=S—NH), 10.14 (s, 1H, NH), 7.94 (s, 1H, ArH), 7.70 (d, 1H, ArH), 7.42 (d, 1H, J = 5.2 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 184.02, 158.89, 163.22, 153.17, 150.17, 118.48, 116.07, 110.74. FT-IR (KBr, cm–1): 3057 (−NH), 2941 (−CH aromatic), 1705 (O=C—NH cyclic), 1587 (—C=N). HRMS: m/z 280.3 (M + H+). Anal. Calcd for C10H5FN4OS2: C, 42.85; H, 1.80; F, 6.78; N, 19.99; O, 5.71; S, 22.88. Found: C, 42.76; H, 1.80; F, 6.69; N, 19.99; O, 5.71; S, 22.88.

Synthesis of 4-Chloro-3-((5-mercapto-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IIIe)

Yield 84.0%, mp 208–210 °C. 1H NMR (400 MHz, DMSO): δ 13.12 (s, 1H, O=S—NH), 10.06 (s, 1H, NH), 7.65 (s, 1H, ArH), 7.57 (d, 1H, ArH), 7.28 (d, 1H, J = 5.2 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 184.13, 162.89, 153.19, 150.17, 118.48, 116.07, 110.74. FT-IR (KBr, cm–1): 3196 (−NH), 2922 (−CH aromatic), 1734 (O=C—NH cyclic), 1620 (—C=N). HRMS: m/z 296.78 (M + H+). Anal. Calcd for C10H5ClN4OS2: C, 40.47; H, 1.70; Cl, 11.95; N, 18.88; O, 5.39; S, 21.61 Found: C, 40.42; H, 1.75; Cl, 11.93; N, 18.91; O, 5.28; S, 21.72.

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)-5-methylindolin-2-one (IIIf)

Yield 84.0%, mp 208–210 °C. 1H NMR (400 MHz, DMSO): δ 13.12 (s, 1H, O=S—NH), 10.06 (s, 1H, NH), 7.65 (s, 1H, ArH), 7.57 (d, 1H, ArH), 7.28 (d, 1H, J = 5.2 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 184.13, 162.89, 153.19, 150.17, 118.48, 116.07, 110.74. HRMS: m/z 276.03 (M + H+). Anal. Calcd for C10H5ClN4OS2: C, 40.47; H, 1.70; Cl, 11.95; N, 18.88; O, 5.39; S, 21.61 Found: C, 40.42; H, 1.75; Cl, 11.93; N, 18.91; O, 5.28; S, 21.72.

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)-5-nitroindolin-2-one (IIIg)

Yield 96.0%, mp 184–188 °C. 1H NMR (400 MHz, DMSO): δ 13.26 (s, 1H, O=S—NH), 10.05 (s, 1H, NH), 7.95 (s, 1H, ArH), 8.70 (d, 1H, ArH), 8.42 (d, 1H, ArH). 13C NMR (100 MHz, DMSO): δ 184.07, 163.54, 150.19, 146.23, 142.15, 127.37, 123.13, 118.48. HRMS: m/z 307.98 (M + H+). Anal. Calcd for C10H5N5O3S2: C, 42.85; H, 1.80; F, 6.78; N, 19.99; O, 5.71; S, 22.88. Found: C, 42.86; H, 1.80; F, 6.68; N, 19.97; O, 5.73; S, 22.83.

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)-2-oxoindoline-5-carboxylic acid (IIIh)

Yield 92.0%, mp 202–206 °C. 1H NMR (400 MHz, DMSO): δ 13.32 (s, 1H, O=S—NH), 12.63 (s, 1H, COOH), 10.07 (s, 1H, NH), 8.68 (s, 1H, ArH), 8.41 (d, 1H, ArH), 7.88 (d, 1H, ArH). 13C NMR (100 MHz, DMSO): δ 184.00, 169.32, 163.56, 150.10, 146.42, 132.15, 129.31, 125.17, 121.63, 117.21. HRMS: m/z 306.99 (M + H+). Anal. Calcd for C11H6N4O3S2: C, 43.13; H, 1.97; N, 18.29; O, 15.67; S, 20.94. Found: C, 43.08; H, 2.04; N, 18.29; O, 15.70; S, 20.91.

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)-5,6-dimethoxyindolin-2-one (IIIi)

Yield 72.0%, oily mass. 1H NMR (400 MHz, DMSO): δ 13.24 (s, 1H, O=S—NH), 10.13 (s, 1H, NH), 7.52 (s, 1H, ArH), 3.81 (s, 6H, CH3). 13C NMR (100 MHz, DMSO): δ 184.11, 163.32, 152.29, 150.14, 140.17, 145.53, 114.46, 111.56, 102.54, 56.12. FT-IR (KBr, cm–1): 3344 (−NH), 2923 (−CH aromatic), 1722 (O=C—NH cyclic), 1610 (—C=N). HRMS: m/z 323.02 (M + H+). Anal. Calcd for C12H10N4O3S2: C, 44.71; H, 3.13; N, 17.38; O, 14.89; S, 19.89 Found: C, 44.80; H, 3.08; N, 17.34; O, 14.92; S, 19.86.

Synthesis of Methyl 3-((5-mercapto-1,3,4-thiadiazol-2-yl)imino)-2-oxoindoline-7-carboxylate (IIIj)

Yield 68.0%, oily mass. 1H NMR (400 MHz, DMSO): δ 13.32 (s, 1H, O=S—NH), 10.17 (s, 1H, NH), 8.28 (d, 1H, ArH), 7.92 (d, 1H, ArH), 7.34 (d, 1H, ArH), 3.84 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO): δ 184.02, 168.52, 163.56, 150.10, 139.42, 139.24, 133.67, 132.15, 124.31, 117.21, 115.17. HRMS: m/z 321.00 (M + H+). Anal. Calcd for C12H8N4O3S2: C, 44.99; H, 2.52; N, 17.49; O, 14.98; S, 20.02. Found: C, 45.10; H, 2.41; N, 17.53; O, 14.93; S, 20.01.

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)-5-methoxyindolin-2-one (IIIk)

Yield 76.0%, oily mass. 1H NMR (400 MHz, DMSO): δ 13.17 (s, 1H, O=S—NH), 10.08 (s, 1H, NH), 7.74 (d, 1H, ArH), 7.51(s, 1H, ArH), 7.14 (d, 1H, ArH), 3.76 (s, 3H, CH3). 13C NMR (100 MHz, DMSO): δ 184.01, 163.52, 155.29, 150.16, 133.17, 122.64, 118.73, 116.83, 113.53, 55.89. HRMS: m/z 293.01 (M + H+). Anal. Calcd for C11H8N4O2S2: C, 45.19; H, 2.76; N, 19.17; O, 10.95; S, 21.94; Found: C, 45.22; H, 2.73; N, 19.14; O, 10.98; S, 21.88.

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)-2-oxoindoline-7-carboxylic acid (IIIl)

Yield 94.0%, mp 208–210 °C. 1H NMR (400 MHz, DMSO): δ 13.22 (s, 1H, O=S—NH), 12.83 (s, 1H, COOH), 10.17 (s, 1H, NH), 8.48 (d, 1H, J = 6.4 Hz, ArH), 8.21 (d, 1H, ArH), 7.52 (m, 1H, ArH). 13C NMR (100 MHz, DMSO): δ 184.02, 169.37, 163.56, 150.10, 140.32, 134.67, 132.82, 124.31, 117.21, 115.62. HRMS: m/z 306.99 (M + H+). Anal. Calcd for C11H6N4O3S2: C, 43.13; H, 1.97; N, 18.29; O, 15.67; S, 20.94. Found: C, 43.10; H, 2.02; N, 18.26; O, 15.72; S, 20.92.

Synthesis of 3-((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)-4,5-dimethylindolin-2-one (IIIm)

Yield 78.0%, oily mass. 1H NMR (400 MHz, DMSO): δ 13.14 (s, 1H, O=S—NH), 10.09 (s, 1H, NH), 7.34 (d, 1H, ArH), 6.58 (d, 1H, ArH). 13C NMR (100 MHz, DMSO): δ 184.02, 163.32, 150.29, 143.64, 123.88, 138.67, 132.53, 131.46, 118.56, 13.2, 18.8. FT-IR (KBr, cm–1): 3356 (−NH), 2924 (−CH aromatic), 1712 (O=C—NH cyclic), 1602 (—C=N). HRMS: m/z 291.36 (M + H+). Anal. Calcd for C12H10N4OS2: C, 49.64; H, 3.47; N, 19.30; O, 5.51; S, 22.09; Found: C, 49.61; H, 3.50; N, 19.26; O, 5.55; S, 22.09.

General Protocol for the Preparation of 3-((5-(Phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IVa–m)[62]

Compound IIIa–m (10 mmol), bromobenzene (10 mmol, 1.05 mL), bis(triphenylphosphine)palladium(II)dichloride (0.010 mmol, 0.1 mol %), and EtONa (25 mmol, 1.7 g) were added into two-neck RBF. The vessel was purged with nitrogen gas, followed by the addition of toluene (10 mL). The reaction mixture was allowed to stir at 115 °C in an oil bath overnight. The progress of the reaction was monitored using TLC (EA/Hex) for new spot formation. an excess of ether was added to the reaction mixture. The reaction mixture was filtered and concentrated under reduced pressure. Crude products were further purified by flash chromatography (Combiflash) on silica gel to afford the final purified products with excellent yields.

Yield, Melting Point, And Spectral Characterization of Compounds (IVa–m)

Synthesis of 3-((5-(Phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVa)

Yield 80.0%, mp 184–186 °C. 1H NMR (400 MHz, DMSO): δ 10.59 (s, 1H, NH), 7.76 (d, 1H, J = 7.0 Hz, ArH), 7.54 (d, 2H, ArH), 7.49 (t, 1H, J = 6.0 Hz, ArH), 7.32 (m, 5H, ArH). 13C NMR (100 MHz, DMSO): δ 164.24, 163.99, 150.08, 140.04, 129.79, 129.25, 122.07, 121.34, 117.48. FT-IR (KBr, cm–1): 3196 (−NH), 2922 (−CH aromatic), 1732 (O=C—NH cyclic), 1620 (—C=N). HRMS: m/z 337.2 (M + H+). Anal. Calcd for C16H10N4OS2: C, 56.79; H, 2.98; N, 16.56; O, 4.73; S, 18.95. Found: C, 56.75; H, 2.98; N, 16.60; O, 4.69; S, 18.99.

Synthesis of 5-Chloro-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVb)

Yield 90.0%, mp 192–194 °C. 1H NMR (400 MHz, DMSO): δ 10.59 (s, 1H, NH), 7.75 (d, 1H, J = 6.0 Hz, ArH), 7.72–7.54 (m, 5H, ArH), 7.49–7.42 (m, 2H, ArH), 7.32 (d, 1H, ArH). 13C NMR (100 MHz, DMSO): δ 168.30, 163.59, 139.12, 136.23, 131.50, 129.59, 129.25, 125.52, 119.18. FT-IR (KBr, cm–1): 3207 (−NH), 2924 (−CH aromatic), 1734 (O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 372.09 (M + H+). Anal. Calcd for C16H9ClN4OS2: C, 51.54; H, 2.43; Cl, 9.51; N, 15.03; O, 4.29; S, 17.20. Found: C, 51.34; H, 2.53; Cl, 9.61; N, 15.13; O, 4.34; S, 17.25.

Synthesis of 5-fluoro-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVc)

Yield 84.0%, mp 186–188 °C. 1H NMR (400 MHz, DMSO): δ 10.58 (s, 1H, NH), 7.82 (d, 2H, ArH), 7.42 (d, 1H, J = 7.0 Hz, ArH), 7.39 (d, 2H, J = 6.0 Hz, ArH), 7.27–23 (m, 3H, ArH). 13C NMR (100 MHz, DMSO): δ 167.30, 162.14, 158.6, 154.6, 136.28, 129.63, 129.21, 125.52, 118.18; 116.12, 112.40, 111.20. FT-IR (KBr, cm–1): 3184 (−NH), 2922 (−CH aromatic), 1724 (O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 356.40 (M + H+). Anal. Calcd for C16H9FN4OS2: C, 53.92; H, 2.55; F, 5.33; N, 15.72; O, 4.49; S, 17.99. Found: C, 53.84; H, 2.63; F, 5.43; N, 15.62; O, 4.56; S, 17.92.

Synthesis of 5-Bromo-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVd)

Yield 82.0%, mp 188–190 °C. 1H NMR (400 MHz, DMSO): δ 10.60 (s, 1H, NH), 8.82 (s, 1H, ArH), 7.76–7.72 (dd, 2H, J = 9.0 Hz, ArH), 7.39 (d, 2H, J = 6.0 Hz, ArH), 7.26–24 (m, 3H, ArH). 13C NMR (100 MHz, DMSO): δ 167.20, 163.18, 150.3, 136.65, 129.63, 129.21, 125.52, 118.18; 114.41. FT-IR (KBr, cm–1): 3173 (−NH), 2999 (−CH aromatic), 1734 (O=C—NH cyclic), 1600 (—C=N). HRMS: m/z 417.30 (M + H+). Anal. Calcd for C16H9BrN4OS2: C, 46.05; H, 2.17; Br, 19.15; N, 13.43; O, 3.83; S, 15.37. Found: C, 46.12; H, 2.10; Br, 19.22; N, 13.50; O, 3.76; S, 15.30.

Synthesis of 4-Chloro-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVe)

Yield 82.0%, mp 218–220 °C. 1H NMR (400 MHz, DMSO): δ 10.03 (s, 1H, NH), 7.62 (d, 1H, ArH), 7.52 (t, 1H, ArH), 7.47 (d, 2H, J = 4.7 Hz, ArH), 7.42 (t, 1H, ArH), 7.38–7.36 (m, 3H, ArH). 13C NMR (100 MHz, DMSO): 167.20, 163.18, 150.3, 148.42, 136.53, 134.65, 132.34, 129.53, 129.11, 125.18, 123.76, 117.18. HRMS: m/z 372.4 (M + H+). Anal. Calcd for C16ClH9N4OS2: C, 51.54; H, 2.43; Cl, 9.51; N, 15.03; O, 4.29; S, 17.20. Found: C, 51.49; H, 2.48; Cl, 9.51; N, 15.02; O, 4.30; S, 17.20.

Synthesis of 5-Methyl-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVf)

Yield 70.0%, mp 178–280 °C. 1H NMR (400 MHz, DMSO): δ 10.03 (s, 1H, NH), 8.32 (s, 1H, ArH), 7.40 (d, 2H, ArH), 7.28–7.25 (m, 3H, ArH), 6.76 (d, 1H, J = 4.8 Hz, ArH), 2.28 (s, 3H, CH3). 13C NMR (100 MHz, DMSO): δ 167.10, 163.28, 150.2, 138.42, 136.17, 134.65, 134.34, 131.56, 129.63, 129.41, 121.6, 125.54, 117.18, 21.4. HRMS: m/z 352.42 (M + H+). Anal. Calcd for C17H12N4OS2: C, 57.93; H, 3.43; N, 15.90; O, 4.54; S, 18.20 Found: C, 57.90; H, 3.46; N, 15.80; O, 4.63; S, 18.22.

Synthesis of 5-Nitro-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVg)

Yield 92.0%, mp 194–196 °C. 1H NMR (400 MHz, DMSO): δ 10.05 (s, 1H, NH), 8.52 (s, 1H, ArH), 8.12 (d, 2H, J = 8.4 Hz, ArH), 7.40 (d, 2H, J = 6.8, Hz ArH), 7.28–7.25 (m, 3H, ArH). 13C NMR (100 MHz, DMSO): δ 167.10, 163.58, 150.2, 147.34, 138.42, 136.47, 129.63, 129.38, 126.16, 125.54, 123.54, 122.68, 118.60. FT-IR (KBr, cm–1): 3201 (−NH), 2922 (−CH aromatic), 1732 (O=C—NH cyclic), 1620 (—C=N). HRMS: m/z 383.32 (M + H+). Anal. Calcd for C16H9N5O3S2: C, 50.12; H, 2.37; N, 18.27; O, 12.52; S, 16.73. Found: C, 50.18; H, 2.31; N, 18.23; O, 12.54; S, 16.75.

Synthesis of 2-Oxo-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indoline-5-carboxylic acid (IVh)

Yield 90.0%, mp 212–214 °C. 1H NMR (400 MHz, DMSO): δ 12.71 (s, 1H, COOH), 10.96 (s, 1H, NH), 7.42–7.34 (m, 5H, ArH), 7.04 (d, 2H, ArH), 6.94 (d, 1H, J = 7.3 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 169.48, 167.89, 163.24, 150.10, 146.78, 136.45, 132.15, 129.31, 129.14, 129.0, 125.17, 121.63, 117.21. FT-IR (KBr, cm–1): 3414 (−OH), 2928 (−CH aromatic), 1758 (O=C—OH carboxylic acid), 1620 (—C=N). HRMS: m/z 382.3 (M + H+). Anal. Calcd for C17H10N4O3S2: C, 53.39; H, 2.64; N, 14.65; O, 12.55; S, 16.77. Found: C, 53.48; H, 2.74; N, 14.55; O, 12.51; S, 16.72.

Synthesis of 5,6-Dimethoxy-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVi)

Yield 72.0%, mp 156–158 °C. 1H NMR (400 MHz, DMSO): 10.10 (s, 1H, NH), 7.38–7.36 (m, 3H, ArH), 7.27–7.24 (m, 3H, ArH), 3.81 (s, 6H, CH3). 13C NMR (100 MHz, DMSO): δ 167.12, 163.46, 152.32, 150.10, 145.10, 140.23, 136.40, 129.34, 129.10, 129.0, 125.48, 114.36, 102.14, 56.15. FT-IR (KBr, cm–1): 3192 (−NH), 2922 (−CH aromatic), 1732 (O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 398.0 (M + H+). Anal. Calcd for C18H14N4O3S2: C, 54.26; H, 3.54; N, 14.06; O, 12.05; S, 16.09 Found: C, 54.22; H, 3.58; N, 14.16; O, 12.00; S, 16.04.

Synthesis of Methyl 2-Oxo-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indoline-7-carboxylate (IVj)

Oily mass. 1H NMR (400 MHz, DMSO): δ 10.05 (s, 1H, NH), 8.28 (d, 1H, ArH), 7.94 (d, 1H, ArH), 7.38–7.36 (m, 3H, ArH), 7.27–7.24 (m, 3H, ArH), 3.84 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO): δ 168.50, 167.16, 163.48, 150.10, 140.18, 136.78, 133.76, 132.80, 129.34, 129.10, 129.0, 125.52, 124.3, 117.21, 115.22, 51.02. FT-IR (KBr, cm–1): 3192 (−NH), 2920 (−CH aromatic), 1732 (O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 396.2 (M + H+). Anal. Calcd for C18H12N4O3S2: C, 54.53; H, 3.05; N, 14.13; O, 12.11; S, 16.18. Found: C, 54.58; H, 3.02; N, 14.10; O, 12.17; S, 16.13.

Synthesis of 5-Methoxy-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVk)

Yield 74.0%, mp 174–176. 1H NMR (400 MHz, DMSO): δ 10.08 (s, 1H, NH), 7.72 (d, 1H, J = 6.3 Hz, ArH), 7.50–7.48 (m, 3H, ArH), 7.42 (t, 1H, J = 4.1 Hz, ArH), 7.36 (d, 2H, J = 4.2 Hz, ArH), 7.12 (d, 1H, ArH), 3.74 (s, 3H, CH3). 13C NMR (100 MHz, DMSO): δ 167.10, 163.52, 156.32, 150.14, 136.78, 133.56, 133.40, 129.43, 129.10, 129.0, 125.54, 122.28, 118.74, 116.39, 113.42, 55.76. FT-IR (KBr, cm–1): 3188 (−NH), 2922 (−CH aromatic), 1730 (O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 368.04 (M + H+). Anal. Calcd for C17H12N4O2S2: C, 55.42; H, 3.28; N, 15.21; O, 8.68; S, 17.40; Found: C, 55.30; H, 3.38; N, 15.23; O, 8.60; S, 17.48.

Synthesis of 2-Oxo-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indoline-7-carboxylic acid (IVl)

Yield 90.0%, mp 218–220 °C. 1H NMR (400 MHz, DMSO): δ 12.78 (s, 1H, COOH), 10.16 (s, 1H, NH), 8.44 (d, 1H, J= 6.4 Hz, ArH), 8.22 (d, 1H, J= 4.8 Hz, ArH), 7.58 (t, 1H, ArH), 7.48–7.46 (m, 3H, ArH), 7.36 (d, 2H, J = 6.8 Hz, ArH). 13C NMR (100 MHz, DMSO): δ 169.42, 163.52, 150.13, 140.44, 136.52, 134.60, 132.72, 129.44, 129.12, 129.0, 124.38, 122.28, 117.64, 124.31, 117.61, 115.70. HRMS: m/z 382.1 (M + H+). Anal. Calcd for C17H10N4O3S2: C, 53.39; H, 2.64; N, 14.65; O, 12.55; S, 16.77. Found: C, 53.42; H, 2.61; N, 14.56; O, 12.60; S, 16.81.

Synthesis of 4,5-Dimethyl-3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-one (IVm)

Yield 80.0%, mp 180–182 °C. 1H NMR (400 MHz, DMSO): δ 10.05 (s, 1H, NH), 7.38 (d, 2H, J = 6.4 Hz, ArH), 7.28 (t, 2H, ArH), 7.22 (t, 1H, ArH), 6.58–6.55 (d, 2H, ArH), 2.36 (m, 3H, CH3). 2.20 (m, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 167.02, 163.52, 150.23, 143.76, 138.14, 136.40, 132.54, 131.42, 129.44, 129.02, 125.52, 118.58, 18.82, 13.24. FT-IR (KBr, cm–1): 3184 (−NH), 3090 (−CH aromatic), 1726 (O=C—NH cyclic), 1618 (—C=N). HRMS: m/z 366.1 (M + H+). Anal. Calcd for C18H14N4OS2: C, 58.99; H, 3.85; N, 15.29; O, 4.37; S, 17.50; Found: C, 58.86; H, 3.85; N, 15.34; O, 4.41; S, 17.54.

Synthesis of Methyl 1-acetylaziridine-2-carboxylate (V) (Scheme )

Procedure for the Synthesis of Methyl 2-Acetamido-3-hydroxypropanoate[43]

In an inert atmosphere, N-acetyl-dl-serine (9.59 mmol) was solubilized in MeOH (2 mL/mmol). The solution solution was stirred at 0 °C (salt/ice). Then, to the solution was slowly added thionyl chloride (56.69 mmol, 5.9eq ) while the temperature was maintained at 0 °C. After the complete addition of thionyl chloride, the reaction mixture was refluxed for 3 h. TLC was monitored in a BuOH/acetic acid/water (3:1:1) system. The mixture was then concentrated and coevaporated with ether. The product was obtained as a white crystalline solid. Yield 90%, mp 166–168 °C. Product was confirmed by FT-IR.

Protocol for the Synthesis of Methyl 1-Acetylaziridine-2-carboxylate (V) (Scheme )[44]

The methyl ester of N-acetyl-dl-serine (8.3 mmol) was added to a two-neck RBF. In presence of nitrogen gas, dry toluene (18 mL) and triethyl amine (24.9 mmol, 3 equiv) were added to the RBF. The reaction mixture was stirred at −30 °C and then sulfuryl chloride (9.96 mmol, 1.2 equiv) was slowly added dropwise with the help of a syringe. The reaction mixture stirred at room temperature, and the pH was monitored at alkaline conditions.

Reaction completion was confirmed by TLC (EA/Hex) until an UV and ninhydrin-active spot was visible. At the end of reaction, to the mixture was added an excess of ethyl acetate, and the reaction mixture was washed with brine. The nonpolar layer was dried over Na2SO4, then evaporated under vacuum. The crude product was further purified by column chromatography (silica G 100–200) using EA/Hex as the mobile phase. Yield 76.0%, crude oil. 1H NMR (400 MHz, CDCl3): δ 3.76 (s, 3H, −O–CH3), 3.04 (t, 1H, −CH), 2.12 (dd, 1H, −CH2), 2.34 (s, 3H, −CH3), 1.82 (dd, 1H, −CH2). 13C NMR (DMSO, 400 MHz): δ 172.58, 171.34, 51.94, 35.46, 29.57, 20.67. HRMS: m/z 143.2 (M + H+). Anal. Calcd for C6H9NO3: C, 50.35; H, 6.34; N, 9.79; O, 33.53; Found: C, 50.42; H, 6.27; N, 9.83; O, 33.49.

Synthesis of Methyl 1-(1-((2-oxoindolin-3-ylidene)hydrazono)ethyl)aziridine-2-carboxylates (VIa and VIc) (Scheme )

Synthesis of 3-Hydrazineylideneindolin-2-ones (Vx and Vy)

Hydrazone derivatives of isatin (Vx and Vy) were synthesized by the microwave irradiation of Isatin with 55% hydrazine and ethylene glycol at medium power for 30 s. Following a workup procedure conducted per the reported protocol, final products were obtained with the yields of 81.5% (Vx) and 83% (Vy) and melting points 218–220 °C (lit. 219 °C).[42]

General Protocol for Synthesis of Methyl 1-(1-((2-Oxoindolin-3-ylidene)hydrazono)ethyl)aziridine-2-carboxylates (VIa and VIc)

3-Hydrazineylideneindolin-2-one (1 equiv) (Vx or Vy) in methanol (1 equiv) and warmed solution of β-cyclodextrin-SO3H (1.3 equiv) in water were stirred at warm temperature. To the mixutre was then added methyl 1-acetylaziridine-2-carboxylate (1 equiv). The reaction mixture was refluxed with stirring for 15–20 min until the completion of the reaction. The reaction mixture was allowed to cool to room temperature. The product was extracted in ether. The nonpolar layer was dried over anhydrous sodium sulfate, followed by evaporation under vacuum, to obtain solid or semisolid derivatives. The resulting products were further purified by column chromatography.

Methyl 1-(1-((2-Oxoindolin-3-ylidene)hydrazineylidene)ethyl)aziridine-2-carboxylate (VIa)

Yield 78.0%, mp 160–162 °C. 1H NMR (400 MHz, DMSO): δ 10.02 (s, 1H, NH), 7.83 (d, 2H, J = 5.8 Hz, ArH), 7.54 (t, 1H, ArH), 7.28 (t, 1H, ArH), 3.68 (s, 3H, −O–CH3), 3.34 (s, 3H, −CH3), 2.58 (t, 1H, −CH), 2.10 (dd, 1H, −CH2), 1.86 (dd, 1H, −CH2). 13C NMR (100 MHz, DMSO): δ 171.68, 169.02, 163.52, 156.45, 141.26, 138.34, 129.47, 131.42, 124.18, 119.22, 117.49, 52.13, 35.10, 29.83, 14.64. HRMS: m/z 286.1 (M + H+). Anal. Calcd for C14H14N4O3: C, 58.74; H, 4.93; N, 19.57; O, 16.77; Found: C, 58.24; H, 5.03; N, 19.86; O, 16.88.

Methyl 1-(1-((5-Fluoro-2-oxoindolin-3-ylidene)hydrazineylidene)ethyl)aziridine-2-carboxylate (VIc)

Yield 76.0%, mp 172–174 °C. 1H NMR (400 MHz, DMSO): δ 10.03 (s, 1H, NH), 7.93 (s, 1H, ArH), 7.73 (d, 2H, J = 4.6 Hz, ArH), 7.42 (d, 1H, J = 4.3 Hz, ArH), 3.64 (s, 3H, −O–CH3), 2.70 (s, 3H, −CH3), 2.41 (t, 1H, −CH), 2.12 (dd, 1H, −CH2), 1.88 (dd, 1H, −CH2). 13C NMR (100 MHz, DMSO): δ 171.55, 169.00, 163.52, 158.60, 156.28, 138.14, 136.85, 129.47, 119.35, 118.04, 112.74, 51.93, 35.58, 29.86, 14.71. HRMS: m/z 304.1 (M + H+). Anal. Calcd for C14H13FN4O3: C, 55.26; H, 4.31; F, 6.24; N, 18.41; O, 15.77; Found: C, 55.18; H, 4.35; F, 6.28; N, 18.35; O, 15.83.

Experiment for Biological Activity

Anticancer activity study was studied through the National Institute of Health’s (NIH) NCI-60 human cancer cell line screening program[63] at the Chemotherapeutic Agents Repository, c/o Fisher Bio Services, 20301, Century Boulevard, Building 6, Suite 800, Germantown, MD 20874, United States. The National Cancer Institute (NCI) selected 20 new compound structures from all synthesized compounds for a single-dose screen. All 20 compounds were selected from all schemes, they were sent in the desirable purified form to NCI for a preliminary human cancer single-dose study. The screening was done using the sulforhodamine-B (SRB) assay.[64] All the compounds were tested against 60 cancer cell lines of nine panels at a 10 μM dose. A graph of the mean growth percent of the tested cells was obtained as a result of the single-dose screening. The resulting values for growth inhibition (0 to 100) and cytotoxicity (<0) were interpreted by the COMPARE program.[65]

The compounds that exhibited broad-spectrum anticancer activity in the single-dose screening further proceeded to the five-dose screening toward 60 human cancer cell lines. In the five-dose screening test, the compound under investigation was exposed to cancer cells for a period of 48 h to estimate the cell growth using the SRB protein assay. The experimental details of the assay and the measured activity pattern across all cancer subpanels have been reported.[66,67] Anticancer activities for the two compounds in the five-dose screen were interpreted using GI50 (the compound’s molar concentration at which 50% net cell proliferation is inhibited), TGI (the total growth inhibition that represents the cytostatic activity), and LC50 (cytotoxic activity which counts 50% net cell death) calculations. Dose response parameters were calculated for each individual cell line, including GI50, TGI, and LC50.[68]

Conclusions

Three novel series of indolin-2-ones hybrids (IIIa–m, IVa–m, VIa, and VIc) were designed by virtual screening based on the stem cell factor receptor c-KIT using a prevalidated 3D pharmacophore model (ADHRR.24). Obtained hits were further checked for drug-likeness and in silico ADME properties. The privileged anticancer scaffold indolin-2-one was successfully hybridized with other anticancer structural frameworks such as 1,3,4-thiadiazole and aziridine. Schiff bases of indolin-2-ones (IIIa–m, VIa, and VIc) were prepared with excellent yields via a novel green synthetic route in water utilizing β-CD-SO3H as a reusable supramolecular catalyst. The inclusion complex formation suggested that the noncovalent interaction of β-CD-SO3H with the starting materials facilitates the Schiff base formation in water. Indolin-2-one and 1,3,4-thiadiazole hybrids (IIIa–m) were further extended to aryl thioetherification to afford compounds IVa–m. C–S bond formation was successfully achieved using bis(triphenylphosphine)palladium(II)dichloride (0.1 mol %) as a catalyst. A docking study on the c-KIT kinase protein revealed that compounds VIc and IVc interact with GLU671 and CYS673, two key amino acid residues, with docking scores of −10.195 and −9.662, respectively.

Most of the target compounds showed good bioactivities against the tested human cancer cell lines at a single-dose concentration of 10–5 m. Based on their broad spectrum of antiproliferative activity, compounds IVc and VIc were further evaluated via the five-dose screening. Compound IVc had inhibitory concentrations of 0.71 and 1.04 μM against MDA-MB-231/ATCC and MCF7 cell lines, respectively, with an average activity of 1.47 μM IC50 against the breast cancer subpanel. Compound VIc showed potent activity of IC50 1.40 μM toward colon cancer cell lines. Cytochrome P450 and phase I and II metabolism predictions show that compounds IVc and VIc provide suitable regioselectivity for various metabolic reactions involving aliphatic hydroxylation, aromatic hydroxylation, and S-oxidation. Compounds IVc and VIc have the best results for antiproliferative activity, pharmacophore alignment, the docking simulation, ADME, and metabolite predictions, suggesting that these two compounds could be a promising candidates for cancer treatment.