A Mini Review on Isatin, an Anticancer Scaffold with Potential Activities against Neglected Tropical Diseases (NTDs)

Isatin, chemically an indole-1H-2,3-dione, is recognised as one of the most attractive therapeutic fragments in drug design and development. The template has turned out to be exceptionally useful for developing new anticancer scaffolds, as evidenced by the increasing number of isatin-based molecules which are either in clinical use or in trials. Apart from its promising antiproliferative properties, isatin has shown potential in treating Neglected Tropical Diseases (NTDs) not only as a parent core, but also by attenuating the activities of various pharmacophores. The objective of this mini-review is to keep readers up to date on the latest developments in the biological potential of isatin-based scaffolds, targeting cancer and NTDs such as tuberculosis, malaria, and microbial infections.


Introduction
Neglected Tropical Diseases cause health, social, and economic problems in tropical and subtropical regions and affect the world's poorest and most vulnerable people. They are most common in the tropical and subtropical areas of Africa, Asia, and Latin America, where hot and humid climatic conditions promote vector growth [1]. These are commonly referred to as NTDs because they receive little attention in terms of surveillance, prevention, and treatment. Poor sanitation and hygiene, the development of resistant strains, and the toxicity of current antiprotozoal regimens are the primary causes of the rise in the global burden of protozoan diseases. According to WHO statistics, this broad group of ailments affects more than one billion people worldwide and is found in 149 tropical and subtropical countries. NTDs restrict afflicted communities severely, resulting in a slew of illnesses, pain, disability, and death, as well as significant social, economic, and psychological implications for millions of people [2]. Initiatives from all over the world have been critical in the fight against NTDs. The Drugs for Neglected Diseases Initiative (DNDi) is a non-profit research organisation dedicated to the development of novel medications for people suffering from neglected diseases [3]. Aside from de novo drug discovery, the repositioning and re-engineering of existing drugs or drug-like molecules with known pharmacokinetics and established target profiles are excellent starting points for identifying new chemical entities.
Isatin, also known as 1H-indol-2,3-dione, is a natural heterocyclic compound extracted as red-orange powder from a variety of plants worldwide, including Isatis tinctoria, Couroupita guianensis Aubl, Melochia tomentosa, and Boronia koniamboensis [4]. Moreover, it is also found in the secretion of the parotid gland of Bufo frogs as well as in the Australian mollusc Dicathais orbita [5]. In humans, it is identified as a metabolite of tryptophan or epinephrine and is largely distributed in the central nervous system (CNS), peripheral tissues, as well as body fluids. Substituted isatins can also be found in plants such as melosatin alkaloids (methoxy phenyl isatins) isolated from the Caribbean tumourigenic

Anticancer Activities of Functionalized Isatins
In every country on this planet, cancer is the main source of mortality and a key hindrance to extending life expectancy. Cancer is the primary or second largest cause of death among people under the age of seventy, according to the World Health Organization (WHO). Globally, 19.3 million new cancer cases are expected to be diagnosed in 2020, with about 10.0 million cancer deaths [10]. Female breast cancer has superseded lung cancer as the most frequently diagnosed cancer, with an estimated 2.3 million new cases, followed by lung, colorectal, prostate, and stomach cancers. The unprecedented diversity of cancer continues to provide insights to the underlying factors, but it also perpetuates the need for a global intensification of measures to control the disease [11].
A number of isatin-based compounds have entered into clinical trials, including two compounds, namely Sunitinib and Toceranib, that have been approved for clinical use against tumours (Figure 1). Sunitinib suppresses the catalytic activity of kinases in the phosphorylation of proteins by reversibly binding to their ATP binding sites. Toceranib, a Sunitinib-like molecule, acts as a selective inhibitor of specific receptor tyrosine kinases (RTKs), thus triggering tumour cells' apoptosis in vivo. Other derivatives, including Nintedanib, Semaxinib, and Orantinib, are currently undergoing clinical trials for their anticancer potential ( Figure 1). These promising molecules have demonstrated the capacity to slow or stop the growth of tumours via modulating cell growth, proliferation, survival, and migration. However, some of these anticancer candidates also display side effects, such as limited efficacy, diarrhoea, hypertension, vomiting, hand-foot syndrome, and neutropenia, which provides an impetus for the identification of new candidates with promising activities [12].

Isatin-Based Amides and Sulphonamides
Gao et al. synthesized a series of compounds, having isatin as caps and o-phenylenediamine as a zinc-binding functionality. The synthesized scaffolds were evaluated for their HDAC inhibition using HeLa nuclear extract. Compound 1 proved to be the most active among the series, with comparable HDAC inhibition and antiproliferative activities with entinostat (MS-275) ( Figure 2). However, 1 exhibited moderate HDAC1 selectivity over HDAC2 and HDAC3, as compared to entinostat (MS-275) [13]. Abo-Ashour et al. synthesized a series of hydrazide-linked isatin-sulfonamides and evaluated various carbonic anhydrase isoforms viz. hCA I, II, IX, and XII. The synthesized scaffolds inhibited the tested hCAs to varying degrees; for example, the KIs on hCA I ranged from 671.8 to 3549.5 nM, while, for hCA II, hCA IX, and hCA XII, it ranged from 36.8 to 892.4; 8.9 to 264.5, and 9.0 to 78.1 nM, respectively. The isatin-sulfonamide 2 proved to be the most active among the synthesized series, exhibiting KIs of 8.9 and 9.2 nM on hCA IX and XII, respectively ( Figure 2). Molecular docking studies of 2 within the hCA II, IX, and XII active sites were also performed in order to rationalize the observed results [14].
The authors further extended the study and synthesized a series of isatinbenzenesulfonamide hybrids linked via a hydrazine linker and evaluated them against tumour-associated human carbonic anhydrase isoforms, hCA I, II, IX, and XII, along with their antiproliferative evaluation on a panel of sixty cancer lines according to US-NCI protocol. The synthesized compounds exhibited inhibitory activities with K 1 ranging from 28.3 to 692.2; 8.3 to 65.4; and 11.9 to 72.9 nM on hCA II, IX, and XII isoforms, respectively. The promising compound among the synthesized series, 3 (K 1 from 7.8-32.6 nM), exhibited broad spectrum activity on various cancer lines ( Figure 2). Molecular modelling studies were also carried out in order to ascertain the possible binding interactions of the promising scaffolds in the active sites of isoforms II and IX [15].
Eldehna et al. synthesized a series of amido/ureido-tethered isatin-benzene sulfonamide hybrids and evaluated the in vitro inhibitory activity against a panel of hCA I and II (cytosolic) and IX and XII (transmembrane, tumour-associated) isoforms. Most of the synthesized hybrids inhibited the tested isoforms in variable degrees, with the best activity being observed on tumour-associated isoform hCA XII, with K 1 values ranging from 0.47 to 2.83 nM. Among the amide-linked hybrids, the presence of either an electron-withdrawing (NO 2 ) or electron-releasing (OCH 3 ) substituent improved the activity profiles, as evidenced by hybrids 4a and 4b exhibiting Ki (inhibition constant) values of 0.69 and 0.47 nM, respectively ( Figure 2). Among urea-linked hybrids, compound 4c, with −NO 2 substituent at the C-5 position of the isatin ring, proved to be promising, with a K i value of 0.64 nM [16].
Further, a series of sulfonamide incorporating substituted indolin-2-ones through aminoethyl or (4-oxothiazolidin-2-ylidene) aminoethyl linkers was synthesized and evaluated in vitro on human carbonic anhydrase isoforms hCA I, II, IV, and VII. The synthesized scaffolds inhibited the tested hCAs in variable degrees. In the case of hCA I, the scaffolds exhibited Ki's ranging from 42 to 8550.9 nM, while the Ki's in case of hCA II, hCA IV, and hCA VII were in the range of 5.9-761; 4.0-2069.5; and 13.2-694 nM, respectively. The scaffold 5 proved to be most the promising hCA II inhibitors, with Ki's in the range of 5.9 to 9.4 nM ( Figure 2) [17].
In another report, Eldehna et al. disclosed the synthesis and in vitro evaluation of a series of indolinone-based sulfonamides as inhibitors of carbonic anhydrase isoforms, namely hCA IX and XII. The synthesized sulfonamides exhibited inhibitory activities in low nM concentration towards both hCA IX (Ki's: 6.2-64.8 nM) and XII (Ki's: 7.1-55.6 nM) isoforms. Further, the synthesized scaffolds were also evaluated for in vitro antiproliferative activities on HCT-116 and MCF-7 cells. Compound 6 proved to be most active on HCT-116, with an IC 50 of 3.67 µM, and was further assayed for cell cycle disturbance and apoptosis induction in HCT-116 cells (Figure 2). The compound exhibited cell cycle arrest at the G2-M stage, along with an alteration of the Sub-G1 phase and induced intrinsic apoptotic mitochondrial pathway through down-regulation of the antiapoptotic protein Bcl-2 level [18].
The above authors further reported the synthesis of N-substituted isatin-SLC-0111 hybrids along with their evaluation on physiologically relevant hCA isoforms viz. hCA I, II, IX, and XII using a stopped flow CO 2 hydrase assay. The synthesized hybrids efficiently inhibited hCA IX and XII, with Ki's ranging from 4.7 to 86.1 nM and 1.3 to 80.9 nM, respectively. The promising hybrids were then evaluated for their antiproliferative activities on MDA-MB-231 and MCF-7 cells under hypoxic conditions and identified 7 as the most active hybrid with IC 50 s of 7.43 and 12.90 µM ( Figure 2). Further, 7 disrupted the MDA-MB-231 cell cycle through the modification of the sub-G1 phase and arrested the G2-M phase. Additionally, 7 exhibited a substantial increase in annexinV-FITC positive apoptotic cells from 1.03 to 18.54%, along with potent VEGFR-2 inhibitory activity, with an IC 50 of 260.64 nM [19].
Wang et al. synthesized a series of isatin-inspired α,β-unsaturated ketones and evaluated their antiproliferative activities on a panel of cancer cell lines using an MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay. The initial screening revealed 8 as active on all the cell lines tested, being most active on BGC-823, SGC-7901 and NCI-H460 cells with IC 50 s of 3.6, 5.7, and 3.2 µM, respectively ( Figure 2). Compound 8 effectively inhibited the growth of NCI-H460 cells in a concentrated and time-dependent manner, with cell cycle arrest in G2/M phase. Additionally, 8 inhibited the growth of an NCI-H460 xenograft tumour in vivo [20].
Yu et al. synthesized eighteen indoline-2,3-diones and evaluated their antiproliferative activities on mantle cell lymphoma (MCL) cells. The synthesized compounds displayed good antiproliferative activity, with compound 9 being most potent, with an IC 50 of 0.4-0.7 µM on MCL cells ( Figure 2). The compound induced cell apoptosis and cell cycle arrest in the G2/M phase in a dose-dependent manner [21].
George et al. disclosed the synthesis of isatin-benzenesulfonamides for assessing their inhibitory activities on human CA isoforms viz. hCA I, hCA II, hCA IX, and hCA XII. Most of the synthesized scaffolds displayed substantial inhibitory activities on tumour-associated isoforms IX and XII. Compound 10 revealed the highest activity among the synthesized scaffolds, with a K i of 68.3 and 21.5 nM on hCA IX and hCA XII, respectively ( Figure 2).
Docking studies was carried out in order to understand the interactions between 10 and the four isoforms studied. As expected, 10 exhibited stronger interactions with isoform XII, while few interactions were observed with isoform IX. Further, the poor interactions of 10 with isoforms I and II confirmed its poor activity profiles [22].
Panga et al. synthesized a series of isatin-pomalidomide hybrids and assayed for in vitro antiproliferative activities against U266B1 and RPMI 8226 on multiple myeloma cells using an MTT assay with pomalidomide as the reference drug. Most of the synthesized hybrids exhibited moderate to good cytotoxicity on the tested cell lines. Among these, compound 11 exhibited an IC 50 of 2.5 µM on U266B1 and 6.7 µM on RPMI8226 cells, which are values better than that of the physical mixture of 5,7-di-bromoisatin and pomalidomide (PM + DBIS) ( Figure 2) [23].
Eldehna et al. aimed at developing a new set of small molecules featuring the isatin framework tethered with a thiazolo [3,2-a]benzimidazole (TBI) motif via a cleavable hydrazide linker as potential anticancer CDK2 inhibitors. Within the CDK2 binding region, the vast tricyclic TBI motif is expected to form plenty of hydrophobic contacts. Most of the synthesized hybrids substantially hindered the development of the two cell lines under investigation. Notably, 12 was the most potent moiety among these, with IC 50 = 2.60 ± 1.47 µM against MDA-MB-231 and IC 50 = 3.01 ± 0.22 µM against the MCF-7 cell line ( Figure 2). The TBI ring's capacity to well accommodate and form extensive hydrophobic interactions within a hydrophobic pocket in the CDK2 binding site was shown by docking simulations, as predicted. In addition, docking modelling emphasized the importance of hydrazide linkage and isatin unsubstituted (NH) functionality in H-bonding interactions [24].

1H-1,2,3-Triazole-Tethered Isatin Hybrids
Yu et al. synthesized a series of isatin-triazole hybrids and evaluated their antiproliferative activities against a panel of cancer cell lines. The synthesized hybrids exhibited moderate to good antiproliferative activities on tested cells, with selectivity against MGC-803. The most promising scaffold of the synthesized library, 13, displayed an IC 50 of 9.78 µM (MGC-803) and was less cytotoxic on normal cell lines viz. HL-7702 and GES-1, with IC 50 s of 40.27 and 35.97 µM, respectively ( Figure 3). The hybrid 13 instigated morphological changes on the tested cell lines along with the induction of cell cycle arrest (G2/M phase), the generation of cellular ROS, and the inhibition of the migration of cells in a concentration-dependent manner [25].
Nagarsenkar et al. reported the synthesis and antiproliferative evaluation of a series of triazole-tethered 3-benzylidene-isatin hybrids. The synthesized hybrids were assessed for their cytotoxic potential on DU145 (prostate), PC-3 (prostate), MDA-MB-231 (breast), BT549 (breast), A549 (lung), and HeLa (cervical) human cancer cell lines using MTT assay. Among the synthesized series, compound 14 proved to be most potent, with an IC 50 of 3.7 µM against DU145 cells ( Figure 3). Its safety profile on normal prostate cells (RWPE-1) confirmed it to be non-cytotoxic. Compound 14 induced apoptosis on DU145 cells, as confirmed by AO/EB staining and Annexin V binding assay and DAPI nuclear staining with a cell cycle arrest at G2/M phase. The compound additionally resulted in the collapse of mitochondrial membrane potential and increased intracellular ROS levels in DU145 cells [26].
Kumar et al. reported a series of triazole-linked ospemifene-isatins and ospemifenespiroisatins along with their antiproliferative evaluation on MCF-7 and MDA-MB-231 cells. SAR studies disclosed that the hybrids with bromo-substituent at the C-5 and C-7 positions of the isatin core, along with an ethyl/propyl as a spacer, were promising on MCF-7 cells. The compound 15 proved to be the most promising, with an IC 50 value of 1.56 µM against MCF-7 ( Figure 3) [27].
Aneja et al. reported the synthesis of isatin-triazole hydrazones as potent inhibitors of microtubule affinity-regulating kinase 4 (MARK4) on the basis of a toxicity analysis on MCF-7, HepG2, and MDA-MB-435s cells. Among the synthesized hydrazones, the compound 16 exhibited good binding affinity along with easy transportation, as confirmed by a Human Serum Albumin (HSA) binding assay ( Figure 3). The compound induced apoptosis on MCF-7, MDA-MB-435s, and HepG2 cells with IC 50 s of 6.22, 9.94, and 8.14 µM, respectively. Additionally, the cell-based assay validated the cell promotion, apoptosis, and enhanced ROS generation by 16 [28]. Yu et al. synthesized a series of 1H-1,2,3-triazole-tethered isatin-steroidal hybrids and evaluated their antiproliferative activities on a panel of different cancer cell lines using an MTT assay. The preliminary evaluation results indicated that the hybrids with a terminal isatin motif exhibited substantial growth inhibition of SH-SY5Y cells. Among the synthesized series, compound 17 proved to be the most active, and exhibited an IC 50 of 4.06 µM against SH-SY5Y cells, comparable to that of 5-FU ( Figure 3). Additionally, 17 arrested the cell cycle growth at the G2/M phase along with induction of apoptosis and subsequent lowering of mitochondrial membrane potential. The observed activity of 17 was further substantiated using Docking studies [29].
Singh et al. synthesized 1H-1,2,3-triazole-tethered isatin conjugates and evaluated for cytotoxicity on four human cancer cell lines. The results showed that 18 was twice as effective as 5-fluorouracil on the THP-1 cell line and had an IC 50 of <1 µM against all cell lines except Caco-2 ( Figure 3). Activity profiles showed a reliance on the substituents on isatin rings, with preference for hydrogen, while an electron-withdrawing fluoro and nitro substituents on either ring diminished the antiproliferative activity [30].
Sharma et al. synthesized 3,5-diaryl-N-acetyl-pyrazolines(DNAP)-isatin hybrids via Cu-promoted click reaction. The synthesized hybrids were assayed for their antiproliferative activity on four human cancer cell lines using a sulforhodamine B assay. The most potent hybrid 19 displayed an IC 50 value of 1.3 µM (HeLa cells) with ten-fold selectivity towards HeLa cell lines ( Figure 3). SAR studies revealed that both the electronic effects and the length of the linker remarkably affected the cytotoxic activity of the synthesized hybrids. The inclusion of a propyl chain as a spacer and methoxy substituents on the phenyl ring of N-acetyl pyrazoline were considered optimum for the good antiproliferative activities of the synthesized hybrids [31].
Further, a series of 1H-1,2,3-triazole-tethered curcumin-isatin hybrids were synthesized by Sharma et al. and assayed for their cytotoxicity against THP-1, COLO-205, HCT-116, A549, HeLa, CAKI-I, PC-3, and MiaPaca-2 human cancer cells. The most potent compounds were also assayed for tubulin inhibition. Compound 20 was found to substantially inhibit the tubulin polymerization (IC 50 = 2.87, 4.15, 1.2, and 5.67 µM against THP-1, COLO-205, HCT-116, and PC-3, respectively) ( Figure 3). Additionally, 20 led to the disruption of microtubules, as affirmed by immunofluorescence studies. The docking study confirmed that 20 fits well at the interface of β1 and α2 subunits of tubulin and stabilized by H-bonds, polar, and van der Walls interactions [32].  (Figure 4). The scaffold induced cell cycle arrest in the G0/G1 phase along with a reduction in Cdk4 expression level, as indicated by flow cytometric and Western blot analysis, respectively. Further studies such as cell cycle analysis, mitochondrial potential assay, annexin V-FITC assay, and Western blot analysis of 21 confirmed apoptosis as its possible mechanism of action [33]. Nunes et al. disclosed the synthesis and antiproliferative activities of a series of spiropyrazoline oxindoles on the HCT-116 p53 human colon cancer cell line. Out of the synthesized library, eight derivatives exhibited good activities with IC 50 s less than 15 µM. Among these potent compounds, two derivatives viz. 24a (IC 50 13.1 µM) and 24b (IC 50 10.9 µM) were selected to delineate the mechanism of action ( Figure 4). Both scaffolds induced apoptosis with cell arrest at the G0/G1 phase along with up-regulation of p53 steady state levels. Further, the combination of 24a with 5-fluorouracil (5-FU) fashioned a synergistic inhibitory effect on the proliferation of colon cancer cells [36].

Spiro Compounds Based on Isatins
Senwar et al. developed a concise method for the synthesis of spirooxindole-derived morpholine-fused-1,2,3-triazoles. The methodology included the regiospecific ring-opening of isatin-spiro-epoxides by azide as a nucleophile, followed by successive O-propargylation and intramolecular 1,3-dipolar cycloaddition. The synthesized compounds were assessed for their antiproliferative activities on various human cancer cell lines. Among these, compound 25 (IC 50 = 1.87 µM) showed good growth inhibition on A549 cells ( Figure 4). Flow cytometry investigation demonstrated that compound 25 arrested the cells at the G2/M period of the cell cycle. The apoptosis-inducing effect of these compounds was examined with Hoechst nuclear staining and AO/EB staining. Additionally, compound 25 prompted apoptosis in A549 cells through the breakdown of mitochondrial membrane potential and the elevation of intracellular responsive oxygen species levels [37].

Urea/Thiourea-Based Isatin-Derivatives
Eldehna et al. disclosed the synthesis of a series of amide/urea-based indoline-2-ones via conjugating type IIA (sorafenib) and IIB (sunitinib and nintedanib) as PTK inhibitors. The synthesized hybrids were evaluated for multi-kinase inhibitory activity on VEGFR2, PDGFR-b and FGFR-1 and for antiproliferative activity on HepG2, MCF-7, A549, and A498 cancer cells. The hybrid 26a proved to be the most potent multi-kinase inhibitor among the synthesized ureido series, with IC 50 s of 0.18, 0.23, and 0.10 µM on VEGFR-2, FGFR-1, and PDGFR-b, respectively ( Figure 5). For antiproliferative activities, 26a proved to be most potent on HepG2 cells (IC 50 = 2.67 ± 0.14 µM), while 26b was most potent on A498 cells (IC 50 = 0.78 ± 0.02 µM). Further, the synthesized compounds proved to be non-cytotoxic over human normal melanocyte (HFB4). The synthesized compounds also exhibited promising pharmacokinetic and drug-likeness properties, as confirmed by an ADME prediction study [38].    Figure 6). Additionally, EGFR and tubulin inhibition assays were carried out for the promising hybrids, and outstanding results were observed compared to the reference drugs. Molecular docking studies were further performed in order to delineate the mechanism of action of the promising compounds in the EGFR binding site [43].

Isatin-Based Schiff's Bases and Oximes
Nam et al. designed and synthesized two series of isatin-3'-oxime and isatin-3'methoxime-based hydroxamic acids as analogues of SAHA approved by the FDA for treating cutaneous T-cell lymphoma. Among the synthesized series, the compounds that either had or lacked a halogen (F or Cl) substituent at the 5th or 7th position on the isatin core exhibited hindrance against histone-H3 and histone-H4 deacetylation. In addition, compound 32 showed potent cytotoxicity against five cancer cell lines, with IC 50 s of 0.64, 0.79, 0.98, 1.10, and 0.89 µM against SW620, MCF-7, PC-3, AsPC-1, and NCIH460, respectively ( Figure 6). A docking study performed with the selected compound 32 revealed that these compounds bind to HDAC8 with higher affinities than SAHA. Furthermore, the carbonyl oxygen of the isatin moiety in compound 32 was assessed to associate with the backbone amide through hydrogen bonds [44].
Dweedar et al. synthesized a series of indoline-2,3-dione hydrazones via treating indoline-2,3-diones with hydrazine to yield 3-hydrazonoindolin-2-ones, which were reacted with the appropriate aldehydes. From the biological assay results, it was found that some compounds showed notable activity, with IC 50 values in the range of 6.25-25.8 µM, among which 33 gave the most extreme activity, with an IC 50 of 6.25 µM ( Figure 6). The docking analysis of the GSK-3b receptor for both indirubin-3'-oxime and our suggested compounds, taking 33 as representative example, showed that they interacted through hydrogen bonds [45]. Liang

Metal Complexes of Isatin Containing Ligands
Ali et al. synthesized and characterized six tridentate Cu(II) complexes of thiosemicarbazone with isatin (CuL1-CuL6) and evaluated their antiproliferative activities. Gel electrophoresis indicated that these complexes could prompt the cleavage of plasmid DNA. It was revealed that the DNA cleavage by these complexes is concentration-dependent. CuL1 and CuL2 exhibited oxidative behaviour towards DNA, while CuL3-CuL6 complexes induced DNA cleavage via oxidative and hydrolytic pathways. In vitro antiproliferative activity on human colon cancer cell line (HCT-116) showed concentration-dependence with low IC 50 s (0.08-8.6 µM). The complex 35 proved to be most potent, with an IC 50 of 0.08 µM against human colorectal cancer (HCT116) cells, which is more pronounced than the reference drug, 5-fluorouracil (IC 50  Balachandran et al. synthesized N-substituted isatin thiosemicarbazone-based complexes along with an assessment of their antiproliferative profiles on a panel of four human cancer cells, HepG-2 (liver), MOLM-14 (acute monocytic leukemia), U937 (histiocytic lymphoma), and IM-9 (myeloma). Complex 37 displayed promising activity against IM-9 cells, with an IC 50 of 7.92 ± 1.03 µM; however, it had no effect on HepG-2 cells (Figure 7). Its activity on MOLM-14 cells (IC 50 = 45.92 µM) was significant, while good activity was observed on U937 cells (IC 50 = 56.35 µM). Mechanistically, 37 accelerated apoptotic cell death in the IM-9 cells, causing cell cycle arrest at the G1 phase. It also downregulated Bcl-2 (b-cell lymphoma-2), upregulated Bax (bcl-2 associated X protein), released cytochrome c, and triggered caspases-3 in IM-9 cells [49]. Haribabu et al. synthesized and assayed mono-and bi-nuclear copper(II) complexes containing N-substituted isatin-thiosemicarbazones for their antiproliferative activity. Complex 39 showed promising cytotoxic activity against the Jurkat (leukaemia) and HeLa S3 (cervical) cell lines, with IC 50 s of 5.83 and 3.53 µM, respectively. The complex 39 was nine-fold more active than cisplatin against HeLa S3 and Jurkat cell lines, while it proved to be non-cytotoxic to the IMR90 cell line, as indicated by its high IC 50 values (>100 µM) (Figure 7) [51].
The group further extended the approach and synthesized a series of nickel(II) complexes [Ni(L) 2 ] of N-substituted isatin thiosemicarbazones. In vitro cytotoxicity evaluation revealed substantial activities on human breast (MCF7) and lung (A549) cancer cells, with the best outcomes being recorded for complexes 40a and 40b, exhibiting IC 50 < 0.1 µM (Figure 7). Complexes 40a and 40b showed a stronger DNA binding affinity than the other complexes, which could be attributed to the presence of benzyl and allyl groups [52].
Youssef et al. synthesized and characterized the ligand (1Z,3Z)-N'1,N'3-bis((E)-2hydroxy-3H-indol-3-ylidene)malonohydrazonic acid and its Ni(II) complexes. These complexes were tested for their in vitro cytotoxicity against hepatocellular carcinoma human tumour cells (HePG-2). It was found that the Ni(II) complex (42)   Teng et al. disclosed the synthesis and antiproliferative evaluation of a series of di-or trisubstituted isatins on the human tumour cell line Jurkat using MTT assay. The presence of benzyl-substituent at N-1, trans-2-(methoxycarbonyl)ethen-1-yl at C-5, and an intact carbonyl functionality at the C-3 position of the isatin ring greatly enhanced the cytotoxic activity. Among the synthesized scaffolds, compound 47 proved to be most cytotoxic, with an IC 50 of 0.03 µM, >330-fold higher than the parent molecule ( Figure 8). Compound 47 exhibited antiproliferative activities via inducing mitochondrial apoptosis, as confirmed by cell morphology changes, a annexin-V/PI-staining study, and the activation of caspase-3 [59]. Lozinskaya and co-workers have reported the synthesis of 3-arylidene-2-oxindoles as Glycogen synthase kinase 3β (GSK-3β) inhibitors. The most promising compound among the synthesized series, 50, was found to inhibit GSK-3β, with an IC 50 of 4.19 nM (Figure 8). Compound 50 was found to effectively occupy the hydrophobic cleft of the allosteric binding site. The pyridine moiety is oriented toward the solvent-accessible region of the pocket, exposing it to polar substituents that influence the compound's solubility and pharmacokinetic properties [62].
Zhang et al. synthesized a series of N-1 benzyl substituted 5-arylisatins and evaluated their antiproliferative activities on human leukemia K562 and liver cancer HepG2 cells using an MTT assay. The presence of N-benzyl and C-5 phenyl substituents along with an intact carbonyl functionality at the C-3 position proved to be important for good antiproliferative activities. Compound 51 proved to be the most potent among the series, exhibiting the highest antiproliferative activity against K562 cells, with an IC 50  Han et al. reported the synthesis of di-or tri-substituted isatins along with their in vitro antiproliferative evaluation on three human tumour cell lines, K562, HepG2, and HT-29, using an MTT test. SAR studies revealed that the presence of 1-benzyl and 5-[trans-2-(methoxycarbonyl)ethen-1-yl], along with an intact carbonyl at the C-3 positions, were prerequisites for good cytotoxic activity. In acrylate-containing isatin analogues, the size and orientation for N-substitution is important for their anticancer activity. The compounds 57a and 57b were identified as the two promising compounds among the synthesized series on human leukemia K562 cells, with an IC 50 of 3 and 6 nM, respectively (Figure 8) [69].

Antimycobacterial/Tubercular Activities of Isatin-Based Scaffolds
After the outbreak of COVID-19, TB is expected to be the second leading cause of death from a single infectious agent. In 2020, the WHO African and South-East Asia regions accounted for over 84% of HIV-negative TB deaths and 85% of all TB deaths among HIVnegative and HIV-positive adults. [70]. India accounted for 38% of global TB deaths among HIV-negative people and 34% of the total number of TB deaths among HIV-negative and HIV-positive people combined [71]. The rapid emergence of multi drug resistant (MDR) tuberculosis, combined with Mycobacterium tuberculosis (Mtb)'s prominent ability to enter a dormant state, known as latent TB infection, poses significant challenges for tuberculosis control [72,73]. As a result, the identification of novel targets and the development of new agents capable of combating drug-resistant tuberculosis and latent tuberculosis infection remain urgent priorities [74].  (Figure 9). SAR studies implied that the inclusion of -Cl and -F substituents at the C-5 position of the isatin ring improved the antimycobacterial activity, while the hybrids having a semicarbazone moiety at the C-5 position proved to be more active than the corresponding thiosemicarbazone analogs. The synthesized hybrids (CC 50 : 2-8 µg/mL) were, however, more cytotoxic than the parent MXFX (CC 50 : 128 µg/mL) drug [76].

Ciprofloxacin-Isatin and Moxifloxacin-Isatin Hybrids
Yan et al. synthesized a series of 1H-1,2,3-triazole-tethered isatin-moxifloxacin hybrids and assayed them for their in vitro antimycobacterial activities against drug sensitive and multidrug-resistant Mtb, while the cytotoxicity was assessed on VERO cells. Most of the synthesized compounds exhibited excellent activities, with MICs in the range from 0.05 to 2.0 µg/mL. The most potent hybrid, 60 (MIC of 0.05 and 0.06 µg/mL on Mtb H 37 Rv and MDR-TB), was two to eight-fold more potent than the reference drugs, moxifloxacin, and rifampicin on H 37 Rv, while 2 to >2048-fold more active than moxifloxacin, rifampicin, and isoniazid on MDR-TB (Figure 9). The synthesized hybrids were, however, more cytotoxic than the moxifloxacin against the VERO cell line [77]. A series of amide-tethered ciprofloxacin-isatin hybrids were synthesized by Chen and co-workers, who evaluated their in vitro antimycobacterial activity. The synthesized hybrids displayed excellent in vitro activity on both H 37 Rv and MDR strains, with MIC values ranging from 0.12 to 32 µg/mL. Hybrid 61 proved to be most active among the synthesized series, with an MIC of 0.5 µg/mL (H 37 Rv) and 0.12 µg/mL (MDR-TB), comparable to that of isoniazid (0.12 µg/mL) and rifampicin (0.25 µg/mL) (Figure 9) [78]. Mhiri et al. synthesized a series of dispirooxindolopyrrolidines and dispirooxindolopyrrolothiazoles via a three-component 1,3-dipolar cycloaddition of (Z)-3arylidenebenzofuran-2-ones, substituted isatins, and α-aminoacids. X-ray diffraction analysis was used to confirm the stereochemistry of the synthesized spiro adducts. The synthesized scaffolds were assessed in vitro on the Mtb H 37 Rv strain along with cytotoxicity assessment on RAW 264.7 cells. Among these, the potent compounds displayed antiTB activities, with MIC values ranging from 1.56 to 6.25 µg/mL. Specifically, dispirooxin-dolopyrrolothiazoles, 64a and 64b, were proved to be the most promising compounds, with an MIC of 1.56 µg/mL ( Figure 10) [81].

Schiff's Bases and Oximes of Functionalized Isatins
Abdu-Allah et al. synthesized a series of 1H-1,2,3-triazolylsalicylhydrazones of isatins using para-amino salicylic acid (PAS) as the core scaffold. The synthesized scaffolds were assayed for their antimycobacterial activities on Mtb H 37 Rv and exhibited good to high activity, with an MIC of 0.39-1.5 µg/mL. The promising compound among the series, 67, proved to be ten-fold more potent than PAS and equipotent to rifampicin (MIC = 0.39 µg/mL), with a selectivity index of >128 ( Figure 11). Additionally, the synthesized compound also proved to be active against persistent forms of mycobacteria comparable to standard drugs in the nutrient starvation model. A study was also conducted in order to explain the observed activity and to serve as a tool for further development [84].  37 Rv and MDR-TB, respectively, being two-fold more active than rifampicin and isoniazid on MDR-TB ( Figure 11). The promising compound 69 was further assessed for its metabolic stability and in vivo pharmacokinetic profiles [86]. Eldehna et al. synthesized a series of nicotinic acid hydrazides and assayed for their antimycobacterial activity. The activity results revealed that the synthesized isatin-hydrazides were more active than the parent hydrazides. SAR studies revealed the lipophilicity as a crucial element for accounting for antimycobacterial activity, with a preference of halogen over the hydrogen as substituent. Compound 76, with a bromo substituent at the C-5 position of the isatin core, displayed an MIC of 6.25 µg/mL ( Figure 11) [93].
Karunanidhi et al. synthesized and characterized novel isatin hydrazones and their thiomorpholine-tethered analogues. Under level-I testing, all of the synthesized compounds were assayed for antimycobacterial activity against the H 37 Rv strain of Mtb. The most active compounds, 77a and 77b, exhibited IC 50 s of 1.9 and 3.9 µM, respectively ( Figure 11). These compounds were then investigated against five drug-resistant strains of Mtb, comprising isoniazid-resistant strains (INH-R1 and INH-R2), rifampicin-resistant strains (RIF-R1 and RIF-R2), and a fluoroquinolone-resistant strain (FQ-R1). Interestingly, 77a and 77b were shown to be the most efficacious compounds against the RIF-R1 MTB strain, with IC 50 values of 3.6 µM and 1.9 µM, respectively, followed by the INH-R1 MTB strain, with IC 50 values of 3.5 µM and 3.4 µM, respectively. With an IC 50 of 5.9 µM and 4.9 µM, respectively, the lead compounds 77a and 77b demonstrated effective suppression against the FQ-R1 MTB strain, indicating broad-spectrum efficacy. The electron-withdrawing (NO 2 ) substituent on the electron-rich 5-membered heterocyclic system contributed significantly towards the achievement of enhanced antimycobacterial potency in both 77a and 77b [94].
Elsayed et al. reported the design and synthesis of isatin-nicotinohydrazide hybrids as promising antimycobacterial and antibacterial agents. A drug-susceptible Mtb strain (ATCC 27294) was used to evaluate the target hybrids' antitubercular activity, and hybrid 78 was shown to be as effective as INH, with an MIC of 0.24 µM (Figure 11). In terms of the structure-activity relationship, it was discovered that the N-substitution and the presence of a halogen at the C-5 position of the oxindole ring impacted the antimycobacterial activity of halogen-bearing compounds. Compounds with a Br substituent have higher activity than Cl-substituted and -unsubstituted compounds [95].
Johansen et al. designed and synthesized, isatin-mono/bis-isoniazid hybrids with potential antimycobacterial activity. Compound 79 displayed high activity against drugsusceptible Mtb, with MICs ranging from 0.195 to 0.39 µg/mL ( Figure 11). Moreover, these compounds were found to be well tolerated on Vero kidney cells at high concentrations (200 µg/mL), resulting in high selectivity indices. These compounds significantly suppressed mycolic acid biosynthesis and inhibited FAS-II components without affecting FAS-I. Mechanistically, these compounds shared a similar mode of action as INH, i.e., they required KatG in order to exhibit biological effects. However, the synthesized compounds were surprisingly more bactericidal and displayed a delayed resistance development compared to the standard drug INH when tested on the bacterial loads [96].

Ferrocene-Tethered Isatin Hybrids
Kumar et al. disclosed the synthesis and antimycobacterial evaluation of isatinferrocene and isatin-ferrocenyl chalcone hybrids obtained via Cu-promoted click reaction. Antimycobacterial SAR revealed the reliance of activity on the C-5 substituent of the isatin ring and the length of the alkyl chain, with a preference for halogen substituents (F, Cl) and a propyl chain length. The inclusion of a chalcone core among the isatin-ferrocene hybrids, 80a-h, enhanced the antimycobacterial activities irrespective of the nature of the substituent at the C-5 situation (Figure 12) [97]. The authors further extended the work and synthesized a series of 1H-1,2,3-triazolelinked spiroisatin-ferrocene and isatin-ferrocenes. The synthesized hybrids 81a-x and 82a-p were examined for their antimycobacterial properties, showing improvement in activity profiles with the inclusion of a ferrocene core in contrast to their organic precursors ( Figure 12). The observed SAR confirmed the independence of the length of the alkyl chain, nature of the substituent at C-5, and the presence of either a keto-carbonyl or ketal unit at the C-3 position of the isatin ring on antimycobacterial activities [98].

Miscellaneous Isatin Derivatives with Antimycobacterial Potential
Shaikh et al. synthesized a series of triazole-based isatins using click reaction and evaluated their antimycobacterial activities against Mtb H 37 Rv. Compound 83 exhibited maximum antimycobacterial activity, with IC 90s of 7.56-8.09 µg/mL against H 37 Rv ( Figure 13). Cytotoxicity studies proved that the promising scaffolds have low cytotoxicity on human cancer cell lines A549 and PANC-1. Molecular docking studies have shown the high affinity of the synthesized compounds towards cytochrome P450 lanosterol 14αdemethylase via van der Waals interactions with amino acid residues. Additionally, ADME parameters of the synthesized compounds confirmed their good drug-like properties [99].

Antiplasmodial/Malarial Activities of Isatin-Based Scaffolds
P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi are members of the Plasmodium family of protozoan parasites that cause malaria. P. falciparum and P. vivax are the most virulent and are primarily responsible for the disease's morbidity and mortality [102]. An estimated 241 million malaria cases were reported in 85 malaria-endemic countries in 2020, an increase from 227 million in 2019, with the WHO African Region accounting for the majority of the increase. The WHO African Region accounted for nearly 95 percent of all cases in 2020, with an estimated 228 million cases. India accounted for 83 percent of the cases in the Southeast Asian region. Malaria deaths increased by 11% in 2020 compared to 2019 to an estimated 627,000; of the additional 69,000 deaths, an estimated 47,000 (68%) were caused by service disruptions during the COVID-19 pandemic [103]. The continuous evolving drug resistance to both the conventional (quinolines) as well as contemporary (Artemisinin combination therapy) drugs has exacerbated the need for identifying new entities with promising antiplasmodial activities. A number of reports have shown the potential of isatin-based compounds as promising antiplasmodials [104,105].

Isatin-7-Chloroquinoline Hybrids
Nisha et al. synthesized a series of β-amino alcohol-linked 4-aminoquinoline-isatin hybrids and evaluated their antiplasmodial activities. The hybrids 86a and 86b displayed comparable activities to the antimalarial drug, Chloroquine (CQ), with IC 50 s of 11.8 and 13.5 nM, respectively on a CQ-resistant (W2) strain of P. falciparum and were devoid of any cytotoxicity on normal cells ( Figure 14) [106]. Raj et al. synthesized a series of 1H-1,2,3-triazole-tethered 7-chloroquinoline-isatin hybrids and assessed their activities on a CQ-resistant W2 strain of P. falciparum. SAR revealed the dependence of activity on the nature of the substituent on the isatin core as well as the length of alkyl chain introduced as a linker between the two pharmacophores. The C-5 unsubstituted hybrid proved to be the least active among the synthesized series, while the inclusion of chloro substituent improved the antiplasmodial activities. Compound 87, with an optimum combination of propyl chain as spacer and chloro substituent at the C-5 position of the isatin core proved to be the most potent among the series, exhibiting an IC 50 of 1.21 µM (Figure 14) [107].
Further, the synthesis and antiplasmodial evaluation of 1H-1,2,3-triazole-tethered isatin-7-chloroquinoline and 3-hydroxy-indole-7-chloroquinoline hybrids was reported by the same authors. The antiplasmodial activities displayed the dependence of activity on the length of the alkyl chain but independence on the nature of the substituent present at the C-5 position of the isatin or indole ring. The most potent hybrid of the synthesized series, 88, exhibited an IC 50 of 69 nM, comparable to that of CQ on the CQ-resistant strain of P. falciparum (Figure 14 Kumar et al. disclosed the synthesis and antiplasmodial and cytotoxic evaluation of 7-chloroquinoline-based hybrids with isatins/indoles/nitroimidazoles obtained via Cupromoted click reaction. Among the synthesized series, the hybrids with a shorter alkyl chain length (ethyl/butyl) as spacer displayed better antiplasmodial activities than the ones with longer chain lengths (hexyl/octyl). The most promising hybrid among the synthesized series, 90, exhibited an IC 50 of 0.33 µM on the tested strain ( Figure 14) [110].

Isatin-Based Schiff's Bases
Akhaja et al. designed, synthesized, and evaluated in vitro tetrahydropyrimidineisatin hybrids on a CQ-sensitive 3D7 strain of P. falciparum. SAR studies revealed that the introduction of a nitro (91a, MIC = 0.177 µg/mL) or chloro (91b, MIC = 0.035 µg/mL) substituent improved the antiplasmodial activities, with the substitution pattern profiling as NO 2 > F > Br > H for favourable activity (Figure 15) [111].  Figure 15). A SAR study showed that the scaffolds that had acetylated or diacetonide galactose units exhibited better activity on both strains than the ones with acetylated glucose or acetonide xylose units [112].
Synthesis of N-alkylated 3-glycoconjugated-oxopropylidene oxindoles along with their in vitro antiplasmodial evaluation on both CQ-sensitive and CQ-resistant strains of P. falciparum were reported by Thakur

Miscellaneous Antiplasmodial Isatin Derivatives
A series of thiolactone-isatin hybrids was synthesized by Hans et al. and resulted in the identification of tetracyclic byproducts with superior antiplasmodial potential. The SAR studies ascertained the most potent compound of the series, 94, with an IC 50 of 6.92 µM against the CQ-resistant (W2) strain of P. falciparum (Figure 16) [114].
Kumar et al. synthesized a series of 1H-1,2,3-triazole-tethered isatin-ferrocene hybrids using Cu-promoted click reaction and assayed for their antiplasmodial activities on both 3D7 and W2 strains of P. falciparum. Hybrid 95, along with an optimum combination of halogen substituents at the C-5 position of the isatin ring and a propyl chain as spacer, were the most potent and non-cytotoxic among the series, with IC 50 values of 3.76 and 5.97 µM against 3D7 and W2 strains, respectively ( Figure 16) [115]. Ladani et al. described an efficient single-pot, three-component reaction of substituted isatin, enaminones, and active methylene, yielding diversely functionalised spiro-indolinone that incorporated 1,2,4-triazolo [1,5-a] quinolones, using L-proline as a catalyst. The synthesized compounds were evaluated for their in vitro antibacterial, antiTB, and antiplasmodial activities, respectively. The synthesized scaffolds exhibited IC 50 s in the range of 0.122 to 0.454 µM against P. falciparum. The synthesized scaffold, 96, exhibited an IC 50 of 0.122 µM, comparable to that of CQ ( Figure 16) [116].

Antimicrobial Activities of Isatin-Based Scaffolds
Antibiotics are unquestionably a blessing to human civilization, having saved millions of lives by combating infections or microbes. Various antibiotics have been used for therapeutic purposes over the years. In the mid-twentieth century, antibiotics were regarded as the "wonder drug". There was an idealistic belief at the time that communicable disease was on its way out. Antibiotics were thought to be a magic bullet that selectively targeted microbes responsible for disease eradication. Antibiotic resistance progresses rapidly and is therefore a major source of concern. A growing number of infections, such as pneumonia and gonorrhoea, are becoming more difficult and, in some cases, impossible to treat, while antibiotics have become less effective [117].  Ma et al. synthesized and evaluated a series of propylene and butylene-tethered di-isatin heteronuclear hybrids for assessing their antibacterial activities. Most of the synthesized hybrids were active on both Gram-positive and Gram-negative strains, while few of them displayed substantial activities on drug-resistant strains. In particular, compound 104 (MIC = 32-512 µg/mL) proved to be more active than vancomycin on Gram-negative pathogens, while its inhibitory activity was higher than mono-isatin E. coli DNA gyrase ( Figure 17) [125].
Kandile et al. reported the synthesis of isoxazolyl-isatin-based benzenesulphonamides and explored their in vitro antimicrobial and antifungal activities on a few pathogenic bacterial strains. In vitro evaluation studies revealed compounds 106a and 106b to be the most promising of the synthesized series ( Figure 17). Compound 106b (MIC = 5 µg/mL), with the inclusion of chlorine at the C-5 position of the oxindole core, presented more potent antifungal characteristics than 106a (MIC = 15 µg/mL). Additionally, 106b demonstrated an MIC = 5 µg/mL against S. dysenterie and B. cereus, comparable to a standard drug such as sulphamethoxazole [127].
Farag et al. synthesized a series of 5-(morpholinosulfonyl)isatins as well as their corresponding hybrids with amino-thiazoles for evaluating their antibacterial activities on a panel of Gram-positive and Gram-negative strains. Among the synthesized scaffolds, 5-(morpholinosulfonyl)isatin (107a) showed significant activity against the screened microbes, with MIC values ranging from 0.007 to 0.49 µg/mL ( Figure 17). Compound 107a proved to be four-fold more active than amphotricin B in inhibiting the growth of A. fumigates (MIC = 0.24 µg/mL) and displayed two-fold more potency than amphotricin B on A. clavatus (MIC = 0.98 µg/mL). Among the thiazole-linked hybrids, compounds 107b (MIC = 0.03-0.12 µg/mL) and 107c (MIC = 0.06-0.49 µg/mL) proved to be the most effective on the tested bacterial strains [128].
Zhang et al. reported a series of antibacterial isatin-β-thiosemicarbazones with excellent activity against a clinically isolated MRSA (methicillin-resistant Staphylococcus aureus) strain, while few of them exhibited potent inhibition against the VRE (vancomycin-resistant enterococcus) strain. Compound 108a proved to be the most promising of the synthesized series, exhibiting MICs of 0.78, 1.56, and ≤0.78 mg/L on MRSA, S. aureus, and B. subtilis, respectively ( Figure 17). From an SAR perspective, the presence of a halogen atom at the C-7 position of the isatin ring (R 1 ) H on the phenyl ring (R 2 ) improved the activities of the synthesized scaffolds. On this rationale, the library was extended, and a series of 41 isatin-β-thiosemicarbazones were synthesized. Compound 108b had an MIC value of 0.39 mg/L against three MRSA strains [129].
The fabrication of a ferrocene-affixed isatin-2,4-thiazolidinedione molecular hybrid connected through a triazole moiety was described by Yagnam et al. A simple copper catalysed alkyne-azide 1,3-dipolar cycloaddition synthesized an isatin-coupled 2,4thiazolidinedione moiety via a triazole unit. All of the novel entities were screened for antimicrobial efficacy against a variety of ram-positive and Gram-negative bacteria. The  compounds 109a, 109b, 109c, and 109d exhibited MIC values of 4 µg/mL against bacterial strains and 32 µg/mL against fungal strains ( Figure 17) [130].
Tehrani et al. synthesized a series of N-benzylated isatin-Schiff bases and evaluated their antibacterial activities on a series of Gram-positive and Gram-negative bacterial strains using a microtiter plate method. Among the synthesized series, 110a and 110b were the most potent compounds on Pseudomonas aeruginosa, with the MIC = 6.25 µg/mL ( Figure 17). The SAR analysis revealed that the inclusion of (thio)urea-moiety led to the identification of active compounds with a broader spectrum of antibacterial activity. Additionally, the compounds with high lipophilicity did not exhibit any measurable antibacterial activity, suggestive of the fact that optimal lipophilicity could be significant for the activity of the synthesized compounds [131].

Miscellaneous Isatin Scaffolds with Antimicrobial Activities
Salem et al. synthesized a series of 2-oxospiro[indoline-3,4'-pyran] derivatives via the single pot reaction of substituted indole-2,3-diones, appropriate nitriles, and β-dicarbonyl compounds, and evaluated for their in vitro antibacterial, antifungal, and immunomodulatory activity. Most of the synthesized compounds displayed high activity in killing pathogens, with a good MBC value against norfloxacin, and were also investigated on an extended panel of MDR bacteria. The most potent compound of the series, 113, displayed an increase in the intracellular-killing activity of neutrophils ( Figure 19). It showed that MIC = 0.78 µg/mL against S. aureus ATCC 33,591 and MIC = 1.95 µg/mL against P. aeruginosa ATCC BAA-2111 [134]. Bhagat et al. synthesized and evaluated a series of indolindione-coumarin hybrids for their antimicrobial activities on Gram-negative (E. coli and S. enterica), Gram-positive (S. aureus and M. smegmatis), and fungal strains (C. albicans, A. mali, Penicillium sp., and F. oxysporum). The compounds 114a and 114b proved to be most promising, with MIC 30 and 312 µg/mL for Penicillium sp. and S. aureus, respectively ( Figure 19). SAR studies revealed that the electronic nature of substituents on the isatin core as well as the length of the alkyl chain between the two pharmacophores substantially affected the antimicrobial properties of the synthesized hybrids. Molecular docking studies of 114b in the active site of S. aureus dihydrofolate reductase confirmed Van der Waal's π-π stacking and H-bonding interactions and deciphered its mechanism of action [135].
Singh et al. synthesized and evaluated a series of 1H-1,2,3-triazole-tethered curcumincoumarin and curcumin-isatin hybrids for their antibacterial activities on Gram-positive (E. faecalis and S. aureus) and Gram-negative (P. aeruginosa and E. coli) bacterial strains. Among the synthesized hybrids, 115 proved to be the most promising, with an MIC of 6.25 µg/mL ( Figure 19). SAR studies revealed the presence of an ethyl chain as a spacer along with the bromo substituent at the C-5 position of the isatin ring, -chloro at the C-4 position of ring-A, and -OCH 3 at C-2 on ring-B of curcumin to be optimum for antibacterial activity. Molecular modelling studies of 115 were also carried out in the active site of DHFR so as to study the various modes of binding interactions [136]. Khatoon et al. reported the in silico and in vitro evaluations and synthesis of coumarinincorporated isatin hydrazones as antileishmanial agents. Molecular docking was first used to determine the binding confirmation of lead molecules to the target protein. Only three of the docked compounds demonstrated high-binding affinities. These compounds were then tested for antileishmanial activity against Leishmania tropica promastigotes and amastigotes and were found to be active, with IC 50 values ranging from 0.1 to 4.13 µmol/L. Compound 116 was found to be the most effective, with IC 50 values of 0.10 and 0.87 µmol/L against L. tropica promastigote and axenic amastigote forms, respectively ( Figure 19) [137].
Freitas et al. synthesized thiazolyl-isatin derivatives from thiosemicarbazone or phenyl-thiosemicarbazone and tested their activity against Trypanosoma cruzi. Compounds 117a (IC 50 = 4.12 µM) and 117b (1.72 µM) exhibited the best anti-T. cruzi activity for the trypomastigote form, with a selectivity index higher than benznidazole (BZN) (Figure 19). SEM analysis revealed that T. cruzi trypomastigote cells treated with compound 117b induced changes in the shape, flagella, and surface of the body, resulting in parasite death. Compounds 117a (IC 50 = 7.36 and 7.97 µM, respectively) and 117b (6.17 and 6.04 µM, respectively) demonstrated the best activity for the promastigote form among the series, as well as a higher selectivity than Miltefosine [138].

Conclusions
Isatin is a highly promising scaffold in drug discovery due to its ubiquitous presence in biological systems; it is a molecular architecture that can be easily modulated in addition to a plethora of biological activities. Of course, isatin's anticancer potential is overwhelming, as evidenced by the number of isatin-based compounds in use as therapeutics or in various stages of clinical trials. Isatin derivatives have demonstrated promising antiproliferative attributes against various cancer cells, targeting specific biomolecules or organelles as free ligands or those coordinated to metal ions. Adherence to metal ions frequently enhances its bioactivities, indicating a synergistic mechanism comprising the metal and the ligand. They also disclose a variety of modes of action, including the ability to bind DNA, generate reactive species that induce oxidative damage, and suppress specific proteins. Isatinpharmacophore hybrids have the potential to overcome drug resistance and provide new functional entities with multiple mechanisms of action and good safety profiles. In the case of antimycobacterials, the inclusion of isatin with isoniazid has not only improved the lipophilicity as well as the activity of the hybrids, but also minimized the frequency of the development of resistance. The inclusion of isatin core with quinoline core afforded hybrids better antiplasmodial profiles than CQ itself against the CQ-resistant species of P. falciparum. The current evidence therefore suggests that further exploiting this promising moiety can provide efficient clinical candidates with a reduced incidence of drug resistance. We anticipate that introducing isatin into various drugs/organic moieties will significantly impact the treatment of various diseases in the near future, given the multitude of biological activities and mechanistic pathways provided by isatin-based compounds.
Author Contributions: Conceptualization: V.K.; funding acquisition, writing: S.C., S. and A.A.; review and editing V.K. All authors have read and agreed to the published version of the manuscript.