Chemistry of trisindolines: natural occurrence, synthesis and bioactivity

Heterocyclic nitrogen compounds are privileged structures with many applications in the pharmaceutical and nutraceutical industries since they possess wide bioactivities. Trisindolines are heterocyclic nitrogen compounds consisting of an isatin core bearing two indole moieties. Trisindolines have been synthesized by reacting isatins with indoles using various routes and the yield greatly depends on the catalyst used, reaction conditions, and the substituents on both the isatin and indole moieties. Amongst the synthetic routes, acid-catalyzed condensation reaction between isatins and indoles are the most useful due to high yield, wide scope and short reaction times. Trisindolines are biologically active compounds and show anticancer, antimicrobial, antitubercular, antifungal, anticonvulsant, spermicidal, and antioxidant activities, among others. Trisindolines have not previously been reviewed. Therefore, this review aims to provide a comprehensive account of trisindolines including their natural occurrence, routes of synthesis, and biological activities. It aims to inspire the discovery of lead trisindoline drug candidates for further development.


Introduction
Heterocyclic compounds are ubiquitous in nature and possess many bioactivities, making them targets for drug development, health supplements and as highly functional materials. According to the Food and Drug Administration (FDA), 59% of the drug molecules are heterocyclic compounds containing at least one nitrogen atom. 1 One such compound is the indole 1 ( Fig. 1) scaffold with a bicyclic structure incorporating a benzene ring fused to a pyrrole ring. 2 It is among the top ten First Ambar Wati is a doctoral student in the Department of Chemistry, Institut Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia. She completed her undergraduate study in Chemistry at the same institute in 2016. Her research interests include organic synthesis and medicinal chemistry. Currently, she is screening several natural products as antidiabetic, anticancer, and antitubercular candidates at the laboratory of Natural Product and Synthetic Chemistry (NPSC) at ITS.
Dr. Mardi Santoso undertook undergraduate program in chemistry at Institut Teknologi Sepuluh Nopember, Indonesia; and master leading to PhD program in organic chemistry, School of Chemistry, The nitrogen heterocyclic scaffolds used for constructing drug molecules. 1 Examples of indole-based drugs include indomethacin, indoxole, and pindolol. Several good reviews covering the chemistry and bioactivity of indoles have been published. [3][4][5] Trisindolines are compounds containing two indole units connected to the 1H-indol-2,3-dione 2 (also known as isatin) unit either at its C-3 position to form 3,3-di(3-indolyl)-2indolone 3 or at its C-2 position to form a 2,2-di(3-indolyl)-3indolone 4 regioisomeric structure (Fig. 1). Since their rst discovery, 6 trisindolines have attracted considerable attention due to their wide biological activities including anticancer, [7][8][9] antimicrobial, 10 antimycobacterial, 11 antifungal, 10 anticonvulsant, 10 a-glucosidase inhibition, 12 and spermicidal 13 activities. The 3,3-di(3-indolyl)-2-indolone 3 isomer is far more interesting than the 2,2-di(3-indolyl)-3-indolone 4 isomer due to its higher potency and potential as a drug lead compound. 6 It is worth noting that the widely occurring 1H-indol-2,3-dione 2 (isatin) framework is an indole ring bearing two carbonyl groups at C-2 and C-3 ( Fig. 1). 14,15 Isatin forms a core structure of several biologically active molecules and commercial drugs including Sunitinib, Nintedanib, and Semaxanib. 15,16 The chemistry and bioactivity of isatins have also been covered in several reviews. 15,17 To date, no reviews have been published about trisindolines. Hence, this comprehensive review covers the period of 1980-2020 and focuses on the natural occurrence, synthesis, and bioactivities of trisindolines. It highlights the scope, advantages, and limitations of various syntheses of trisindolines. It gives a comprehensive account of the activities reported for Dr. Ziad Moussa undertook undergraduate and postgraduate studies in chemistry at the University of Calgary (BSc 1998, PhD 2003. His PhD research was conducted under the supervision of Professor Thomas Back and focused on the synthesis and evaluation of some novel selenium-based chiral auxiliaries and glutathione peroxidase mimetics. Following postdoctoral research at Texas A&M University (2003)(2004)(2005)(2006) in the laboratories of Professor Daniel Romo, Ziad joined the department of chemistry at Taibah University as an Assistant Professor (2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018). Currently, he is an Associate Professor of organic chemistry at United Arab Emirates University (UAEU). His main research interests include the development of synthetic methodologies and the synthesis and characterization of novel bioactive organic and heterocyclic molecules.

Dr. Arif Fadlan is an Assistant
Professor in the Department of Chemistry, Institut Teknologi Sepuluh Nopember (ITS) Surabaya, Indonesia. He obtained his BS and MS degree from ITS, and his DSc (Materials Science) (2018) degree from Nara Institute of Science and Technology (NAIST), Japan. His current research interests include small molecule and sugar-modied active compounds, essential oils, and pharmacology properties of neglected but promisingplant extracts.
trisindolines with a special focus on the structure-activity relationship (SAR) where possible. The review is also meant to be a one-stop reference work for researchers interested in the development of trisindolines as lead drug candidates. Since trisindolines have two isomeric structures, 3,3-di(3-indolyl)-2indolone 3, and 2,2-di(3-indolyl)-3-indolone 4, we will limit our discussion to the more common and more biologically active 3,3-di(3-indolyl)-2-indolone 3.

Synthesis of trisindolines
Trisindolines were synthesized (vide infra) following several routes. Efficient synthesis of trisindolines depends on the catalyst used, reaction conditions and the reactivity of the indoles and isatins which primarily depends on the position and type (electron donating group or electron withdrawing group) of the substituents on these rings. Additionally, the NH substituents on indoles and isatins rings also impact the efficiency of the reaction. The following sections will highlight these synthetic routes in detail.
The synthetic routes based on acid-catalyzed reaction are the most researched and most efficient with respect to yield, selectivity and reaction time. 13 Trisindolines have also been synthesized in protic solvents and in the complete absence of acid catalysts. Acid-catalyzed indolylation of isatin proceeds through a Friedel-Cras electrophilic aromatic substitution mechanism. Although the indole itself undergoes faster electrophilic aromatic substitution than benzene, a catalyst is usually required for the indolylation to proceed and give reasonable yields. 26 The indolylation is made efficient by increasing the nucleophilicity of the indole ring via appropriately-positioned substituents and by acid-catalyzed activation of the C-3 carbonyl of isatin. 27 . Mechanistically, the reaction involves two steps where 3-hydroxy-3-indolyl-2-indolone 7 is formed rst and then is converted to 3,3-di(3-indolyl)-2indolone 3 following the addition of a second equivalent of indole 1 (Scheme 1 and 2). Strong acid catalysts directly afford 3,3-diindolyl-2-oxindole 3. 26 The proposed general mechanism of acid-catalyzed formation of trisindoline 3 through reaction between isatin 2 and indole 1 is shown in Scheme 2. Formation of 3,3-di(3-indolyl)-2indolone 3 proceeds through a pathway in which the C-3 position of isatin 2 is activated to give intermediate 8 which undergoes nucleophilic attack by the indole 1 to generate 9. Deprotonation of the tertiary alcohol 9 to 3-hydroxy-3-(1H-indol-3-yl)indolin-2-one 7 is followed by protonation to form 10. Dehydration of 10 generates a,b-unsaturated iminium ion 11 which is followed by addition of a second molecule of indole 1 and re-aromatization of 12, affording trisindoline 3.
Since many common symmetrical and unsymmetrical trisindolines have been prepared by different routes, we categorized them in Table 1 and 2 and assigned them numbers for easy reference throughout this review. In symmetrical trisindolines, the isatin unit bears the same indole moieties. However, in unsymmetrical trisindolines, the isatin unit bears different indole moieties.
In the below sections, the grouping of the catalysts under different headings is not very strict and is meant for easy reference. This is because some catalysts can fall under several groupings.

Synthesis of trisindolines from isatin as a coupling partner
Trisindolines can be synthesized from several coupling partners, the most notable of which is indole 1 and isatin 2. Several reagents and catalysts that promote the coupling process are discussed in the next sub-sections. 3.
The Wells-Dawson heteropoly acid H 6 P 2 W 18 O 62 is wellknown for its super acidity, stability in solution, and its recyclability. Trisindolines 3, 5, 17-19 and 22-29 were successfully synthesized in 86-95% yields using 5 mol% H 6 P 2 W 18 O 62 in water at 60 C within 30 minutes. N-Alkyl and N-benzyl isatins and indoles also reacted to give high yields. 31 Heteropoly acid silicotungstic acid (H 4 SiW 12 O 40 , 0.1 mol%) successfully catalyzed the synthesis of trisindolines 3, 5, 15, 17, 18, 22-25 and 27-29 in 85-96% yields within 5-70 minutes at room temperature in methanol. However, 5-bromoisatin and 5bromoindole reacted at a slower rate, giving lower yields. 32 3.1.1.3 Polyphosphoric acid (PPA) catalyzed synthesis. Polyphosphoric acid (PPA) is a mineral acid catalyst used in several organic transformations. However, owing to its high viscosity, the acid exhibited limited use. 33 On a different note, perlite is universal name for amorphous volcanic glass that mainly comprises of alumina-silicate. Perlite expands 4-20 times of its original volume once heated to 760-980 C. Due to its high surface area, low density, and inertness, expanded perlite can be used as solid support for heterogeneous catalysts. 34 Esmaielpour et al. (2017) fabricated expanded perlite-polyphosphoric acid (EP-PPA) (Scheme 5) and evaluated its catalytic activity for bisindolylmethane (BIM) synthesis. The BIMs were obtained in excellent yields within short reaction times. Nevertheless, trisindoline 3 was obtained in 65% yield within 40 minutes. 33 3.1.2 Organic acids-based catalysts. The synthetic routes based on organic acid-catalyzed reactions to afford 3,3diindolyl-2-oxindoles are generally selective and give high yields under short reaction time. The next subsections discuss the use of various organic acids in this regard.
3.1.2.1 p-Toluenesulfonic acid (p-TSA)-catalyzed reactions. p-Toluenesulfonic acid (p-TSA) is a nontoxic, affordable, safe, and readily available catalyst used frequently in many organic Scheme 3 Trisindoline synthesis using sulfuric acid as catalyst.  36 In comparison, although the yields of the products are similar, reactions in dichloromethane proceeded at a much faster rate than in acetonitrile (less than 1 h vs. several hours).
3.1.2.3 Succinimide-N-sulfonic acid catalyzed reactions. Trisindolines 3 and 22 were successfully synthesized in 96% and 92% yields, respectively, using 5 mol% succinimide-N-sulfonic acid in acetonitrile. 38 3.1.2.4 Sulfamic acid-catalyzed reactions. An efficient and simple synthesis of trisindolines 3, 5, 15-18, 20, 27, 29, 31, 32 and 54-66 was disclosed by Brahmachari et al. (2014) using sulfamic acid (NH 2 SO 3 H). Reaction between indoles and isatins using 20 mol% sulfamic acid in ethanol : water (1 : 1, v/v) at ambient temperature gave the desired trisindolines in 84-94% yields within 2-7 h. The electron decient 5-cyanoindole was unreactive. The catalyst was also reused with a slight decrease in efficiency in the third cycle. 39 In a competitive experiment (Scheme 8), when the indole was treated with several carbonyl  compounds including benzil, isatin, acetophenone, and benzanilide, it selectively reacted with isatin to produce trisindoline 3 in 81% yield. Sharma et al. (2016) adopted Brahmachari et al. method to synthesize 56 in 87% yield. 40 3.1.2.5 N 0 -isopropylbenzohydrazide hydrochloride-catalyzed reactions. The Friedel-Cras reaction between isatin 2 and indole 1 was successfully catalyzed using 10 mol% N 0 -isopropylbenzohydrazide hydrochloride 196 aminocatalyst in methanol at room temperature to afford trisindoline 3 in 72% yield. 41 No variations of the indoles or isatins were reported (Fig. 3). (1 mol%) to catalyze the solvent-free condensation reaction between indoles and isatins under grinding conditions (Scheme 9). The reaction at ambient temperature gave trisindolines 3, 15, 16, 22 and 67 in yields ranging from 82-90% aer 10 h. Under these conditions, 5-methoxyindole and 5bromoindole as well as N-ethylindole gave high yields of the trisindolines 16, 15 and 67. 2-Methylindoles also reacted but gave 82% yield of 22. Notably, the catalytic activity of the recovered catalyst remained high, giving trisindoline 3 in 96% yield in the rst cycle and in 78% yield aer ten cycles. 42 3.1.2.7 Dodecylsulphonic acid catalyzed reactions. Hazarika et al. (2008) used 10 mol% dodecylsulphonic acid (DCA) as a catalyst and water-solubilising agent to catalyze the reaction of indole 1 and isatin 2 to obtain trisindoline 3 in 87% yield within 20 minutes at room temperature. No other variations of the isatins and indoles were evaluated. 43 3.1.2.8 Photoacids catalyzed reactions. Photoacids are molecules that become more acidic when absorb light. They can acidify a neutral aqueous solution within very short times (nanoseconds). 44 N,N 0 -Bis[3,5-bis(triuoromethyl)phenyl]thiourea 198 also known as Schreiner's thiourea belongs to photoacid organocatalysts that function as proton donors. Trisindole 32 was synthesized in 79% yield using a blue LEDs-activated Schreiner's thiourea 198 in 1,4-dioxane at room temperature for 18 h. 45 Under these conditions, but using 370 nm LEDs, trisindolines 3 and 18 were synthesized in 69% and 65% yields, respectively. Unfortunately, the synthesis of trisindoline 32 required 4 equivalents of the indole which is much higher than other routes. This nding reveals that light irradiation plays a prominent role in initiating the reaction.  46 HFIP activates the C 3 carbonyl group of isatin in the rst step and is also involved in subsequent proton transfer reactions during the isomerization and dehydration processes of the next two steps. The reaction conditions work well with Nalkylated indoles but are ineffective with indoles bearing substituents (e.g. methyl, phenyl) on C-2 of the indole ring. Additionally, no desired products were obtained when indole bearing NO 2 or CN was used as substrate. However, isatin ring tolerates a wide range of substituents such as halogen, nitro, methyl and methoxy substituents. 46 3.1.3 Heterogeneous and nanoparticle acidic catalysts 3.1.3.1 Amberlyst-15-catalyzed reactions. Amberlyst-15, an acidic cation-exchange resin, is a heterogeneous catalyst that has been used for several organic transformations. Sarra et al.
(2012) reacted various isatins and indoles in the presence of Amberlyst-15 in water (reaction of N-benzylisatin using H 2 -O : acetone (4 : 1)) at 70 C for 30 minutes, furnishing 88-95% yields of the trisindolines 3, 5, 17-19, 22-29, 84 and 85. 5-Nitroand 5-bromo isatins reacted well with either indole or 5-methylindole while derivatization of the NH of the isatin or indole with electron-donating group did not have any signicant effects on reaction rate and product yields. 47 The catalyst showed outstanding reusable activity. Amberlyst-15 was also found to catalyze the electrophilic substitution reaction of 3methylindole with isatins to afford the corresponding 2,2-diaryloxindole 4 in high yield. This is an added advantage since the number of reports on the reaction of isatin with 3-substituted indoles is scant. 47 3. g À1 ) offers a remarkably higher surface area compared to KSF (around 10 m 2 g À1 ) 48 and has been shown to catalyze trisindolines formation much faster than KSF. Excellent yields of 85-93% for symmetrical and 88-91% for unsymmetrical trisindolines were obtained. 2-Methylindole enhanced the reactivity and the yield. 5-Br isatins showed enhanced reactivity in some cases compared to their chloro analogues. Unsymmetrical trisindolines 174, 175 and 176 were prepared by initially reuxing equimolar amounts of isatin and indole substrates, followed by adding a second equivalent of a different indole. Without KSF, the reaction was unsuccessful even when heated at reux for 12 h. The catalyst was easily recovered and reused without signicant loss of reactivity. 50 3.
other side, the grinding method was conducted by mortar and pestle (0.1 mol%, 0.06 g catalyst/1 mmol of isatins), requiring considerably shorter reaction times (1-6.5 minutes) than the preceding method. However, yields were relatively lower ranging from 63 to 98% yields. In the grinding technique, the starting materials were pulverized to ne powder which generated local heat that sped the reaction. Interestingly, nano-SiO 2 catalyst produced best yields of trisindolines (98% for both techniques) with 5-nitroisatin 28, while N-Bn isatins 25 and 24 relatively gave better yield under magnetic stirring (98% yields). 52 As a notable feature of the protocol and while the attempted multi-step one pot preparation of unsymmetrical 3,3di(indolyl)indolin-2-ones failed to produce the desired products, nano-SiO 2 efficiently catalyzed the synthesis of various unsymmetrical trisindolines 174 and 177-179 using the two reported methods (Scheme 12). 52,53 Another mechanical synthesis was achieved using ballmilling technique. The method was introduced as an ecofriendly solvent-free method for the synthesis of trisindoline in the presence of silica gel (SiO 2 ) as acid catalyst and grinding medium. 54 Trisindoline 3 was afforded aer 7 h of grinding in 62% yield, compared to the grinding technique by mortar and pastel using nano-SiO 2 as a catalyst. 52 Azizian Both EDG and EWG attached to C-5 isatin did not impact the yield appreciably but substrates with EWG required longer reaction time than those with EDG. Methyl on C-2 of the indole ring and EDG or EWG substituents on the NH of isatin did not affect the yield either. Further, the catalyst was recycled ve times without loss in yield. 55 Similarly, Chakrabarty et al. (2006) further demonstrated the application of H 3 PO 4 -SiO 2 in the synthesis of trisindoline 3 (80% yield). 56 Nikoofar et al.  (Table 3). Both methods gave 80-96% yields. However, the reaction using grinding method proceeded within 2-5 minutes while the stirring method required 20-40 minutes. Under both methods, 5-nitroisatin was the most sluggish substrate while trisindoline 3 was produced in >94% yield albeit it required longer reaction times. Unsymmetrical trisindolines 174 and 177-179 were also successfully prepared by reacting 1 eq. of isatin and 2 eq. of different indoles. Grinding technique afforded products 174 and 177-179 in 72-78% yields within 5-10 minutes, while stirring technique afforded the same in 71-80% within 5-60 minutes (Scheme 12). 53 Compared to a previous method by the same authors using nano-SiO 2 catalyst, 52 this catalyst was comparable in terms of yield and reaction time for both techniques (Table 3).  . SAMSNs was prepared by incorporating a mercaptopropyl moiety into mesoporous silica nanoparticles, followed by oxidation of the SH group with H 2 O 2 to SO 3 H. The catalyst showed wide scope for substituted isatins with outstanding yields of 90-98%. Trisindoline 3 gave the best yield within 10 minutes. Generally, isatins bearing EWG gave better yield within short reaction time than methyl-substituted isatin. The catalyst also worked well for bulky isatins, N-benzyl-5,7-dibromoisatin and 5,7-dibromoisatin. 57 SBA-15 (SBA: Santa Barbara Amorphous) is mesoporous silica which possess uniform-sized nanopores with a large surface area and high thermal stability. 58  . Substitution of NH of indoles with methyl groups reduces the reaction time and/or enhances the yields. EWG groups (halogen, nitro) on C-5 of isatin did not affect the reaction time or yield appreciably. N-allyl trisindoline was not formed even aer 48 h while N-benzyl trisindoline 25 was obtained within 3 minutes in 90% yield. Unexpectedly, instead of forming the desired trisindoline 22, compound 200 was obtained in 95% yield when 2-methylindole was employed as a substrate. 59 The modication of silica gel with 3-aminopropyltriethoxy silane results in aminopropylsilica gel (APSG). Subsequent anchoring of indium(III) acetylacetonate complex to aminopropylsilica gel (APSG) gave In(acac) 3 -APSG which was investigated as a reusable heterogeneous catalyst for oxindole synthesis. Sharma and Sharma (2010) utilized this catalyst (10 wt%) to prepare trisindolines 3, 5, 15, 16, 22, 27, 42, 85 and 88 in 81-93% yield in water/acetonitrile (4 : 1) solvent system within 2.5-6 h (Scheme 15). Indoles with EDGs showed better reactivity than those with EWGs (5-nitroindole) which showed complete inertness when reacted with 5-nitro or 5-bromoisatin. Additionally, 5-bromoindole did not undergo reaction with 5nitroisatin. 60 3.1.3.5 Nano-SiO 2 supported boron triuoride catalyzed reactions. Boron triuoroide-etherate (BF 3 $OEt 2 ) is a Lewis acid homogeneous catalyst which has been used in many organic transformations. Heterogenization of BF 3 is possible if anchored on solid materials. One such example is the nano-SiO 2 -BF 3 À CH 3 OH 2 + which has been synthesized by mixing BF 3 $OEt 2 and preheated silica gel in MeOH and stirring for 3 h at room temperature. The catalyst was further applied for surpassed 60 wt%, the chemical yields of the trisindolines declined, suggesting that the excess amounts of PWA blocked the MCM-pores, thus highlighting the role of the pores in facilitating the coupling reaction. Unsubstituted trisindoline 3 was formed in excellent 99% yield. Having a methyl group on various positions of the indoles (C-2 or C-5 or C-6) as well as a nitro group on C-5 or C-6 gave moderately lower yields (55-80%) than indoles bearing uorine, and chlorine substituents on C-5 or C-6 positions (96-98%). On the contrary, 1-methylisatin and 5-methylisatin, as well as 5-chloro, 7-uoro, or 4bromo-isatins reacted smoothly with indoles, furnishing trisindolines in excellent 60-99% yields. Interestingly, unlike its 5and 7-halogenated analogues, 4-bromoisatin showed the least reactivity affording the product 95 in only 60% yield. The catalyst exhibited high activity and was recycled and reused over six cycles with an overall drop of 9% in yield. 63 3.1.3.7 Graphene oxide catalyzed reactions. Graphene oxide catalyzed the reaction between indoles and isatins in water media under ambient temperature to give trisindolines 3, 5, 15, 18, 21-25, 27, 31, 84-88, 99 and 100 in 65-98% yields within 1.5-5 h. 64 The best yields were obtained when the indoles were substituted with EDG on C-2 and/or isatins were substituted with EWG on C-5. EWG on C-5 of the indole decreased the yield and/or prolonged the reaction time. N-Methylisatin exhibited better reactivity than N-benzylisatin. minutes. Both EDG and EWG substituents attached to isatin were well-tolerated. Furthermore, 2-methylindoles were more reactive than indole itself. Interestingly, the heterogeneous catalyst could be reused up to 8 times albeit with a slight 5% drop in the yield. 65 In addition, the catalyst is characterized by very low leaching of L-proline during 8 cycles. Another notable feature of this catalyst is its superiority compared to other catalysts in terms of yield and reaction times.
3.1.5 Ionic liquids and related catalysts. Ionic liquids have been used extensively as environmentally benign and green reaction solvents and catalysts in many organic transformations due to their special physicochemical characteristics. 66 Several ionic liquids successfully catalyzed the reaction between indoles and isatins to afford trisindolines.   69 The reaction showed a wide substrate scope where substituted indoles (halogen, methoxy) and isatins (halogen, nitro, methyl) gave the desired trisindolines in excellent yields. However, the reaction between 4-bromoisatin and indole required longer reaction time of 2 h to afford 81% yield of 95. 5-Nitroisatin and 5methylisatin reacted smoothly with indole and gave better yield (96% of 27 and 98% of 33, respectively) than in the case when [(CH 2 ) 4 SO 3 HMIM][HSO 4 ] was used. 67 The catalyst 205 was recycled and reused six times with a 10% overall decrease in the yield of 3. 69 3.1.5.5 Prolinium triate catalyzed reactions. Since several ionic liquids are air and moisture sensitive, their catalytic activity tends to decrease over time. Protic prolinium triate (PrOTf), obtained by treating aqueous L-proline with triic acid, was proposed as water-tolerant ionic liquid. Shiri et al. (2013) demonstrated the usefulness of 10 mol% prolinium triate as a homogeneous catalyst in acetonitrile for the synthesis of trisindolines 3, 18 and 22 in 90%, 89% and 94% yields, respectively. 70 The reaction proceeded at room temperature but took 5 h for completion.
3.1.5.6 Low transition temperature mixtures (LTTMs) catalyzed reactions. Synthesis of trisindolines is also possible using low transition temperature mixtures (LTTMs), also called deep eutectic solvents (DES), comprising oxalic acid dihydrate and L-proline as the solvent/catalyst (Scheme 22). The reaction tolerates wide substrate scope and produces trisindolines 3, 5, 15,16,18,22,27,31,42,54 and 86 in 80-93% yields within 14-18 minutes, even in the presence of EWGs such as CN and NO 2 (Scheme 23). 71 This is not surprising as the reactions between isatins and 5-cyanoindole or 5-nitroindole are oen difficult and fail to proceed satisfactorily even with catalysts due to poor nucleophilicity. 39,60,72 LTTM could be recycled and reused ve cycles without signicant loss of reactivity. 71 3.1.6 Metallic species-based catalysts. Several metal-based catalysts were used to catalyze the reaction between isatins and indoles to trisindolines. The following sub-sections highlight some of the important reagents used.  3.1.6.3 Metal triates catalyzed reactions. Metal triates such as CuOTf 2 , ZnOTf 2 and BiOTf 2 successfully catalyze the reaction between indoles and isatins to give trisindolines. For example, 2 mol% BiOTf 2 catalyzed the reaction between indoles and isatins at room temperature within 2.5-4 h to give 82-95% yields of trisindolines 3, 15, 16, 22, 119, 122 and 123. Both EWGs and EDGs attached at any position of the indole gave considerably good yields. 75 Praveen et al. examined CuOTf 2 (5 mol%) 10 and ZnOTf 2 (1 mol%) 76 and found them to be suitable catalysts especially when the NH of the indoles and isatins was substituted with various groups (Scheme 27). Both catalysts worked well to give similar high yields but the reaction with ZnOTf 2 proceeded at a much faster rate within 5 minutes. Moreover, the use of ZnOTf 2 did not cause isomerization of the (E)-cinnamyl substituents. 10,76 Metal triates seem to be ideal catalysts when NH substituted trisindolines are required.
The mechanism for the generation of biomolecule capped Pd nanoparticles is shown in Fig. 5. The biomolecules in the aqueous Artemisia annua leaf extract coordinate with the Pd 2+ ions to produce metal complexes, which are subsequently reduced to seed Pd 0 particles. The seed particles agglomerate to clusters, which serve as nucleation centres where remaining metal ions get reduced catalytically (Fig. 5).
3.1.7.2 Nickel oxide nanoparticle catalyzed reactions. Nasseri et al. (2015) used nickel oxide nanoparticle (average particle size diameter of 11 nm) to catalyze the synthesis of 3,3-diindolyloxindoles by the condensation of indoles with isatin derivatives in water at 70 C for 0.5-1.5 h. 85 The reaction was best catalyzed using 0.004 g NiO for every mmol of isatin. NiO nanoparticles were prepared via reaction of nickel(II) nitrate hexahydrate with urea in water under heating conditions (115 C, 1.5 h), followed by calcination at 400 C for 1 h. The use of nano NiO was advantageous in improving the yield of 1 to 98%, compared to bare NiO (45% yield) and various other catalytic systems (10-68% yield). Furthermore, the NiO catalyst could be recycled at least ve times. The optimized reaction conditions gave fair to excellent yields of 3,3-diindolyloxindoles 3, 5, 14, 18, 22, 24, 25, 27, 85, 102 and 149-152 (60-98%). Isatin derivatives bearing electron withdrawing group on C-5 showed higher yield and shorter reaction time than those with EDG at the same position. conditions. While quantitative yields for a model reaction was obtained in water as solvent, organic solvents performed relatively poorly, producing less than 80% yields aer 1.5 h. The optimized conditions tolerated a wide scope of substrates with yields ranging from 95-99%. All isatins with EDG or EWG substituents on C-5 were reactive as well as those containing halogens on C-5 of the indole unit. Allyl or benzyl substituents attached to N-isatin barely affected the yields. 72 It is noted though, 5-cyano, N-methyl, and azo indoles failed to generate the desired products. equimolar amounts of indoles and isatins substrates, unsymmetrical products 175, 177-179 and 188 were simply prepared by using a 1 : 1 equimolar mixture of two different indoles with the appropriate matching total molar amount of isatins (Scheme 32). It is noteworthy that the preceding one-step, threecomponent condensation for the preparation of unsymmetrical oxindoles derivatives from indoles and isatins has rarely been reported in the literature and no by-products of symmetrical oxindoles were observed. Various indoles and isatins with EDGs and EWGs underwent successful condensation. The substituent on isatin (H, CH 3 , Bn) had no noticeable effect on the yield or regiochemistry. Interestingly though, blocking the C-3 position forced the condensation reaction to occur at the less reactive C-2 position. 86 The preparation of MgAl 2 O 4 catalyst is shown in Scheme 33.
3.1.7.5 Cupric tungstate (CuWO 4 ) nanoparticle catalyzed reactions. Paplal et al. (2020) synthesized a series of symmetrical trisindolines 3, 21, 24, 25, 100 and 154-160 by treating isatins and indoles with CuWO 4 nanoparticle catalyst (10 mol%) in water at 60 C for approximately 1 h (Scheme 34). Reaction temperature was critical to the preparation of the trisindolines as room-temperature reactions led to the formation of monosubstituted indolinones (3-hydroxy-3-(indol-3-yl)indolin-2ones) as sole products. However, this notable feature was exploited to prepare unsymmetrical 3,3 0 -bis-indolyl-2-oxindoles in good yields (80-85%) (Scheme 35). As such, running controlled experiments with various starting indoles to produce mono-substituted products at room temperature, followed by further treatment with another indole at 60 C gave unsymmetrical 3,3 0 -bis-indolyl-2-oxindoles 179 and 189-191 (80-85% yields). In this study, the reactivity of N-substituted isatin, 5bromo, and 2-methylindole was explored. Trisindolines 3, 21, 24, 25, 100 and 154-160 were obtained in excellent yields (88-99%), whereas trisindoline 3 (R 1 -R 3 ]H) was isolated in highest yield (99%) (Scheme 34). The substituents on the N atom of isatin ring (N-benzyl, N-allyl, N-propyl, N-propargyl, N-methyl) and the bromo group attached to C-5 position of indole did not impact the reactivity of substrates or yield of the products. The catalyst was recycled up to 6 cycles without loss of the catalytic   activity. XRD analysis of the recovered CuWO 4 aer six cycles revealed no change in its morphology suggesting potential for re-use. 87 3.1.7.6 Zinc oxide (ZnO) nanoparticle catalyzed reactions. Nano zinc oxide (ZnO) has been exploited as catalyst for many organic transformations. Since the surface of nanostructured-ZnO consists of both Lewis acid (Zn 2+ ) and Lewis base (O 2À ) sites, it is suitable for catalyzing organic reactions (Fig. 6). Recently, Zn 2+ located on the surface of ZnO disc has been shown to play important role in activating the C-3 carbonyl group of isatin to induce 3-indolylation. To test the importance of surface catalyst, ZnO was covered with hydrophobic stearic acid and the results showed that the coated ZnO-catalyzed reaction did not generate the desired product. Nanodisc ZnO was utilized to prepare a series of trisindolines under solventfree system at 100 C within 2 h to produce the desired trisindolines 3, 16-18 and 54 in 82-88% yields. Interestingly, the reaction of isatin with deactivated indole (bearing cyano group) displayed higher 86% yield compared to the reaction with activated indole (bearing methoxy substituent). N-Methylindole or N-methylisatin gave 83% and 88% yields, respectively. 88 3.1. The yields were modest to excellent where various substrates reacted completely within 5-45 minutes, except for sluggish 5nitroindole which required 130 minutes for complete conversion. Indole substrate with electron donating group had slightly better yield than that with halogen group (EWD). The presence of 5-methoxy group on indole, a strong electron donating group, accelerated the reaction (complete conversion within 10 minutes) and had an opposite effect to the 5-nitro group. 5-Bromoisatin was comparably less reactive than 5-chloroisatin, conceivably due to the steric factors associated with the bulky size of the bromo group. Sulfonated b-cyclodextrin displays better solubility in water than b-cyclodextrin owing to the polar sulfonate and hence shows enhanced reactivity prole. b-CD-SO 3 H also successfully catalyzed the synthesis of 3-hydroxy-3indolylindoline-2-ones in water at reux temperature (86-96% yield). These intermediate products are useful for the synthesis of unsymmetrical trisindolines. 89 3.1.8. Interestingly, indoles bearing EDGs such as methoxy or methyl, and EWG such as bromo gave similar yields. The catalyst was recycled ve times without loss of catalytic ability. 90 The interaction between b-cyclodextrin and the starting materials gave bcyclodextrin-substrate complexes through non-covalent bond interactions. 90, 91 3.1.8.3 Sulfonated polyethylene glycol (PEG-OSO 3 H) catalyzed reactions. Sulfonated polyethylene glycol (PEG-OSO 3 H) (10 mol%) catalyzed synthesis of trisindolines 3, 5, 15, 18, 21-25, 27, 31, 84-88, 99 and 100 in acetonitrile at room temperature for 1.5-5 h, affording 65-98% yields. N-Methyl-or N-benzylisatins gave lower yields compared to others, especially when treated with 5-bromoindole. Consistent with what has been observed with other catalysts, isatin bearing a 5-nitro group required the longest reaction time and afforded the highest yields. PEG-OSO 3 H catalyst could be re-obtained and re-used with no signicant loss of catalytic activity up to ve cycles. 92 3.1.9 Magnetic nanoparticle-based catalysts. Catalysis using magnetic materials has recently attracted intensive research as it offers facile separation of catalysts that exhibit magnetic properties by using external magnet, avoiding ltration, centrifugation, or other techniques to separate the reusable catalyst. 93 As such, nano magnetic sulfonic acid-supported  (Fig. 7). The optimum amount of catalyst was 0.15 g Scheme 38 The proposed mechanism of trisindoline 3 synthesis catalyzed by DABCO-3@FSMNPs. mol À1 isatin 95 . 5-Bromo substituted isatin improved the yield, while 2-methyl on indole and/or benzyl on N atom of isatin reacted smoothly to produce good yields. Generally, compound 3 was obtained in 93% yield within lower reaction time compared to other methods 94 possibly because ultrasonic irradiation promoted faster and higher yielding reactions. 95 Another fabricated supported-Fe 3 O 4 @SiO 2 MNPs, a carboxylic acid-embedded Fe 3 O 4 @SiO 2 (Fe 3 O 4 @SiO 2 @COOH), gave trisindolines 3,5,18,22,23,85,162 and 163 in 60-98% yields at 80 C in aqueous solvent within 30-60 minutes. As many reports utilizing ferrite-silica (Fe 3 O 4 ) based catalysts, 5-bromoisatin was excellent electrophile. Furthermore, it produced the highest yield when coupled with the highly nucleophilic 2-methylindole. EDG (methyl, morpholinomethyl) attached on N-isatin reduced the reactivity. The catalyst possesses high density of acidic sites and magnetic behaviour. 96 Fe 3 O 4 @SiO 2 @Bi 2 O 3 MNPs was used to prepare trisindolines 3, 5, 14, 18, 22-25, 85, 151 and 164 in 65-97% yields within 25-90 minutes. Methyl or benzyl attached to N-isatin render it less electrophilic, promoting slower reactivity and requiring prolonged reaction time to improve the yield. However, the presence of 5-bromo (as electron withdrawing group) on isatin ring led to a remarkably improved yield. 97 Gupta et al. (2019) fabricated silica-coated magnetitenanoparticle anchored DABCO-derived and acidfunctionalized ionic liquid (DABCO-3@FSMNPs) and applied it as recyclable nanocatalyst to prepare a library of trisindolines. The reactions were carried out in H 2 O at 90 C for 2 h to afford products 3, 5, 15, 16, 31, 32, 42, 60, 62, 86, 101 and 111 in 85-98% yields. Both electron-decient and electron-rich indoles reacted well with either isatin or halo-substituted isatin. However, 2-methylindole was inert and did not afford any products. 98 The three-component reaction was found to proceed exclusively via H-bonding intermolecular interactions between isatin and indole substrates and the nanocatalyst. The catalyst could be recycled easily without any signicant loss in catalytic activity.
The plausible pathway of trisindoline formation catalyzed by DABCO-3@FSMNPs is illustrated in Scheme 38. Hydrogen bonds are formed by the interaction of both cationic and anionic species of the ionic liquid catalyst with the reactants (indole and isatin) (IV). The carbonyl (C-3) of isatin 2 is activated by the proton-donor of the sulfonic acid, making it more electrophilic. At same time, indole 1 is more nucleophilic due to the participation of triuoroacetate as proton acceptor from the N-H of indole 1. Hence, it allows the nucleophilic attack of C-3 indole 1 onto activated carbon of C-3 isatin 2 to render V. Intermediate VII, generated by the dehydration of V to the corresponding a,b-unsaturated iminium ion, is attacked by a second indole 1 molecule to produce trisindoline 3.
3.1.10 Nanocomposite materials based catalysts 3.1.10.1 TiO 2 -impregnated SiO 2 catalyzed reactions. Nanocomposite materials possess large surface areas and unique properties when compared to free nanomaterials. These materials consist of a matrix (graphene or graphene-like) and llers, which can be metals, metal oxides, etc99. Haghighi and Nikoofar (2014) employed nanocomposite TiO 2 -impregnated SiO 2 as a Lewis acid to catalyze the synthesis of a series of symmetrical trisindolines under neat conditions at 50 C for 15-120 minutes, producing the corresponding products 3, 5, 15, 17, 18, 22-25, 27 and 28 in 72-93% yields. The catalyst is prepared by mixing column chromatography grade SiO 2 60 in CHCl 3 with nano TiO 2 and stirring at room temperature for 1.5 h. Simple evaporation of the solvent at room temperature affords 50% (w/ w) nano TiO 2 /SiO 2 as white solid. The reaction of 2-methylindole and unsubstituted isatin exhibited the fastest reaction time and the highest yield (93%). Bromo substituent attached to C-5 of either isatin or indole gave longer reaction and the lowest product yield. N-benzylated and 5-Br, 5-NO 2 -isatins were less reactive and required longer reaction times for complete conversion. Under similar reaction conditions, the catalytic activity of non-supported free nano TiO 2 (70% yield) or SiO 2 (60% yield) did not afford better yields of 3 in comparison with nano TiO 2 @SiO 2 (85% yield). 100 3.   (Table 4). Indoles with electron-releasing substituents at C-2 and isatins bearing EWG at C-5 position reacted synergistically to give the best yields. Although nano-SiO 2 (20 mg) catalyzed the synthesis of trisindolines more efficiently than SiO 2 @g-C 3 N 4 (60 mg), a tedious separation of nano-SiO 2 from the reaction media as well as agglomeration of nano-SiO 2 rendered it impractical. The remarkable catalytic behavior of SiO 2 @g-C 3 N 4 nanocomposite has been attributed to the uniformly distributed SiO 2 nanoparticles where size distribution centered at a value of 17.6 nm. 102 3.1.10.4 CuO@g-C 3 N 4 nanocomposite catalyzed reactions. Another doped graphitic carbon nitride catalyst, nanocomposite CuO@g-C 3 N 4, was also investigated and applied for trisindoline synthesis by Allahresani (2017) (Scheme 39). The reactions were conducted at room temperature in water to produce the targeted trisindolines 3, 15, 17, 18, 21, 23-25, 27, 29, 31, 84, 86-88, 99, 100 and 165 within 45-75 minutes with yields ranging from 80-95% (Table 4). 103 The best yields were obtained with 85 mg of the catalyst per mole of isatin. The synthesis of the same trisindolines required longer time than those prepared using SiO 2 @g-C 3 N 4 . Indole bearing electron withdrawing group at C-5 position led to longer reaction time and lower yields (82-83%). The catalyst was however able to match the optimal yields obtained in ref. 102 with isatin bearing EWG on C-5 and indoles having EDG on the C-1 position. The catalytic activity of CuO@g-C 3 N 4 has been attributed to the uniformly distributed CuO nanoparticles impregnated into the pi-conjugated graphitic carbon nitride nanosheets assist in C-C bond formation. One noted disadvantage involves the aggregation of the nanoparticles as observed in the second reuse of SiO 2 @g-C 3 N 4 catalyst. 103 3.1.10.5 Fe(III)@g-C 3 N 4 nanocomposite catalyzed reactions. Recyclable Fe(III)@g-C 3 N 4 is another nanocomposite catalyst Allahresani and co-workers (2018) studied for oxindoles synthesis (Table 4). Initially, g-C 3 N 4 nanosheets were prepared by the oxidation of melamine powder in a heated furnace (550 C) over 4 h. The catalyst is then prepared by stirring a mixture of FeCl 3 and g-C 3 N 4 nanosheets in EtOH at 40 C for further 6 h. The most effective microscopic and spectroscopic characterization techniques for such nanocomposites include XRD and TEM. For instance, XRD analysis for pure g-C 3 N 4 established the hexagonal phase of the nanosheets by two observed distinctive peaks at 13.1 and 27.4 . The signals indicate interlayer stacking and repeated units. Absence of such peaks for Fe(III)@g-C 3 N 4 suggests a breakdown of the nanosheet hexagonal phase, interlayer stacking, and planar repeated units due to the incorporation of FeCl 3 . On the other hand, while the TEM image of pure g-C 3 N 4 shows the expected agglomerated nanosheet, that of Fe(III)@g-C 3 N 4 clearly shows the presence of FeCl 3 throughout the surface of the nanocomposite sheets. The condensation of indoles and isatin molecules to give trisindolines 3, 15, 17, 18, 21, 23-25, 27, 29, 31, 84, 86-88, 99, 100 and 165 in 79-96% yields proceeded under reux in water using 35 mol% Fe(III)@g-C 3 N 4 catalyst, and it required shorter reaction time (27-55 minutes). Trisindoline 17 was obtained in 96% yield in just 27 minutes. It is noted that among a dozen other g-C 3 N 4 -based catalysts, Fe(III)@g-C 3 N 4 furnished the highest yield of trisindolines. 99 In summary, the presence of g-C 3 N 4 support limits metal and metal oxide nanoparticles from accumulation to increase the activity and selectivity of the catalyst. Metal or metal oxide nanoparticles exhibits a problem in its aggregation, and this problem can be overcome by doping with g-C 3 N 4 . 103 Among above g-C 3 N 4 support catalyst, Fe(III)@g-C 3 N 4 required shorter reaction times, and no signicant differences in the yields of product was observed. Reactions of indoles substituted by either EDG or EWG at C-5 with isatin produced notably excellent 87-95% yields of trisindolines 3,5,15,16,30,42,56 and 166 (Scheme 40). The 5nitroindole substrate, however, required higher reaction temperature of 90 C. 104 3.1.10.7 Ru(III)-exchanged zeolite Y nanocomposite catalyzed reactions. Khorshidi and Tabatabaeian (2010) treated a mixture of indoles and isatins with Ru(III)-exchanged zeolite Y as a catalyst (10 mol%) in 1,2-dichloroethane for 20-75 minutes to afford trisindolines 3,15,17,19,22,54 and 167 in 60-98% yields. The catalyst was prepared from zeolite FAU-Y and ruthenium chloride hydrate by stirring a mixture thereof at room temperature for 1 day. EDG on the indoles (N atom, C-2 or C-3) enhanced the yield or contributed to faster reaction than indoles with EWG. 5-Cyanoindole reacted well with isatin and yielded 80% of 54. However, reacting 5-cyanoindole with 5cyanoisatin resulted in the longest reaction time and lowest yield of 167. 105 In addition, another mild synthesis of trisindoline 3 catalyzed by NaY zeolite functionalized by sulfamic acid/ Cu(OAc) 2 (NaY zeolite-NHSO 3 H/Cu(OAc) 2 ) in acetonitrile for 50 minutes gave 96% yield. 106 3.1.11 Biocatalyzed reactions. a-Chymotrypsin from bovine pancreas (BPC) was utilized as an enzymatic catalyst for the synthesis of trisindolines by Xue et al. (2016). Indoles and isatins were treated with 0.93 kU of bovine pancreas (BPC) in methanol with 20% water at 30 C for 60-96 h to yield trisindolines 3, 15-18, 20, 27-29, 32, 38, 56-66, 68 and 69 in 63-97% yields. When polar protic solvent was changed to aprotic solvent, 7 was formed instead of 3. Trisindoline 3 was obtained in 96% yield aer 72 h (Scheme 41). Isatins bearing EDGs or EWGs worked well with the indole and its substituted form. Nevertheless, 5-nitroisatin and 1-methylindole were less reactive, producing relatively lower yields. 107 In general biocatalysts require longer reaction time in comparison with other catalysts.
3.1.12 Electrochemical-based reactions. Trisindoline nanorods 3, 5, 15, 17, 18, 22, 25 and 27 were synthesized in excellent yields (90-96%) by electrochemical methods. Reaction of isatins and indoles were conducted in acetonitrile in unseparated cell under constant current (20 mA) at ambient temperature for 5-150 minutes. LiClO 4 was used as electrolyte, and graphite rods as the cathode and the anode. Overall, the reactions of substituted indoles with isatin proceeded in shorter reaction times and gave better yields than those with indole and substituted isatins. Trisindoline 18 and 25 obtained from Nalkylated and N-benzylated isatins gave the highest yields of 96%. The proposed mechanism of the reaction producing trisindoline 3 is displayed in Scheme 42. 108 Deprotonation of the indole at the cathode produces indole anion which undergoes the usual nucleophilic addition at the C 3 carbon of isatin to produce, aer proton transfer and elimination of hydroxide ion, an a,b-unsaturated imine intermediate. A second molecule of indole anion reacts with the imine, followed by protonation of the indole nitrogen by protons produced at the anode and a nal isomerization step to form the product. 3.1.13 Catalyst-free based synthesis. The catalyst-free reaction between isatins and indoles in water under reux condition gave trisindolines 3,5,15,22,32,33,40,85,88,101,104 and 115 in 80-93% yields. 109 The synthesis was also achieved in water/ethanol (7 : 3) mixture within 5-38 minutes to give 55-86% trisindolines 3, 15, 17-19, 22, 23, 69, 153, 158 and 168-171. 110 In both cases, the reactivity and yield of the trisindolines increased with EDG on C-5 of the indole ring and decreased in the presence of EWG such as bromine. Furthermore, substituents attached to the NH of isatins decreased the reactivity and yields.
3.2 Synthesis of trisindolines from isatin-imine as coupling partner 3.2.1 Ru(III)$nH 2 O catalyzed reactions. Ru(III)-catalyst was also used for condensation reactions of CF 3 -attached isatinimine 213 with several indoles in methanol for 1-60 minutes.

Antimicrobial activity
Trisindoline 3 showed antibacterial activity against E. coli, B. subtilis, S. aureus at 10 mg per disk with inhibition zone of 16, 17, and 10 mm respectively. 6 Trisindoline 3, unlike its analogues, possessed better activity against Gram-negative bacteria compared to Gram-positive bacteria. Trisindolines 172 with 5carboxylic acid indole and 173 with 4-hydroxyindole did not show any inhibitions while trisindoline 15 with 5-bromoindole exhibited weak activity (at the concentration of 30 mg per disk) against E. coli. 6 Trisindoline 3 was found to have excellent antibacterial activity against B. cereus, displaying inhibition zone of 20 mm at 10 mg per paper. 18 The antimicrobial activity of trisindoline 3 and N-benzyl-substituted trisindoline 25 was assayed by disc diffusion method using 6 mm paper discs. Compounds 3 and 25 were found to possess inhibition zones of 25 mm and 26 mm against B. subtilis respectively, while chloramphenicol and gentamycin (antibiotics) showed inhibition zones of 26 mm and 28 mm respectively. Trisindolines 5, 31 and 27 with halogen or nitro groups on the isatin moiety caused 11-18 mm inhibition zone, while trisindolines 17, 57 and 29 with methyl group on the nitrogen did not show inhibition zones. Compounds that were active against B. subtilis, also possessed antimicrobial activity against S. aureus, but with lower inhibition zone of 12-16 mm than that of gentamycin with 20 mm inhibition zone. All the Fig. 9 Effect of various substituents on the anticancer activity of trisindolines. Trisindolines 14 with 5-bromo and N-benzyl moieties on isatin showed promising antibacterial activity against S. aureus with inhibition zone of 23 mm and MIC of 1.25 mg mL À1 . Trisindoline 5 without the N-benzyl moiety on isatin and trisindoline 161 with 2-methyl on the indole unit exhibited lower activity with inhibition zones of 12 mm (MIC of 10 mg mL À1 ) and 19 mm (MIC of 2.5 mg mL À1 ), respectively. However, trisindolines 5, 14 and 161 were inactive against E. coli and P. aeruginosa. 95 Trisindolines 125-137 inhibited E. coli and S. aureus successfully. Trisindoline 135 with N-benzyl group in the isatin and N-propargyl group in indole rings emerged as the most active against E. coli with 17 mm inhibition zone as well as the most active against S. aureus with 15 mm inhibition zone. Trisindolines 125-134, 136 and 137 showed zone of inhibitions of 10-15 mm against E. coli and 10-15 mm against S. aureus. Trisindolines 129, 133 and 137 were the most potent with 15 mm inhibition zone against S. aureus. Under the same conditions, Amikacin exhibited 18-and 17 mm zone of inhibition for E. coli and S. aureus, respectively. 10 The presence of substituents on the nitrogen of isatin or indole seems to contribute positively to the antibacterial activity against E. coli.
The antibacterial activity of trisindolines 5 and 6 was examined against S. aureus, B. subtilis, E. coli, and P. aeruginosa by disc diffusion method (Table 6). 23 Trisindoline 5 possessed wider inhibition zone against S. aureus (17.5 AE 0.8 mm) and B. subtilis (18 AE 0.1 mm) than trisindoline 6, and both were weaker than amikacin. However, trisindolines 5 and 6 showed better inhibition against S. aureus and B. subtilis than gentamicin.
Trisindoline 5 with the 5-bromine on isatin is more potent than 6 having 6-bromine. However, both compounds were inactive against Gram-negative bacteria (E. coli, and P. aeruginosa). 19 The structure-activity relationship (SAR) of the trisindolines as antibacterial agents is seen in Fig. 10.

Antimycobacterial activity
The potency of trisindoline 3 and its analogues 13 and 14 as antitubercular agents was examined using the resazurin microtiter assay (REMA) method. 11 Trisindoline 3 was inactive (MIC > 25 mg mL À1 ) while trisindolines 13 and 14 inhibited the growth of Mycobacterium tuberculosis H 37 Rv with MIC values of 12.5 and <6.25 mg mL À1 , respectively. Rifampicin reference showed MIC of <6.25 mg mL À1 under the same conditions. 11

Antifungal activity
Trisindolines 3, 5, 17, 25, 27, 29, 31 and 57 were found inactive against Candida albicans by the disc diffusion method. 59 Compounds 5, 14 and 161 were assayed in vitro using USP 29-NF25 cylinder plate assay against Candida albicans, and none of them were active as antifungal agents. 95 Surprisingly, trisindolines 125-137 displayed inhibitions against Candida albicans by cup plate method. Compounds 126 and 137 showed the best inhibition (15 mm zone of inhibition) while ketonazole as standard antifungal agent showed 16 mm inhibition zone. Other analogues 125 and 127-136 displayed 10-14 mm inhibition zone. The presence of methoxy group on C-5 of the indole increased the activity against Candida albicans. 10 The inactivity of trisindolines to inhibit the growth of C. albicans was presumably caused by the absence of substituents on the nitrogen atom in either isatin or indole.

Anticonvulsant activity
A series of trisindolines 125-137 were tested in vivo to examine their anticonvulsant activity by observing seizure in rat. 10 All the tested compounds 125-137 were active at a dose of 20 mg kg À1 and halted convulsion. Compounds 127, 128, 130, 133, 136, 137 markedly showed excellent activities with extensor time of 31.60-31.80 s while standard drug phenytoin's extensor time was 43.00 AE 0.40 s ( Table 7). The presence of N-propargyl on indole or isatin moieties as well as N-allyl moiety reduced the time of tonic extensor remarkably. Methyl substituent on C-2 of the indole increased the activity. Trisindoline 125 was less promising as anticonvulsant since it possessed longer extensor time than phenytoin. 10

a-Glucosidase inhibition activity
Trisindolines 3, 5, 17, 31, 33 and 45-53 inhibited a-glucosidase activities and possessed signicantly lower IC 50 values than commercial drug acarbose (Fig. 11). 12 The presence of bromo substituent on isatin ring enhanced the activity. Trisindoles with N-benzyl and N-substituted benzyl isatins were more active than N-alkyl analogues. Trisindoline 3 displayed the lowest inhibition activity and yet was superior to acarbose (Fig. 11).

Spermicidal activity
Trisindoline 3 and its analogues 15-17, 22 and 105 were investigated for their effects on sperm mobility ( Table 8). The minimum effective concentrations (MECs) were dened as the minimum concentration that led 100% sperm immobility within 20 seconds without awakening the next motility in Baker's buffer aer 1 h of incubation at 37 C. Trisindoline 16 showed the best inhibition activity with MEC of 0.34 AE 0.018 mg mL À1 . In comparison, nonoxynol-9 (N-9) as standard showed lower MEC of $0.5. 13

Miscellaneous activities
Compound 3 displayed moderate inhibition activity against xanthine oxidase with IC 50 of 179.6 AE 0.04 mg mL À1 while standard inhibitor allopurinol showed IC 50 value of 7.4 AE 0.07 mg mL À1 . 25 Xanthine oxidase is an enzyme responsible for catalyzing the transformation of xanthine to uric acid as well as reducing oxygen (O 2 ) into reactive oxygen species (ROS). The abundance of uric acid causes oxidized lipid membrane, leading to hyperuricemia and obesity. 114 Trisindoline 3 showed promising bioactivity as tyrosinase inhibitor with IC 50 value of 17.34 AE 0.04 mg mL À1 while reference L-mimosine showed IC 50 value of 37.0 AE 0.03 mg mL À1 . 25 Tyrosinase enzyme is critical to melanogenesis as it catalyzes the production of melanin in skin, hair, and eye pigmentation. 115 Trisindoline 3 was less potent as antioxidant since it showed IC 50 value of 431 AE 0.09 mg mL À1 compared with the positive control of 3-tert-butyl-4-hydroxyanisole (BHA) with IC 50 value of 46 AE 0.22 mg mL À1 . 25

Future directions
Various routes and catalysts have been explored for trisindoline synthesis. In majority of cases, acids have been used to catalyze the reaction successfully. Ionic liquid-catalyzed condensation reaction of isatins with indoles have attracted attention since it is considerably environmental benign and requires simple operation giving high yields within short reaction time. Besides, many ionic liquid catalysts could be utilized to synthesize both symmetrical and unsymmetrical trisindolines. On other hand, only a handful of trisindolines have been synthesized using ionic liquids which warrants further exploration. Trisindolines emerge as a new class of bioactive compounds as it displays several promising bioactivities that need developing. However, limited analogues of trisindoline have been well-investigated and the structure activity relationship is reported on limited varieties of compounds especially in when trisindolines were explored as antitubercular and a-glucosidase inhibitors. Interestingly, there is also very limited studies on the biological activity of unsymmetrical trisindolines which should be explored.

Conclusions
Trisindolines are nitrogen heterocyclic structures with promising biological activities. This review provides comprehensive account of trisindolines including their natural occurrence, synthesis, and biological activities. Various routes of synthesis and catalysts used have been discussed in detail. The biological activities of trisindolines have also been discussed with a special focus on the structure activity relationship. This review aims to inspire further development of trisindolines as lead drug candidates.

Conflicts of interest
There are no conicts to declare.