BF3-OEt2 Catalyzed C3-Alkylation of Indole: Synthesis of Indolylsuccinimidesand Their Cytotoxicity Studies

A simple and efficient BF3-OEt2 promoted C3-alkylation of indole has been developed to obtain3-indolylsuccinimidesfrom commercially available indoles and maleimides, with excellent yields under mild reaction conditions. Furthermore, anti-proliferative activity of these conjugates was evaluated against HT-29 (Colorectal), Hepg2 (Liver) and A549 (Lung) human cancer cell lines. One of the compounds, 3w, having N,N-Dimethylatedindolylsuccinimide is a potent congener amongst the series with IC50 value 0.02 µM and 0.8 µM against HT-29 and Hepg2 cell lines, respectively, and compound 3i was most active amongst the series with IC50 value 1.5 µM against A549 cells. Molecular docking study and mechanism of reaction have briefly beendiscussed. This method is better than previous reports in view of yield and substrate scope including electron deficient indoles.

On the other hand, another interesting molecule, the succinimide which is found in many natural products, is also noticed in several clinical drug candidates, indicating that this scaffold plays a vital role in exhibiting a wide range of biological and pharmaceutical activities ( Figure 1) [35][36][37][38]. Further, succinimides can be easily reduced into 5-membered pyrrolidine rings, γ-lactams and lactims, which by themselves are useful natural product motifs and can be oxidized to maleimides [39,40].
Apart from the various indolyl-derivatives synthesized, the preparation of indolylsuccinimidesneeds to be paid attention to as the previously reported methods have several drawbacks, such as the general method of preparation, which is carried out by the reaction of indoles with maleimides by refluxing in acetic acid. However, this reaction requires long reaction time (up to 3-5 days) and the products are obtained in low yields even after use of excess amounts of reactants [76,77]. In case of acid-catalyzed conjugate addition of indoles, careful control of the acidity to prevent side reactions such as dimerization or polymerization is required [78]. Other methods involve the use of Pd and Ru metals for cross-coupling between indole and dihalomaleimides for the synthesis of 3-indolylsuccinimides and 2-indolylsuccinimides, respectively [79][80][81][82]. To the best of our knowledge, only two reactions were reported previously which involves the conjugate addition of indoles to maleimide compounds in the presence of Lewis acids such as ZnCl2/AlCl3(1 mmol) [76][77][78]. However, these reactions were performed in DCE or ni- Apart from the various indolyl-derivatives synthesized, the preparation of indolylsuccinimidesneeds to be paid attention to as the previously reported methods have several drawbacks, such as the general method of preparation, which is carried out by the reaction of indoles with maleimides by refluxing in acetic acid. However, this reaction requires long reaction time (up to 3-5 days) and the products are obtained in low yields even after use of excess amounts of reactants [76,77]. In case of acid-catalyzed conjugate addition of indoles, careful control of the acidity to prevent side reactions such as dimerization or polymerization is required [78]. Other methods involve the use of Pd and Ru metals for cross-coupling between indole and dihalomaleimides for the synthesis of 3-indolylsuccinimides and 2indolylsuccinimides, respectively [79][80][81][82]. To the best of our knowledge, only two reactions were reported previously which involves the conjugate addition of indoles to maleimide compounds in the presence of Lewis acids such as ZnCl 2 /AlCl 3 (1 mmol) [76][77][78]. However, these reactions were performed in DCE or nitromethane under heating or refluxing condition using electron rich indoles. It was observed that the electron-deficient indoles provide unsatisfactory yield and more often very low reaction yield. Furthermore, there are many methods to functionalize the C3 position of the indole nucleus but very few of them allow the use of free N-H indole skeletons.
Thus, an efficient, economical and environmentally benign commercially available or new catalyst is highly desirable for this procedure. In continuation of our work with regard to the preparation of new catalyst for various organic transformations, [79][80][81] we herein report a strategy in which commercially available boron trifluoride diethyl etherfunctions as an effective catalyst [82,83] for the synthesis of indolylsuccinimides (Scheme 1). Interestingly, unlike earlier procedures, it offers a convenient single step reaction and works well with the electron-deficient indoles under mild reaction conditions. These indolylsuccinimide conjugates were evaluated for their cytotoxicity against HT-29, Hepg2 and A549 human cancer cell lines and moreover molecular docking studies were also carried out to understand their binding mode to the receptor.
Molecules 2021, 26, x FOR PEER REVIEW Thus, an efficient, economical and environmentally benign commercially a or new catalyst is highly desirable for this procedure. In continuation of our w regard to the preparation of new catalyst for various organic transformations, [79 herein report a strategy in which commercially available boron trifluoride die erfunctions as an effective catalyst [82,83] for the synthesis of indolylsucc (Scheme 1). Interestingly, unlike earlier procedures, it offers a convenient single action and works well with the electron-deficient indoles under mild reaction con These indolylsuccinimide conjugates were evaluated for their cytotoxicity agains Hepg2 and A549 human cancer cell lines and moreover molecular docking stud also carried out to understand their binding mode to the receptor. Scheme 1. Synthesis of indolylsuccinimides; a Reagents and conditions: BF3OEt2 (50 mol%) acetate, 60 °C,6h.

Chemistry
Initially, 1H-indole (1) (Scheme 2) is taken as a model substrate to study t tion, because the product, 3-indolylsuccinimide 3a is a solidandcan be easily pur shown in Table 1, the reaction between 1H-indole (1a) and maleimide (2a) does place at room temperature in the absence of any catalyst up to 12 h (Table 1, However, use of iodine in ethanol solvent furnished the desired compound w low yield (Table 1,entry 2). Increasing the temperature to 60 °C did not show markable change in the yield. Switching the solvent from ethanol to methanol tonitrile slightly increased the yield (Table 1,entries 3 and 4). Lewis acid catalyz tion of indole to maleimide has been previously reported employingZnCl2, how catalyst is found to be inefficient as it also provided very low yield for the reactio the aforesaid conditions (Table 1,entries 5-7). Moreover, attempts using copper a catalyst also failed to give the desired products in this reaction (Table 1,entrie Similarly, the use of cerium ammonium nitrate (CAN) and boric acid as catalysts not yield the required products (Table 1,entries 13 and 14).
Later, the use of classic Lewis acid boron trifluoride ethyl ether (BF3OEt2) as is attempted and found to be efficient as the desired product is obtained in r good yield (

Chemistry
Initially, 1H-indole (1) (Scheme 2) is taken as a model substrate to study this reaction, because the product, 3-indolylsuccinimide 3a is a solidandcan be easily purified. As shown in Table 1, the reaction between 1H-indole (1a) and maleimide (2a) does not take place at room temperature in the absence of any catalyst up to 12 h (Table 1, entry 1). However, use of iodine in ethanol solvent furnished the desired compound with very low yield ( Table 1, entry 2). Increasing the temperature to 60 • C did not show any remarkable change in the yield. Switching the solvent from ethanol to methanol and acetonitrile slightly increased the yield (Table 1, entries 3 and 4). Lewis acid catalyzed addition of indole to maleimide has been previously reported employingZnCl 2 , however this catalyst is found to be inefficient as it also provided very low yield for the reaction under the aforesaid conditions (Table 1, entries 5-7). Moreover, attempts using copper and silver catalyst also failed to give the desired products in this reaction ( Table 1, entries [8][9][10][11][12]. Similarly, the use of cerium ammonium nitrate (CAN) and boric acid as catalysts too did not yield the required products ( Later, the use of classic Lewis acid boron trifluoride ethyl ether (BF 3 OEt 2 ) as catalyst is attempted and found to be efficient as the desired product is obtained in relatively good yield (Table 1, entry 15) when compared to the other reactions attempted. Moreover, increasing the reaction temperature from room temperatureto 60 • C in acetonitrile as solvent displayed a nearly two-fold increases in the yield (Table 1, entry 16). However, decrease in yield is reported when solvent is changed from acetonitrile to methyl alcohol (entry 17). Further, change in solvent from protic to aprotic viz. 1,2-dichloroethane (entry 18) increased in the product yield is observedA substantial yield i.e.,~78%, of 3-indolylsuccinimide 3a is obtained when ethyl acetate is taken as solvent and the reaction time reduced significantly from 12 h to 6 h ( Table 1, entry 19). Thus, the optimal reaction conditions for the efficient synthesis of 3-indolylsuccinimide 3a, catalyzed by BF 3 OEt 2 (0.5 mmol) requires 1a (1.0 mmol) and 2a (1.0 mmol) wherein ethyl acetate will be used as a solvent at 60 • C upon stirring for 6 h, hence this condition will be extended to the synthesis of a variety of Indolylsuccinimides to afford the desired product in 78% yield.
With the optimized reaction conditions established, the substrate scope of BF 3 OEt 2catalysed C3-alkylation reaction with different indole substrates is investigated. A series of 5-substituted indoles are found to undergo the desired coupling to give the corresponding products in moderate to excellent yield (56−86%, Scheme 3). The structure of resultant compounds is established on the basis of their 1 H, 13 C NMR and HRMS spectra. However, some of the products were reported previously are corroborated with their reported data and found in accordance (Appendix A.) [76,77,84].
The electronic nature of the indoles was shown to have more influence on the reaction efficiency. The presence of electron-donating groups (5-OMe) significantly increased the yield as it is obvious in case of electrophilic substitution reaction of indole that provides 86% of 3b. However, electron-deficient groups (5-CN and 5-NO 2 ) had drastic effect on reactivity, and the corresponding 3-indolylsuccinimides were obtained in relatively low yields (56% and 60% for 3e and 3f) than the electron-neutral group containing compound 3a with 78% yield. The time required for the completion of this reaction is found between 2and 6 h. The optimal reaction conditions are also compatible with halogenated indole (i.e., 5-F and 5Br), with the corresponding products 3c and 3d obtained in 75% and 84% yields (Scheme 3).
We next investigated the scope of N-alkylated maleimides as substrates and the results are displayed in Scheme 4. All the N-alkylated maleimides reacted well, giving the desired products 3g-3i in 78-88% yield. Notably, N-methyl and N-benzyl maleimides exhibited excellent reactivity than N-Phenyl (3h, 78%) to give corresponding products 3g and 3i in 83% and 88% yield. These results indicate that N-protected maleimides havesome effect on the reaction. ER REVIEW 5 of 18 indole (i.e., 5-F and 5Br), with the corresponding products 3c and 3d obtained in 75% and 84% yields (Scheme 3). We next investigated the scope of N-alkylated maleimides as substrates and the results are displayed in Scheme 4. All the N-alkylated maleimides reacted well, giving the desired products 3g−3i in 78-88% yield. Notably, N-methyl and N-benzyl maleimides exhibited excellent reactivity than N-Phenyl (3h, 78%) to give corresponding products 3g and 3i in 83% and 88% yield. These results indicate that N-protected maleimides havesome effect on the reaction.  To extend the scope, we also made substituted indoles products (3j-3x) with good to excellent yield (58-90%). Electron-deficient groups (5-CN and 5-NO 2 ) containing compounds were formed in relatively low yield (3j, 3n, 3r and 3v with 64%, 58%, 68% and 64%) than electron donating group (MeO, 3k, 74%) and halogenated compounds (Br and Cl, 3l-3m, 70% and 72%). Moreover, the reaction of N-benzyl maleimides furnished the products in excellent yield compared to their N-methyl and N-phenyl counterparts. Interestingly, when N-alkylated indole and N-alkylatedmaleimide was investigated, the yield of the products can rise drastically (3w-3x, 84% and 90%) as shown in Scheme 5. To extend the scope, we also made substituted indoles products (3j-3x) with good to excellent yield (58-90%). Electron-deficient groups (5-CN and 5-NO2) containing compounds were formed in relatively low yield (3j, 3n, 3r and 3v with 64%, 58%, 68% and 64%) than electron donating group (MeO, 3k, 74%) and halogenated compounds (Br and Cl, 3l-3m, 70% and 72%). Moreover, the reaction of N-benzyl maleimides furnished the products in excellent yield compared to their N-methyl and N-phenyl counterparts. Interestingly, when N-alkylated indole and N-alkylatedmaleimide was investigated, the yield of the products can rise drastically (3w-3x, 84 and 90%) as shown in Scheme 5.

Biological Evaluation
To access the cytotoxicity, all compounds were screened against three human cancer cell lines namely HT-29, Hepg2 and A549 by MTT assay [87,88]. The compound 3w was found to be the most potent congener amongst the series with IC 50 value 0.02 µM and 0.8 µM against HT-29 and Hepg2 cell lines, respectively and compound 3i was most active amongst the series with IC 50 value 1.5 µM against A549 cells.

Structure Activity Relationship
Indole based bioactive molecules are reported in literature for diversified activity including anti-canceractivity [89].
It is observed from the Table 2 that compounds having unprotected indole and succinimide moieties (3a-3f) showed favorable activity against HT-29 and Hepg-2 cell lines with IC 50 values ranging from 3.6 to 9.1 µM. Interestingly, compound 3b has IC 50 value of 3.6 µM against Hepg-2 cells. However, N-methylated, N-phenylandN-benzylatedsuccinimids with unprotected indoles were relatively less active against these cell lines. Moreover, Nmethylated and N-benzylatedsuccinimides (3i, 3k-3m, 3u and 3v) are more active against A549 cells than N-phenyl compounds with IC 50 ranging from 1.5 to 8.7 µM. Remarkably, compound 3w having N-methyl on both indole as well as succinimide rings showed potential cytotoxicity with IC 50 values of 0.02 and 0.8 µM against HT-29 and Hepg-2 cell lines, respectively. In view of electron rich and deficient indoles no remarkable distintion was observed, however these indoles were more active against HT-29 and Hepg-2 cells than A549 cells. Interestingly, halogenated indoles showed enhanced effect against A549 cells.

Molecular Docking Studies
Molecular docking studies were carried out with a view to understand the site of binding by these compounds. It is evident from the literature that various indolylmaleimidederivatives show cytotoxic properties by inhibiting the cyclin-dependent kinases and therefore molecular docking studies were performed at the ATP binding pocket of CDK2 as a model kinase [90]. A number of structural studies have demonstrated that most of inhibitors bind to CDK2 in a fashion similar to Staurosporine (which is a known kinase inhibitor), the adenine ring of ATP, forming a triplet of hydrogen bonds to the peptide backbone of residues Glu81 and Leu83, which resides in the hydrophilic hinge region at the back of the binding pocket [91]. The crystal structure of Staurosporine in complex with CDK2 provides insight into the interactions responsible for high-affinity binding to a variety of kinases. The crystal structure of the protein was obtained from Protein Data Bank (PDB ID 1AQ1) [31], necessary corrections to the protein were carried out using Protein Preparation Wizard from the Schrodinger package and 3D structures were generated by Schrödinger suite (Schrödinger'sLigPrep program). Molecular docking studies were performed by using a GLIDE docking module of Schrödinger suite and the results were analyzed on the basis of the GLIDE docking score and molecular recognition interactions. All the 3D figures were obtained using Schrödinger Suite 2014-3 [92].
Docking studies were performed on 3c and 3w, which suggests a reasonable binding mode in the ATP-binding pocket of CDK2 (Figure 2). This binding mode is very similar to the Staurosporine binding mode. Likewise, compound 3c hydrogen bond to the backbone carbonyl of Glu81 and to the backbone amine of Leu83 are formed, compound 3w formed hydrogen bond with leu83. The indole ring is located in the hydrophobic cleft formed by the amino acids Ile10, Val18, Lys33, Phe80, Leu134, and Asp145.
Docking studies were performed on 3c and 3w, which suggests a reasonable binding mode in the ATP-binding pocket of CDK2 (Figure 2). This binding mode is very similar to the Staurosporine binding mode. Likewise, compound 3c hydrogen bond to the backbone carbonyl of Glu81 and to the backbone amine of Leu83 are formed, compound 3w formed hydrogen bond with leu83. The indole ring is located in the hydrophobic cleft formed by the amino acids Ile10, Val18, Lys33, Phe80, Leu134, and Asp145.

Preparation of Compounds
The detailed procedure of the synthesis isgiveninAppendix A. section.

Preparation of Compounds
The detailed procedure of the synthesis is given in Appendix A section.

Biological Activity
The detailed procedure employed for the biological activity is given in Appendix A section.

Conclusions
A simple and efficient BF 3 -OEt 2 promoted C3-alkylation of indoles has been developed to access 3-indolylsuccinimidesfrom commercially available indoles and maleimides, in good to excellent yields (64-90%) (under mild reaction conditions. This is an improved protocol compared to the previously reported ones, in view of yield and substrate scope including electron-deficient indoles. Furthermore, anti-proliferative activity of these congeners was evaluated against HT-29, Hepg2 and A549 human cancer cell lines. Compound 3w was found to be the most potent amongst the series with IC 50 value of 0.02 µM and 0.8 µM against HT-29 and Hepg2 cell lines, respectively and compound 3i was most active amongst the series with IC 50 value 1.5 µM against A549 cell line.

Appendix A. Experimental Section
Appendix A.1. Chemistry All reagents, starting materials, and solvents were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA) or Alfa Aesar (Johnson Matthey Company, Ward Hill, MA, USA) and used without further purification. Reactions were monitored by TLC, performed on silica gel glass plates containing 60 F-254, and visualization on TLC was achieved by UV light or using an iodine indicator. Column chromatography was performed with Merck 60-120 mesh silica gel. 1 H and 13 C NMR spectra were recorded with 75, 100, 300, 400, and 500 MHz spectrometer in CDCl 3 and DMSO-d 6 solutions. Chemical shifts (δ) are expressed in ppm relative to the internal standard TMS and multiplicities of NMR signals are represented as singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), triplet of doublet (td) and multiplets (m). High-resolution mass spectra (ESI-HRMS) were obtained by using ESI-QTOF mass spectrometer. Melting points were determined on an Electro-thermal melting point apparatus and are uncorrected.

Appendix A.2. General Procedure for the Synthesis of Compounds (3a-3x)
A mixture of 117 mg (1 mmol) indole, 97 mg (1 mmol) maleimide andapproximately a drop of BF 3 OEt 2 (0.5 mmol) in 5 mL of ethyl acetate solvent was stirred at 60 • C for 6 h. Completion of reaction was monitor by TLC. After completion, the mixture was cooled to room temperature. In some cases where the product succinimide appeared as a solid from the reaction mixture filtered and recrystallized. Otherwise, H 2 O (20 mL) was added to the solution and extracted with EtOAc (2 × 25 mL). The combined organic layer was dried over anhydrous Na 2 SO 4 and concentrated by rotary evaporation to afford crude product which was further purified by column chromatography using EtOAc and hexane as solvent system.