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.

1. Introduction

The properties like anticancer [1,2], antioxidant [3,4,5], antirheumatoidal [6,7] and anti-HIV [8,9,10] has made indole a privileged scaffold and its derivatives such as indolylsuccinimide are important intermediates in organic synthesis and pharmaceuticals [11,12,13,14,15,16,17,18]. The indole ring system is present in many commercially marketed drugs (Figure 1) [19,20,21]. Moreover, different indole derivatives targeting regulation of PI3K/Akt/mTOR/NF-κB and other signaling pathways have been reported [22,23,24]. Maleimide and its N-substituted derivatives are potent and selective telomerase inhibitors suitable for cancer therapy [25]. This moiety is used as a bridge to the disulfide bond present in protein and protein PEGylation [26,27] and as a linker in antibody-drug conjugates to increase their tolerability, intra-tumoral drug delivery as well as to improve the therapeutic efficacy [28,29,30]. Similarly, Ru(II) and Pt(IV) based compounds with maleimide functionality have been prepared to selectively deliver these compounds to the tumor [31,32]. Additionally, maleimide-derived molecule MIRA-1 reactivates mutant p53 in living cells and induces mutant p53-dependent cell death in different human tumor cells [33,34].

Figure 1

Indole and succinimide rings present in natural products and drug candidates.

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].

Thus, importance of developing methods to synthesize indolyl-derivatives can never be overestimated, which is also indicated by the large number of reports appearing consistently in various journals from the past decades [41,42,43,44,45,46]. Most importantly, their synthesis with selective functionalization has become an active research area [47,48,49]. In contrast to the conventional approaches, the direct functionalization of indole has emerged more recently as a preferred methodology as it is more practical and reduces the number of steps [50,51,52,53,54]. Among the several approaches for the direct substitution of indoles, transition metal catalyzed C-H activations are more attractive [55,56,57,58]. However, positions C2 and C3 are the most activated ones, thus direct C3 alkylations are still limited. Therefore, allylic alkylations [59,60,61,62,63,64,65], Baylis–Hillman [66,67,68,69] and more frequently Friedel-Craft type of reactions [70,71,72,73,74,75] are especially useful to achieve this transformation.

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 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.

Scheme 1

Synthesis of indolylsuccinimides; a Reagents and conditions: BF3OEt2 (50 mol%), ethyl acetate, 60 °C, 6 h.

2. Results and Discussion

2.1. 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 employingZnCl2, 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 8–12). Similarly, the use of cerium ammonium nitrate (CAN) and boric acid as catalysts too did not yield the required products (Table 1, entries 13 and 14).

Scheme 2

Optimization of reaction.

Table 1

Optimization of the reaction conditions .

Entry	Catalyst	Solvent *	Temp (°C)	Time (h)	Yield(%) b
1	-	DCE	r.t/60 c	12	0
2	I2	EtOH	r.t/60	12	5
3	I2	MeOH	r.t/60	12	18
4	I2	MeCN	r.t/60	12	25
5	ZnCl2	MeCN	r.t/60	12	trace
6	ZnCl2	DCE	r.t/60	12	10
7	ZnCl2	EtOAc	r.t/60	12	trace
8	Cu(OAc)2	MeCN	r.t/60	12	N.R
9	Cu(OTf)2	MeCN	r.t/60	12	N.R
10	Cu(NO3)2	MeCN	r.t/60	12	trace
11	AgCl	MeCN	r.t/60	12	N.R
12	AgOTf	MeCN	r.t/60	12	N.R
13	CAN	MeCN	r.t/60	12	N.R
14	H3BO3	MeCN	r.t/60	12	0
15	BF3OEt2	MeCN	r.t	12	30
16	BF3OEt2	MeCN	60	12	52
17	BF3OEt2	MeOH	60	12	44
18	BF3OEt2	DCE	60	12	62
19	BF3OEt2	EtOAc	60	6	78

Reaction conditions: 1a (1.0 mmol), 2a (1.0 mmol), catalyst (0.5 mmol), heated in 5 mL of solvent within 12 h. Products were obtained in isolated yields based on indole. Reaction first stirred at r.t. if starting material not consumed totally then temperature shifted to 60 °C. * DCE-dichloroethane, EtOH- ethanol, MeOH-methanol, MeCN-acetonitrile, EtOAc-ethyl acetate.

Later, the use of classic Lewis acid boron trifluoride ethyl ether (BF3OEt2) 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 BF3OEt2 (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 BF3OEt2-catalysed 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 1H, 13C 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].

Scheme 3

Substrate scope of indoles; Reaction conditions: 1a-1f (1.0 mmol), 2a (1.0 mmol), BF3OEt2 (0.5 mmol), heated in 5 mL of solvent within 6 h.

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-NO2) 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.

Scheme 4

Substrate scope of maleimides; Reaction conditions: 1a (1.0 mmol), 2a-2d (1.0 mmol), BF3OEt2 (0.5 mmol), heated in 5 mL of solvent within 6 h.

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.

Scheme 5

Scope of indole and maleimides; Reaction conditions: 1a-1f (1.0 mmol), 2a-2d (1.0 mmol), BF3OEt2 (0.5 mmol), heated in 5 mL of solvent within 6 h.

Plausible Mechanism

BF3-etherate catalyst serves as a source of boron trifluoride via the equilibrium [85,86] shown below. The BF3 coordinate with the oxygen of maleimide, inducing reactions of the resulting adducts A with indole nucleophile to generate intermediate B which on deprotonationconverted to intermediate C hence restoration of aromaticity. Finally, this gives rise to the desire product Scheme 6.

Scheme 6

Plausible mechanism.

2.2. 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 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.

2.2.1. 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 IC50 values ranging from 3.6 to 9.1 µM. Interestingly, compound 3b has IC50 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, N-methylated and N-benzylatedsuccinimides (3i, 3k-3m, 3u and 3v) are more active against A549 cells than N-phenyl compounds with IC50 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 IC50 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.

Table 2

Cytotoxicity of indolylsuccinimide analogues.

Entry	Compound	Cancer Cell Lines (IC50 µ/M) f
		HT-29 g	Hepg2 h	A549 i
1	3a	4.32 (±0.06)	5.2 (±0.03)	10.9 (±0.03)
2	3b	4.67 (±0.16)	03.6 (±0.02)	9.7 (±0.22)
3	3c	4.36 (±0.33)	05.8 (±0.03)	2.1 (±0.20)
4	3d	7.9 (±0.32)	09.1 (±0.04)	6.4 (±0.14)
5	3e	06.2 (±0.23)	05.6 (±0.06)	18.2 (±0.22)
6	3f	05.4 (±0.13)	04.9 (±0.01)	10.7 (±0.05)
7	3g	10.7 (±0.23)	11.4 (±0.03)	9.6 (±0.13)
8	3h	08.4 (±0.06)	15.9 (±0.03)	10.6 (±0.34)
9	3i	24.8 (±0.02)	20.3 (±0.03)	1.5 (±0.45)
10	3j	07.8 (±0.03)	06.9 (±0.11)	9.1 (±0.33)
11	3k	10.6 (±0.12)	13.0 (±0.13)	2.5 (±0.12)
12	3l	10.9(±0.03)	13.6 (±0.035)	3.9 (±0.34)
13	3m	19.9 (±0.12)	14.5 (±0.6)	3.5 (±0.01)
14	3n	09.1(±0.06)	7.8 (±0.21)	12.9 (±0.22)
15	3o	06.6(±0.4)	16.5 (±0.22)	14.4 (±0.03)
16	3p	12.5±0.03	18.7 (±0.20)	11.3 (±0.03)
17	3q	10.2 (±0.33)	5.9 (±0.05)	7.6 (±0.03)
18	3r	10.3 (±0.45)	12.5 (±0.07)	8.7 (±0.06)
19	3s	23.6 (±0.67)	19.5 (±0.56)	15.8 (±0.22)
20	3t	22.5 (±0.34)	18.6 (±0.05)	11.4 (±0.17)
21	3u	10.7 (±0.23)	16.7 (±0.67)	2.4 (±0.14)
22	3v	08.5 (±0.13)	07.2 (±0.09)	3.6 (±0.24)
23	3w	0.02 (±0.02)	0.8 (±0.05)	6.3 (±0.12)
24	3x	28.2 (±0.34)	17.3 (±0.22)	14.7 (±0.12)
25	Doxorubicin	01.2 (±0.03)	01.8 (±0.01)	0.9 (±0.10)

50% Inhibitory concentration after 48 h of compounds treatment and the values are average of three individual experiments. Human colorectal adenocarcinoma cells, Human liver cancer and Human lung cancer.

2.2.2. 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.

Figure 2

(A) Binding pose of compound 3c in ATP binding pocket of CDK2. (B) Binding pose of compound 3w in ATP binding pocket of CDK2. Compounds 3c and 3w shown in stick and colored by the atom type (carbon: grey; oxygen: red; nitrogen: blue; fluorine: green).

3. Materials and Methods

3.1. Preparation of Compounds

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

3.2. Biological Activity

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

4. Conclusions

A simple and efficient BF3-OEt2 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 IC50 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 IC50 value 1.5 µM against A549 cell line.