A greener approach toward N − 1 heteroarylation of indoles: Synthesis and in vitro evaluation of potential anti-proliferative agents

2.1 General methods

Unless stated otherwise, reactions were monitored by thin layer chromatography (TLC) on silica gel plates (60 F₂₅₄), visualizing with ultraviolet light or iodine spray. Column chromatography was performed on silica gel (60–120 mesh) using distilled petroleum ether and ethyl acetate. ¹H and ¹³C NMR spectra were determined in CDCl₃ solution using a Varian 400 MHz spectrometer. Proton chemical shifts (δ) are relative to tetramethylsilane (TMS, δ = 0.0) as internal standard and expressed in parts per million. Spin multiplicities are given as s (singlet), d (doublet), t (triplet), and m (multiplet) as well as b (broad). Coupling constants (J) are given in hertz. Melting points were determined by using a Buchi melting point B-540 apparatus. MS spectra were obtained on a Agilent 6430 series Triple Quard LC-MS/MS spectrometer. HRMS was determined using waters LCT premier XETOF ARE-047 apparatus.

2.2 General method for the synthesis of N-heteroaryl substituted indole derivatives 3

A solution of compound 1 (1.0 mmol), 2 (1.2 mmol) and Cs₂CO₃ (2.0 mmol) in PEG 400 (3 mL) was stirred at 25 °C under ultrasound (using a laboratory ultrasonic bath SONOREX SUPER RK 510H model producing irradiation of 35 kHz) for 2–2.5 h. The temperature of ultrasonic bath was maintained at 25 °C by adding cold water time to time. After completion of the reaction (indicated by TLC) the mixture was diluted with cold water (60 mL) and extracted with EtOAc (3 × 30 mL). The EtOAc layers were collected, combined, washed with brine solution (2 × 30 mL) followed by cold water, dried over anhydrous Na₂SO₄, filtered and concentrated under low vacuum. The crude product isolated was purified by column chromatography over silica gel using 2–5% hexane–EtOAc as eluant.

2.3 5-Methoxy-1-(pyrimidin-2-yl)-1H-indole (3n) (Ackermann and Lygin, 2011)

Ash colored solid; mp 109–110 °C [109–110 °C (Ackermann and Lygin, 2011)]; ¹H NMR (CDCl₃, 400 MHz): δ 8.69 (d, J = 9.0 Hz, 1H), 8.64 (d, J = 5.0 Hz, 2H), 8.24 (d, J = 3.4 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 6.98–6.96 (m, 2H), 6.63 (d, J = 3.4 Hz, 1H), 3.88 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): δ 158.1, 157.6, 155.5, 132.1, 130.3, 126.3, 117.1, 116.0, 112.6, 106.8, 103.1, 55.7; IR (KBr, cm⁻¹): 1578, 15,256 1453; MS (ESI) m/z: 226 (M⁺, 100); HPLC: 95.9%, Column: Symmetry C-18 75 ∗ 4.6 mm, 3.5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: CH₃CN (T/%B): 0/20, 1/20, 4/98, 10/98, 10.5/20, 12/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER (90:10).

2.4 5-Bromo-1-(pyrimidin-2-yl)-1H-indole (3o)

Off white solid; mp 101–103 °C; ¹H NMR (CDCl₃, 400 MHz): δ 8.70 (d, J = 5.0 Hz, 2H), 8.67 (s, 1H), 8.27 (d, J = 4.0 Hz, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 9.0 and 2.0 Hz, 1H), 7.06 (t, J = 5.0 Hz, 1H), 6.62 (d, J = 3.0 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ 158.2, 157.5, 134.0, 133.0, 127.1, 126.4, 123.4, 117.8, 116.5, 115.4, 106.1; IR (KBr, cm⁻¹): 1579, 1522, 1455; MS (ESI) m/z: 274 (M⁺, 100), 276 (M + 2); HPLC: 97.5%, Column: X-Terra RP18 250 × 4.6 mm 5.0 μm, mobile phase A: 0.1% TFA in water, mobile phase B: CH₃CN, gradient (T/%B): 0/20, 3/20, 12/95, 23/95, 25/20, 30/20; flow rate: 1.0 mL/min; Diluent: ACN: WATER (90:10); HRMS (ESI) Calcd for C₁₂H₈BrN₃Na (M + Na)⁺, 295.9799, found 295.9791.

2.5 Preparation of phenyl(1-(pyrimidin-2-yl)-1H-indol-2-yl)methanone (5) (Kumar and Sekar, 2015)

A mixture of indole 3m (1.0 mmol), benzaldehyde 4 (1.5 mmol), PdCl₂ (0.10 mmol), and TBHP (3.0 mmol) in toluene (4 mL) was heated at 90 °C in a reaction tube for 12 h. After completion of the reaction (indicated by TLC) the mixture was cooled to room temperature, treated with EtOAc (50 mL), and washed with saturated NaHCO₃ solution (2 × 20 mL). The organic layer was collected, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue thus obtained was purified by column chromatography over silica gel using 2–5% EtOAc-hexane as eluant to give the desired product; white solid; yield 76%; mp 122–123 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.64 (d, J = 4.3 Hz, 2H), 8.42 (d, J = 8.3, 1H), 7.9 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.62–7.52 (m, 1H), 7.46–7.44 (m, 3H), 7.35–7.27 (m, 1H), 7.14 (s, 1H), 7.07–7.05 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 187.6, 158.0, 157.3, 138.3, 138.0, 137.2, 132.7, 129.5, 128.3, 128.0, 126.5, 122.8, 122.5, 117.3, 115.4, 114.3; MS (ESI) m/z: 300.1 (M⁺, 100).

2.6 Preparation of (1H-indol-2-yl)(phenyl)methanone (6) (Kumar and Sekar, 2015)

A solution of compound 5 (0.8 mmol) and sodium ethoxide (6 equiv.) in DMSO (10 mL) was stirred under nitrogen at room temperature for 5 min and then at 100 °C for 12 h. After completion of the reaction (indicated by TLC) the mixture was diluted with water (60 mL) and extracted with EtOAc. The organic layers were collected, combined, washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated under low vacuum. The residue obtained was purified by column chromatography over silica gel using 2–5% EtOAc–hexane as eluant to give the desired product; off white solid; yield 71%; mp 147–149 °C; ¹H NMR (400 MHz, CDCl₃) δ 9.54 (bs, 1H), 8.09–7.97 (m, 2H), 7.72 (d, J = 8.0 Hz, 1H), 7.66–7.60 (m, 1H), 7.58–7.46 (m, 3H), 7.41–7.34 (m, 1H), 7.20–7.13 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 187.3, 138.1, 137.6, 134.5, 132.4, 129.3, 128.5, 127.8, 126.6, 123.3, 121.1, 113.0, 112.3; MS (ESI) m/z: 221.7 (M⁺, 100).

2.7 Pharmacology

3.1 Chemistry

In order to establish the optimal reaction conditions the reaction of indole (1a) with 2-bromopyridine (2a) was performed under various conditions. Initially, the reaction was performed at room temperature in the presence of Na₂CO₃ in DMSO under ultrasound irradiation using a laboratory ultrasonic bath SONOREX SUPER RK 510H model producing irradiation of 35 kHz. The reaction proceeded under this condition affording the desired product 3a albeit in low yield (entry 1, Table 1). Though replacing Na₂CO₃ with K₂CO₃ did not improve the yield significantly (entry 2, Table 1) the use of Cs₂CO₃ afforded 3a in 61% yield (entry 3, Table 1). We were pleased with this observation that prompted us to investigate the reaction further. To know the role of ultrasound in this C—N bond forming reaction the reaction of 1a with 2a was performed in the absence of any ultrasound irradiation. The product 3a was isolated in poor yield after 12 h (entry 4, Table 1) indicating that ultrasound played a key role in the present reaction. While DMSO was found to be an effective solvent for the present ultrasound assisted reaction there was a need to improve the yield of 3a further. Fortunately, an improvement in yield of 3a was observed when the reaction was performed in PEG 400 for 2 h (entry 5, Table 1). However, no further increase in yield was observed when the reaction was performed for a longer time or at elevated temperature (entry 6 and 7, Table 1). The reaction did not proceed in the absence of a base (entry 8, Table 1). While the reaction proceeded in the absence of ultrasound in PEG 400 the duration of reaction was increased considerably i.e. to 12 h. Thus, the condition of entry 5 was found to be milder as well as optimal and used for further study.

Table 1 The effect of conditions on the reaction of 1a with 2a.^a

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Entry	Base	Solvent	Time (h)	% Yieldb
1.	Na2CO3	DMSO	2	39
2.	K2CO3	DMSO	2	42
3.	Cs2CO3	DMSO	2	61
4.	Cs2CO3	DMSO	12	17c
5.	Cs2CO3	PEG 400	2	69
6.	Cs2CO3	PEG 400	6	70
7.	Cs2CO3	PEG 400	2	71d
8.	–	PEG 400	2	0

a All the reactions were performed using 1a (1 mmol), 2a (1.2 mmol) and a base (2 mmol) in a solvent (3 mL) at 25 °C under ultrasound irradiation as well as open air.

b Isolated yield.

c The reaction was performed in the absence of ultrasound.

d The reaction was performed at 50 °C instead of 25 °C.

Having established the optimal condition for the selective N − 1 heteroarylation of indole it was necessary to expand the generality and scope of this reaction. Thus several indoles (1a–h) were treated with a range of heteroaryl halides (2a–e) under the optimized reaction conditions (Table 2). The reaction proceeded well in all these cases affording good yields of desired products 3a–o. Groups such as Me, OMe, F, Cl and Br on the indole ring were well tolerated under the conditions examined. Notably, though marginal better yields of products were obtained when F, Cl and Br substituents were present on the indole ring (entries 5, 9, 11 and 15, Table 2). Generally, bromo heteroarenes (2a–c) were used as halide reactants in the present ultrasound assisted C—N bond forming reaction. The use of a chloro derivative i.e. 2e was also examined and was successful (entries 13–15, Table 2). One of the products i.e. 3m obtained via this reaction was functionalized further according to a known method. Thus the reaction of 3m with benzaldehyde (4) in the presence of 10 mol% of PdCl₂ and TBHP (tert-Butyl hydroperoxide) in toluene at 90 °C afforded the corresponding 2-acylated product 5 in good yield (Scheme 2) (Kumar and Sekar, 2015; Yan et al., 2014; Li et al., 2015a,b; Wang et al., 2015). The reaction proceeded via a Pd-mediated direct C—H functionalized at C-2 of the indole ring where the pyrimidine ring acted as a C(sp²)—H directing group. This group was easily removed by using EtONa in DMSO to afford the desired 2-benzoyl indole (6) (Scheme 2).

Table 2 Synthesis of N-heteroaryl substituted indole derivatives (3).^a

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Entry	Indole 1	Heteroaryl halide 2	Time (h)	Product 3	% yieldb
1.			2		69
2.		2a	2.5		67
3.		2a	2.5		65
4.		2a	2.5		67
5.		2a	2		72
6.	1b		2.5		69
7.	1b		2.5		67
8.		2c	2.5		69
9.	1e	2c	2		73
10.	1c	2c	2.5		64
11.		2c	2		76
12.	1b		2		69
13.	1a		2		77
14.	1c	2e	2		69
15.		2e	2		70

a All the reactions were performed using 1 (1 mmol), 2 (1.2 mmol) and Cs₂CO₃ (2.0 mmol) in PEG 400 (3 mL) at 25 °C for 2–2.5 h under ultrasound irradiation as well as open air.

b Isolated yield.

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Scheme 2 Synthesis of 2-benzoyl indole (6) via Pd-catalyzed direct C2-acylation of 3m.

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A probable mechanism for the selective N − 1 heteroarylation of indoles under ultrasound irradiation is shown in Scheme 3. The C—N bond forming reaction seemed to proceed via activation of the halide substituent of —N⚌C(X)— moiety of 1. The solvent PEG 400 appeared to play the role of reaction medium as well as an activating agent in the present reaction under the condition employed [indeed the dual role of PEG as a solvent and phase-transfer catalyst (PTC) is known] (Totten et al., 1998; Totten and Clinton, 1988). Thus, H-bond formation of the azomethine nitrogen [—N⚌C(X)—] of 2 with PEG 400 facilitated a nucleophilic attack at the adjacent halogen bearing carbon atom of E-1. Deprotonation of indole 1 afforded the required nucleophile E-2 in situ that on reaction with E-1 afforded E-3. Subsequent departure of halide ion (in the form of CsX) from E-3 generated E-4 that released PEG 400 to complete the reaction cycle along with the generation of desired product 3. Though it is not clear whether all or any particular step was facilitated by the ultrasound irradiation, the ultrasound might have played a role in the generation of anion E-2 (the deprotonation step), its reaction with E-1 (C—N bond formation step) and departure of leaving group from E-3 (the aromatization step). It is to be noted that the reaction also proceeded well in DMSO (Entry 3, Table 1) that unlike PEG 400 was not an H-bond forming agent. However, since dry DMSO was not used in the test reaction, the contaminated moisture might have played the role of activating agent via formation of H-bond with 2a thereby facilitating the reaction. This was further supported by the fact that the reaction of 1a with 2a in the presence of Cs₂CO₃ did not proceed well when performed in dry DMSO affording 3a in 38% yield.

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Scheme 3 Proposed reaction mechanism for the N − 1 heteroarylation of indoles (1) under ultrasound irradiation leading to 3.

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3.2 Pharmacology

The synthesized compounds were tested for their anti-cancer properties in vitro. The cells used for our in vitro studies include human chronic myeloid leukemia cells i.e. K562, human colon carcinoma cells i.e. Colo-205, and non-cancerous human embryonic kidney cells i.e. HEK293. The effect of test compounds on cell viability was measured using a colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after 24 h of treatment in culture medium containing PBS. The percentage inhibition of cell growth for compounds at 10 μM is presented in Table 3. It is evident from Table 3 that compound 3c, 3j and 3n showed promising activities against K562 cells whereas compound 3e, 3i and 3k showed moderate activities. Except the compound 3n none of these compounds showed significant activities against Colo-205 cells. These observations suggested that a substituent such as MeO, Cl, or F at C-5 position of the indole ring was beneficial for the activity against K562 cells among which MeO was found to be the best with the order MeO > Cl > F. Additionally, none of these compounds showed any effect when tested against non-cancerous HEK293 cells indicating their selectivity toward cancer cells especially leukemia. In the view of enormous importance in the identification of new antileukemic agents the present class of molecules is of further interest.

Table 3 In vitro antiproliferative properties of compounds 3 against cancer and non-cancerous cells.^a

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Compounds R1, R2, R3	% Inhibition of cell growth @ 10 μM
	K562 Leukemia	Colo-205 Colon	HEK293b non-cancerous
3a; H, H, H, CH	21.9 ± 1.12	17.1 ± 1.91	0.2
3b; 3-Me, H, H, CH	33.6 ± 1.20	18.6 ± 0.98	−0.2
3c; H, 5-MeO, H, CH	68.8 ± 1.31	20.9 ± 1.83	2.1
3d; H, 6-Me, H, CH	21.7 ± 2.33	21.5 ± 1.85	0.4
3e; H, 5-Cl, H, CH	41.5 ± 1.42	18.4 ± 2.11	−0.9
3f; 3-Me, H, 4-Me, CH	31.8 ± 3.09	13.1 ± 1.23	−2.6
3g; 3-Me, H, 5-Me, CH	30.0 ± 2.10	22.1 ± 2.01	1.8
3h; H, 5-Me, 5-Me, CH	36.2 ± 2.19	18.9 ± 3.03	0.5
3i; H, 5-Cl, 5-Me, CH	39.4 ± 4.35	20.4 ± 2.07	2.1
3j; H, 5-MeO, 5-Me, CH	68.7 ± 3.45	24.9 ± 1.99	1.3
3k; H, 5-F, 5-Me, CH	39.9 ± 2.21	19.2 ± 2.21	−0.7
3l; 3-Me, H, 6-Me, CH	25.3 ± 2.11	21.1 ± 1.15	1.9
3m; H, H, H, N	26.3 ± 3.10	10.9 ± 1.21	0.6
3n; H, 5-MeO, H, N	70.1 ± 2.40	37.5 ± 2.20	2.0
3o; H, 5-Br, H, N	28.5 ± 2.99	16.9 ± 1.84	−0.9

a Data represent the mean values of three independent determinations.

b HEK293 cell line was used as noncancerous cell line.