Design, synthesis and in vitro cytotoxicity studies of novel β-carbolinium bromides

Abstract

A series of novel β-carbolinium bromides has been synthesized from easily accessible β-carbolines and 1-aryl-2-bromoethanones. The newly synthesized compounds were evaluated for their in vitro anticancer activity. Among the synthesized derivatives, compounds 16l, 16o and 16s exhibited potent anticancer activity with IC₅₀ values of <10 μM against tested cancer cell lines. The most potent analogue 16l was broadly active against all the tested cancer cell lines (IC₅₀ = 3.16–7.93 μM). In order to test the mechanism of cell death, we exposed castration resistant prostate cancer cell line (C4–2) to compounds 16l and 16s, which resulted in increased levels of cleaved PARP1 and AO/EB staining, indicating that β-carbolinium salts induce apoptosis in these cells. Additionally, the most potent β-carbolines 16l and 16s were found to inhibit tubulin polymerization.

Introduction

β-Carboline unit containing natural products and synthetic molecules often exhibit a broad spectrum of pharmacological properties including sedative, anxiolytic, hypnotic, antioxidant, anticonvulsant, antitumor, antiviral, antiparasitic and antimicrobial.¹ Particularly, β-carboline analogues have been reported to exhibit significant antitumor activities against several human cancer cells (Fig. 1).^2–4 For example, natural β-carbolines Harmine (1) and Fascaplysin (2) exhibited interesting antiproliferative activity through apoptosis induction, DNA intercalation and inhibition of Cyclin Dependent Kinases.^5–10 Cao research group identified benzyl-β-carbolinium bromide 3 (IC₅₀ = 0.8–8 μM) and 4–5 (IC₅₀ = 0.4–2.7 μM) as potent cytotoxic agent by modification of natural Harmine.^11,12 In 2012, Frederick and co-workers prepared the trisubstituted Harmine derivative 6, with impressive anti-cancer activity (IC₅₀ = 0.7 μM; OE33).¹³

Fig. 1.

Representative β-carboline-based anticancer agents.

Among the various ways to enhance aqueous solubility, alkylation of azaheterocycles may lead to azolium salts with enhanced water solubility.^14,15 In the recent past, many cationic nitrogen heterocycles have been emerged as potent antitumor agents.^16,17 For example, Zhang group reported imidazoliumbromides as potent cytotoxic agents (IC₅₀ < 5.0 μM).¹⁸ Zeng et al. prepared 1-mesityl-3-(2-naphthoyl-methano)-1H-imidazo-lium bromide 7 (IC₅₀ = 0.3–5 μM) with interesting anticancer activity via arresting cell cycle at G1 phase and induced apoptosis in K562 cells.¹⁹ Recently, Xu group identified a series of novel 1-((indol-3-yl) methyl)–1H-imidazolium salts 8 (IC₅₀ = 1.89 μM; HL60) as apoptosis inducing potent anticancer agents.²⁰

In an effort to identify potent cytotoxic agents, recently we designed and prepared synthetic indolyl heterocycles with potent anticancer activities.^21–23 Inspired by the fascinating anticancer properties of β-carbolines and azolium salts, in this paper we designed a diverse series of β-carbolinium salts by incorporating remarkable features of β-carboline and 1-aryl-2-bromoethanones in single molecule as depicted in Fig. 2.

Fig. 2.

Rational design for β-carbolinium bromides.

Substituted β-carboline intermediates 13 were prepared from _L-tryptophan 9 as illustrated in Scheme 1.²⁴ Firstly, the reaction of 9 with formaldehyde solution (3.5 mL, 37%) under basic conditions produced tetrahydro β-carboline-3-carboxylic acid 10. However, under similar reaction conditions substituted tetrahydro β-carbo-line-3-carboxylic acid 11 could not be prepared. Alternatively, carboxylic acid 11 was prepared from the reaction of 9 with aliphatic or aromatic aldehydes under acidic conditions. Next, iodobenzene diacetate-mediated decarboxylative aromatization of 10 or 11 led to β-carbolines 12 in good yields. Reaction of 12 with an appropriate alkyl halide and sodium hydride produced N-alkylated β-carbolines 13 in excellent yields. On the other hand, required 1-aryl-2-bromoethanones 15 were prepared from the reaction of arylethanones 14 with N-bromo-succinimide (NBS) in acetonitrile using p-toluenesulfonic acid (PTSA) as a catalyst in good yields (Scheme 1).²⁵ Finally, the reaction of β-carboline 13 and 1-aryl-2-bromoethanone 15 was performed in refluxing ethanol. After refluxing the reaction contents for 20 h, we isolated the β-carboline salt 16 only in moderate yield (55%). In an attempt to enhance the reaction efficiency and product yield, we performed the alkylation of β-carboline 13 in focused microwave (MW). Reaction of 13 and 1-aryl-2-bromoethanone 15 in focused MW led to β-carbolinium bromide 16a with improved yield and notable reduced time (from hour to min.). MW-assisted organic synthesis has received substantial attention in pharmaceutical industry due to dramatic savings of reaction time and higher product yields.^26,27 Initially, we irradiated the reaction mixtures in MW oven at 50°C for 20 min and obtained 16a in 60% yield. Notably, by increasing reaction temperature from 50°C to 80°C, β-carbolinium bromide salt 16a was produced in 89% yield. The generality of identified reaction conditions was demonstrated by synthesizing an array of β-carbolinium bromides 16a-t in good to excellent yields (75–92%). 1-Aryl-2-bromoethanones with electron-donating (CH₃ and OCH₃) and electron-withdrawing (Br) groups smoothly delivered 16 in high yields.

Scheme 1.

Reagents and conditions: (a) 37% formaldehyde solution, 0.4 N NaOH, 37°C, 3 days, CH₃COOH, rt, 2 days; (b) RCHO, CH₃COOH, 100°C, 12 h; (c) IBD, DMF, rt, 2 h;(d) R¹X, NaH, DMF, rt, 12 h. (e) NBS, CH₃CN, PTSA, reflux, 4–5 h; (f) 13, EtOH, MW, 80°C, 20 min.

Structures of all the synthesized β-carbolinium bromides 16a-t were confirmed by IR, NMR (¹H & ¹³C) and mass spectral data. In ¹H NMR spectrum, two characteristic singlets appeared at ~9.5 and ~6.6 ppm due to C-1 proton of β-carboline and CH₂- of arylacyl moiety at N², respectively. ¹³C NMR of 16 showed a characteristic signal at ~190 ppm for the carbonyl carbon (C=O). A band at 1690 cm⁻¹ in IR spectra of 16 indicated the presence of C=O functional group. The purity of carbolinium bromides 16a–t was found to be greater than 97% by HPLC analysis.

In vitro cytotoxicity of β-carbolinium bromides 16a–t was evaluated against pancreatic (BxPC-3), cervical cancer (HeLa), castration-resistant prostate (C4–2), androgen-independent prostate (PC3), human embryonic kidney 293 (HEK293T) and breast carcinoma (MDA-MB-231) cells by MTT assay. Doxorubicin was used as the reference drug. Activity results in terms of IC₅₀ values are summarized in Table 1. Structure–activity relationship study was carried out by varying substituents on β-carboline (R and R¹¹) and 1-aryl-2-bromoethanone (Ar) moieties. Compound 16a without any substituent on β-carboline and arylacyl moieties was found to be moderately active against a panel of cancer cell lines (IC₅₀ = 21.6–74.9 μM). Replacement of phenyl with a p-tolyl ring in arylacyl part at N² led to 16b with slightly improved cytotoxicity (IC₅₀ = 18.4–31.9 μM, 16a vs 16b). Similarly, p-methoxyphenyl analogue 16c, slightly augmented the growth inhibitory potency when compared to 16a and 16b (IC₅₀ = 13.2–55.8 μM). Dimethoxyphenyl (16d) and trimethoxyphenyl (16e) derivatives were found to be weakly cytotoxic against kidney cells (IC₅₀ = 67.8 and 28.6 μM; HEK293T) and inactive against other cell lines. β-Carbo-line 16f with electron-withdrawing (p-bromophenyl) substituent displayed improved cytotoxicity (IC₅₀ = 18.8–44.1 μM) when compared to compound 16a. 2-Naphthyl analogue 16g showed improved activity with IC₅₀ values ranging 11.1–40.4 μM (16g vs 16a). Replacement of an aryl group with heteroaryl (furyl and thienyl) moiety led to compounds 16h-i with weak cytotoxicity or inactive against the tested cell lines except 16i with moderate cytotoxicity towards kidney (HEK293T) cells (IC₅₀ = 18.2 μM). N-Methylated derivatives 16j–k showed moderate cytotoxicity (IC₅₀ = 14–65 μM) towards tested cancer cell lines. N-(4-Chlorobenzyl) unit is reported to be beneficial for the potency of various indole-based anticancer lead molecules.^28,29 In an efforts to improve anticancer activity of the β-carbolinium bromides, we prepared N-(4-chloro-benzyl) β-carbolinium bromides 16l–n with significant enhanced cytotoxicity against the tested cancer cells compared to β-carbolines with free N-H. Notably, compound 16l found to be the most potent analogue of the series with broad cytotoxicity against all the tested cell lines (IC₅₀ 3.2–7.9 μM). Incorporation of methyl and p-methoxyphenyl substituents at position 1 (16o–s) of β-carboline further increased the growth inhibitory potency (IC₅₀ = 8.7–49.2 μM) when compared to 16c, 16e and 16g (IC₅₀ = 11.1–116.3 μM). N-Benzylation of 16q led to 16t with moderate cytotoxicity (IC₅₀ = 12.6–94.1 μM). Overall, most of the β-carbolinium bromides proved to be active against kidney cells (HEK293T, IC₅₀ = 3.7–67.8 μM). Activity results suggest that substituents at positions 1, 2 and 9 of β-carboline and arylacyl part bearing 4-methoxyphenyl and 2-naphthyl groups are beneficial for the anticancer activity. Notably, the most potent compound 16l with moderate water solubility (86 μg/mL) was found to be 95% stable at pH 4.5 up to 24 h.

Table 1.

In vitro cytotoxicity of β-carbolinium bromides 16a-t against a panel of cancer cells (IC₅₀ in μM).

Compd	R	R1	Ar	BxPC-3	HeLa	C4–2	PC-3	HEK293T	MDA-MB-231
16a	H	H	C6H5	27.8 ± 2.7	37.7 ± 3.4	74.9 ± 5.5	59.6 ± 5.3	21.6 ±2.8	37.2 ±4.1
16b	H	H	4-CH3C6H4	19.9 ± 2.3	27.5 ± 3.1	31.9±3.5	39.1 ± 3.1	18.4±4.1	29.2 ± 2.2
16c	H	H	4-CH3OC6H4	>100	48.7 ± 2.9	45.7 ± 5.4	55.8 ± 4.9	13.2±2.6	>100
16d	H	H	3,4-(CH3O)2C6H3	>100	>100	>100	>100	67.8 ± 7.9	>100
16e	H	H	3,4,5-(CH3O)3C6H2	>100	>100	>100	>100	28.6 ± 3.8	>100
16f	H	H	4-BrC6H4	24.5 ± 3.9	44.5 ± 5.0	33.3 ± 2.9	43.2 ± 5.2	18.8±3.1	44.1 ± 5.3
16g	H	H	2-Naphthyl	15.1 ±2.4	35.2 ± 2.6	40.4±3.9	35.8 ± 3.0	11.1 ±2.2	25.1 ± 3.6
16h	H	H	2-Furyl	>100	>100	>100	>100	>100	>100
16i	H	H	2-Thienyl	38.8 ± 4.9	65.0 ± 4.6	>100	>100	18.2±2.6	48.9 ± 5.8
16j	H	CH3	4-CH3OC6H4	18.0±2.1	55.6 ± 5.4	37.9 ± 4.3	35.2 ± 4.1	14.2±2.8	38.1 ± 2.9
16k	H	CH3	2-Naphthyl	64.9 ± 4.3	33 ± 3.2	25.4±2.2	43.8 ± 3.9	17.6±3.0	54.2 ± 5.4
161	H	4-ClC6H4CH2	4-CH3OC6H4	6.3 ± 1.0	3.2 ± 0.9	7.4 ± 1.2	5.4 ± 0.8	3.8 ± 1.1	7.9 ± 1.1
16m	H	4-ClC6H4CH2	3,4,5-(CH3O)3C6H2	35.3 ± 2.9	13.16 ± 2.5	41.0 ± 3.8	36.5 ± 3.4	11.5±3.1	37.0 ± 4.0
16n	H	4-ClC6H4CH2	2-Naphthyl	36.9 ± 3.6	14.2±2.2	11.6±2.1	15.2 ± 2.7	10.6±2.8	16.2±1.8
16o	CH3	H	4-CH3OC6H4	26.6 ±1.8	23.9 ± 2.9	28.1 ± 2.6	26.7 ± 2.0	9.5 ± 1.1	16.8±3.1
16p	CH3	H	3,4,5-(CH3O)3C6H2	30.9 ± 3.2	54 ± 6.2	49.2 ± 3.4	39.5 ± 3.3	18.9±2.0	40.2 ± 5.2
16q	CH3	H	2-Naphthyl	12.3±2.0	13±2.6	17.7±1.9	19.5±2.1	17.7±2.8	14.1 ± 2.1
16r	4-CH3OC6H4	H	4-CH3OC6H4	20.0 ± 2.5	26.4 ± 1.4	22.1 ± 2.9	28.1 ± 3.4	17.8±2.0	22.4 ± 2.7
16s	4-CH3OC6H4	H	2-Naphthyl	12.2±1.6	15.5±2.0	8.7 ± 1.5	10.1 ±1.1	11.6±1.9	14.1 ± 2.1
16t	CH3	C6H4CH2	2-Naphthyl	94.1 ± 6.4	37.6 ± 2.4	12.6±2.3	15.3±1.4	29.0 ± 4.2	44.1 ± 6.3
Doxorubicin	14.3	4.85	2.5	14.3±2.3	4.8 ± 1.2	2.5 ± 0.8

The activity data represent mean IC₅₀ values of experiments conducted in triplicates.

To determine the preliminary mechanism of action of β-carbolinium bromides, PARP1 cleavage assay for the active compounds 16l and 16s was performed. C4–2 Cells were treated with 16l and 16s for 48 h, and cleaved PARP1 levels was analyzed using immunoblotting. As shown in Fig. 3, exposure of C4–2 cells by either 16l or 16s enhanced the levels of PARP1 cleavage as indicating apoptotic induced cell death in C4–2 cells.

Fig. 3.

PARP1 cleavage (in C4–2 cells) induced by 16l and 16s.

Furthermore, we also conducted acridine orange/ethidium bromide assay to investigate the mechanism of cell death. Acridine orange (AO) stains both live and dead cells, whereas ethidium bromide (EB) only stains dead cells.³⁰ Therefore, AO/EB staining is used to examine whether cell death occurred via apoptosis or necrosis. Effect of β-carbolinium salts on the morphological changes of C4–2 cells is illustrated in Fig. 4. Incubation of compounds 16s (IC₅₀ = 8 μM) and 16l with C4–2 cells (IC₅₀ = 7 μM) for 24 h resulted in typical nuclear fragmentation (red), whereas no visible changes in cell nucleus and cell membrane integrity was observed for the control cells. The results of AO/EB staining revealed that compounds 16l and 16s trigger apoptosis in C4–2 cells, thereby confirming the PARP cleavage data.

Fig. 4.

Apoptosis inducing effects of 16l and 16s in C4–2 cells.

In the recent past, indole-based compounds have been reported for their significant anticancer activity through modulation of tubulin-heterodimer dynamics and binding at colchicine binding sites.³¹ In order to find the theoretical binding sites of novel β-carboline bromides, a docking study for the identified potent compounds 16l and 16s was performed by Molegro Virtual docker program³² according to reported high-resolution crystal structure of the tubulin-DAMA-colchicine (CN-2) complex (PDB ID: 1SA0).³³ Scoring functions and hydrogen bond formed with the surrounding amino acids predicted the binding affinities for 16l and 16s with MolDock Scores of −151.18 and −167.90, respectively. The docking poses of the molecule with the receptor are detailed in Fig. 5. Binding interactions for 16l are strongly stabilized by two hydrogen bonds; first interaction between C=O and Asp251 with hydrogen bond distance of 3.0616 Å; the second one between oxygen of 4-methoxyphenyl group and Cys241 with the bond length of 2.6103 Å. Similarly, compound 16s also showed hydrogen-bonding interactions between the oxygen of 1-(4-methoxyphenyl)-β-carboline and Cys241 with 3.207 Å distance of hydrogen bond. Apart from hydrogen bonding interactions, compounds 16l and 16s also strongly stabilized by steric interactions (red dotted lines) as illustrated in Fig. 5. Additionally, the hydrophobic interactions in the colchicine-binding domain of the tubulin for β-carbolines 16l and 16s are shown in Fig. 6.

Fig. 5.

Binding interactions of 16l and 16s in the colchicine-binding site of tubulin. Hydrogen bonds (green and blue dotted lines) and steric interactions (red dotted lines) are outlined.

Fig. 6.

Hydrophobicity effect of 16l and 16s in the binding pocket of colchicine.

To validate the theoretical molecular-docking hypothesis, we examined the tubulin polymerization activity for the identified two potent compounds 16l and 16s in a cell free system. β-Carbolinium bromides 16l and 16s were found to inhibit tubulin polymerization at 6 μM as shown in Fig. 7.

Fig. 7.

Effect of compounds 16l and 16s on in vitro tubulin polymerization.

In summary, a series of β-carbolinium bromides 16a–t were prepared from easily accessible β-carbolines and 1-aryl-2-bromoethanones using MW irradiation. In vitro anticancer activities of β-carbolinium bromides 16a–t were evaluated against six-human tumor cell lines. β-Carboline 16l displayed most potent cytotoxicity against the tested cancer cell lines with IC₅₀ values ranging 3.16–7.93 μM. The preliminary mechanism of action study revealed that compounds 16l and 16s induced apoptotic cell death by enhancing the level of cleaved PARP1 in C4–2 cells and exhibited their activity through the inhibition of tubulin polymerization. This class of compounds can be further exploited for obtaining highly potent cytotoxic compounds.