Synthesis and anticancer activity studies of indolylisoxazoline analogues

Abstract

A new library of thirteen indolylisoxazolines 6a–m has been synthesized by the treatment of indolylchalcones with hydroxylamine hydrochloride. Evaluation of anticancer activity of indolylisoxazolines 6a–m led to the identification of potent compounds 6c–d, 6i and 6l, with IC₅₀ ranging 2.5–5.0 μM against the tested cancer cell lines. Using a number of complementary techniques such as acridine orange/ethidium bromide staining, PARP1 cleavage and DNA strand breaks assay, we show that the compounds 6c and 6i induce apoptosis in highly aggressive C4–2 cells. Our data further revealed that 6c and 6i inhibited C4–2 cells proliferation without inducing reactive oxygen species (ROS). Finally, we show that compounds 6c and 6i also potently inhibit cell migration, indicating these compounds have the potential to serve as effective anti-cancer agents.

Indole scaffold is frequently present in many important synthetic and natural drug molecules including anticancer, anti-oxidants, anti-inflammatory, analgesics and anti-pyretic drugs.^1,2 Greater versatility and biodiversity of indole nucleus makes it a highly privileged motif for target-based drug design and development of anticancer agents. In the last decade, importance of indole motif in the anticancer drug development is reflected by the identification of many indole-based natural and synthetic anticancer agents with distinct mechanism of actions.^3–5 Among the indole-based compounds, indolylazoles containing five- and six-membered heterocycles linked indole have received greater attention due to their unique properties such as stability and hydrophilic nature, which improves aqueous solubility and thus simplify the formulation for in vivo uses (Fig. 1).⁶ 5-(3′-Indolyl) oxazoles isolated from different micro-organisms are reported to display interesting biological activities. For example, Labradorins 1 and 2 were found to be potential inhibitors (GI₅₀ = 9.6–9.8 μg/mL) of lung cancer cell line.^7,8 Inspired by naturally occurring indolylazoles, we identified 5-(3-indolyl)-1,3,4-oxadiazoles 2 and indolyl-1,2,4-triazoles 3 as potent anticancer agents.⁹ Some of the indolylazoles 3 are reported to show selective cytotoxicity against prostate cancer cell line (IC₅₀ = 0.8 μM) and found to inhibits tubulin polymerization.⁵ Compound A–289099 (4) with oxazoline ring was recognized as an orally active antimitotic agent with most promising anticancer property (IC₅₀ = 6.2 nM) against NCI-H460 cells.¹⁰

Fig. 1.

Representative examples of cytotoxic indolyl(aryl)azoles.

In recent years, accumulating evidences have revealed that isoxazolines possess important biological properties such as anticancer, antimicrobial, fungicidal, anticonvulsant, anti-inflammatory.¹¹ Some of the isoxazolines containing molecules have been found to elicit interesting anticancer activities with improved pharmacokinetics profile. For example, diaryl analogues 5 demonstrated potent cytotoxic activity by blocking most of the cancer cells in G2 phase.¹² Isoxazoline linked dihydro-quinazolinones significantly reduced the growth of cancer cell lines, and disrupted tubulin polymerization.¹³

As a part of our search for novel anticancer agents, in the present study, we designed indole-based heterocycles by linking indole and (hetero) aryl moieties through stable isoxazoline scaffold found in many biologically active molecules and drugs.^14–17 Many anticancer agents with substantial cytotoxicity have been prepared by replacing alkenyl bond of Combrastatin with isoxazoline moiety.¹⁰ With the broad spectrum activities observed for indole derivatives and isoxazolines, we envisioned linking two heterocyclic units in single molecule that could lead to compound with improved efficacy and selectivity.

Indolylisoxazolines 6a–m were synthesized from the reaction of appropriate indolylchalcones 9a–m with hydroxylamine hydro-chloride in ethanol under refluxing conditions as outlined in Scheme 1. First, substituted indole-3-carboxaldehydes 8 were achieved from Vilsmeier-Haack formylation of commercially available indoles, followed by the N-alkylation of 7 with an appropriate alkyl halide in the presence of sodium hydride. This afforded substituted indole-3-carboxaldehydes 8 in excellent yields by incorporating necessary modifications in reported procedures.¹⁸ Next, condensation reaction of aldehyde 8 with diverse acetophenones under basic conditions led to indolyl chalcones 9a–m in good to excellent yields. Finally, treatment of 9a–m with hydroxylamine hydrochloride in the presence of a catalytic amount of anhydrous sodium sulfate in ethanol afforded indolylisoxazolines 6a–m in 60–80% yields.

Scheme 1. Synthesis of indolylisoxazolines 6a–m.

The prepared indolylisoxazolines 6a–m was fully characterized using IR, NMR (¹H & ¹³C) and mass spectral data. In ¹H NMR spectra of 6a–m revealed the appearance of a triplet at δ ~ 6.10 ppm (methyne proton of isoxazoline) in addition to two doublet of doublet at δ ~ 3.82 ppm and 3.65 ppm (methylene protons of isoxazoline). The ¹³C NMR spectra of 6a–m exhibited two characteristic signals at δ ~ 40 ppm and ~ 80 ppm assigned to methyne and methylene carbons of isoxazoline ring. The HRMS spectra of isoxazolines 6a–m showed molecular ion peak in agreement with the calculated mass. Based on HPLC analysis, purity of the synthesized compounds 6a–m was found to be greater than 97%.

Synthesized indolylisoxazolines 6a–m were screened for in vitro cytotoxicity against six human cancer cell lines: castration-resistant prostate cancer cell lines (22Rv1 & C4–2), PTEN−/− prostate cancer cell line (PC3), human embryonic kidney cell line (HEK293), pancreatic cancer cell line (BxPC3) and triple negative breast cancer cell line (MDA-MB-231) using the MTT assay. The IC₅₀ values (in μM) were used to determine the growth inhibition in the presence of indolylisoxazolines 6a–m against the tested cancer cell lines. Doxorubicin was used as a positive control. In our previous report, 3,4,5-trimethoxyphenyl and 4-pyridyl bearing chalcones were found to be potent against a panel of cancer cells.^18,19 In light of favourable effects observed with these derivatives on antitumor activity, isoxazolines 6a–i with similar structural motifs were prepared.¹⁸ Compound 6a without any substituent on indole ring was found to be moderately active against the tested cancer cell lines. Next, introduction of a methoxy group at position-5 of indole ring resulted in analogue 6b with reduced activity. Interestingly, by shifting the methoxy group from position-5 to position-6 in indole ring led to 6c (IC₅₀ = 3.5–16.5 μM) with substantial improvement in cytotoxicity. Replacement of the methoxy group with a fluorine atom was tolerated (MeO: 6c IC₅₀ = 3.5–16 μM vs F: 6d IC₅₀ = 3.66–5 μM). Insertion of a methyl group at position-2 of indole ring (compound 6e) displayed moderate to good cytotoxicity. Recently, chlorobenzylindole and pyridyl motifs have been reported to be beneficial for the cytotoxicity.⁴ By incorporating these important structural units, we prepared isoxazolines 6f–i with cytotoxicity in micromolar range. The analogue 6i with (4-chlorobenzyl)-2-methylindole and pyridyl moieties was found to be the most potent compound with IC₅₀ of 2.5 μM against C4–2 and HEK293 cell lines. In subsequent modifications, we prepared derivatives 6l and 6 m possessing 3,4,5-trimethoxyphenyl fragment which is vital in many anticancer agents, especially in various tubulin binding agents.¹⁸ Compound 6l with indole and 3,4,5-trimethoxyphenyl moieties displayed significant cytotoxicity (IC₅₀ ~ 4.5 μM). Incorporation of C5-methoxy in indole ring (compound 6m) or alteration of any methoxy group in trimethoxyphenyl fragment (compounds 6j & 6k) are detrimental for the activity (Table 1).

Table 1.

In vitro anticancer activity studies of indolylisoxazolines 6a–m (IC₅₀ μM).

Compd	Rl	R2	R3	R4	Ar	22Rv1	C4-2	HEK293	BxPC-3	MDAMB-231	PC3
6a	H	H	H	H	4-pyridyl	> 100	12.75	> 100	30.5	19.66	> 100
6b	H	H	OMe	H	4-pyridyl	35.5	> 100	> 100	68.5	> 100	> 100
6c	H	H	H	OMe	4-pyridyl	16.32	3.5	4.16	9.5	7.38	10.45
6d	H	H	H	F	4-pyridyl	3.66	8	9.5	9.5	5	12.9
6e	H	CH3	H	H	4-pyridyl	17.33	45.1	> 100	95.5	26.6	21.2
6f	4-ClBz	H	H	H	4-pyridyl	9.6	11.42	10.16	> 100	13.66	25.69
6g	4-ClBz	H	OMe	H	4-pyridyl	10.6	> 100	14.16	> 100	26	55.21
6h	4-ClBz	H	H	OMe	4-pyridyl	17.3	> 100	37.16	> 100	> 100	95.1
6i	4-ClBz	CH3	H	H	4-pyridyl	8.33	2.5	2.5	13.5	6.66	11.2
6j	H	H	H	H	3,4-(OCH2O)C6H3	> 100	15.81	> 100	27	26.6	> 100
6k	H	H	H	H	3,4-(OCH3)2C6H3	16.66	65.5	> 100	> 100	> 100	70.14
6l	H	H	H	H	3,4,5-(OCH3)3C6H2	4.51	5	5	13.5	11.66	15.5
6m	H	H	OMe	H	3,4,5-(OCH3)3C6H2	11.33	71.2	45.4	> 100	26	42.58
Doxorubicin						6.32	3.25	3.9	9.12	6.25	9.58

^*The activity data represent mean values of experiments conducted in triplicates at three independent times.

Structure-activity relationship studies of the prepared indolylisoxazolines 6a–m suggested that isoxazolines substituted as 6-methoxyindole, 6-fluoroindole, N-chlorobenzyl-2-methylindole and indole at position-5, and at position-3 with pyridyl and trimethoxyphenyl are favourable for the cytotoxicity (Fig. 2).

Fig. 2.

Identified potent indolylisoxazolines.

To investigate the mechanism of cell death induced by these compounds, we conducted various assays. Acridine orange (AO) stains both live and dead cells, whereas ethidium bromide (EB) stains only dead cells. Therefore, AO/EB staining was used to examine whether cell death occur via apoptosis or necrosis. Effect of indolylisoxazolines on the morphological changes of C4–2 cells is illustrated in Fig 3A. Incubation of compounds 6i (IC₅₀ = 2.5 μM) and 6c with C4–2 cells (IC₅₀ = 3.5 μM) for 48 h resulted in typical nuclear fragmentation (red), whereas no visible changes in cell nucleus and cell integrity was observed for the control cells. The results of AO/EB staining revealed the compounds 6c and 6i triggered apoptosis in C4–2 cells.²⁰

Fig. 3.

Apoptosis inducing effects of 6c and 6i in C4–2 cells.

PARP is a eukaryotic DNA-binding protein that specifically recognizes the single stranded breaks formed during DNA damage. Once PARP detects the single stranded breaks it binds to the DNA, undergoes the structural change and begins the synthesis of a polymeric adenosine diphosphate ribose (poly (ADP – ribose) or PAR) chain which acts as a signal for the other DNA-repairing enzymes.¹⁴ PARP1 cleavage assay (Fig. 3B) shows that indolylisoxazolines 6c and 6i induced apoptosis in C4–2 cells. C4–2 cells were treated with compounds 6c and 6i and level of cleaved PARP was observed. The results revealing the possible occurrence of DNA damage and apoptosis.¹⁰

DNA fragmentation is a key feature of apoptosis, a type of programmed cell death. Apoptosis is characterized by the activation of caspase-3 activated DNase (CAD).²⁰ CAD is normally inhibited by the protein, inhibitor of Caspase Activated DNase (ICAD). During apoptosis, the apoptotic effector caspase, caspase-3, cleaves ICAD and causes CAD to become activated.²¹ Thus, we conducted DNA ladder assay²² to verify that the cell death triggered by compounds 6c and 6i was indeed apoptosis. As shown in Fig. 3C, treatment of C4–2 cells with compounds 6c and 6i at 10 μM concentration for 48 h, triggered the formation of DNA ladder, which confirms that these compounds induce apoptosis.

Previous studies have shown that compounds bearing indole scaf-folds can induce cell death by increasing reactive oxygen species (ROS). ROS can include peroxides, superoxides, hydroxyl radicals and singlet oxygen. ROS are formed as a natural byproduct of the oxygen metabolism. Therefore, we measured whether compounds 6c and 6i induce ROS accumulation using DCFDA staining in C4–2 cells. H₂O₂-treated C4–2 cells were used as a positive control. ROS levels were quantified in untreated and C4–2 cells treated with H₂O₂ (10 μM), 6c (10 μM) or 6i (10 μM) for 48 h. While H₂O₂-treated cells displayed high DCFDA staining, 6c and 6i-treated cells shows similar DCFDA staining, indicating that these compounds do not induce apoptosis via ROS upregulation (Fig. 4).

Fig. 4.

Quantification of ROS levels induced by 6c and 6i.

In solid cancers, metastasis is responsible for 90% of mortality. Therefore, we investigated whether these compounds inhibit cell migration. Wound healing assay is a classical and commonly used method for studying cell migration, which mimics wound healing in vivo.²³ In this assay, a scratch is introduced in a monolayer of cells and then images are taken at the beginning and at regular intervals to access cell migration rates. After cells were wounded, they were further incubated for 12 h, 24 h and 48 h. The cells were fixed and images were captured, which showed that both compounds 6c and 6i potently inhibit C4–2 cell migration (Fig. 5). These results indicate that compounds 6c and 6i have the potential to serve as effective anti-cancer agents.

Fig. 5.

Inhibition of cell migration on C4–2 cells effected by 6c and 6i.

In summary, we have prepared indolylisoxazolines and some of them showed high potency in inhibiting the growth of 22Rv1, C4–2, PC3, HEK293, BxPC-3, and MDA-MB-231 cancer cell Unes at micromolar concentrations. Compounds 6c–d, 6i and 6l were significantly cytotoxic against all six cancer cell lines with selectivity towards C4–2 and HEK293 cells. Importantly, two of the tested compounds 6c and 6i also exhibited strong anti-cell motility property in C4–2 cells. The preliminary anticancer activity studies of indolylisoxazolines revealed that CH₃ group at second position of the indole ring, protection of indole nitrogen with chlorobenzyl group and insertion of OMe group at sixth position of indole ring are beneficial for anticancer activity and selectivity. Identified indolylisoxazolines are potent anticancer agents and could be explored for specific biological effects at molecular level.