Synthesis and Biological Evaluation of FICZ Analogues as Agonists of Aryl Hydrocarbon Receptor

Abstract

The Aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor involved in multiple biological processes including immune cell differentiation, intestinal function and inflammation. Based on the scaffold of naturally occurring AhR ligand 6-formylindolo (3,2-b) carbazole (FICZ, 2), a series of analogues has been designed, synthesized and evaluated by cell-based assays. The structure-activity relationships study has successfully led to the discovery of compound 11e with extremely potent activity.

Graphical Abstract

Aryl hydrocarbon receptor (AhR) is a well conserved helix-loop-helix transcription factor integrating dietary, microbial and metabolic cues to propagate signal transmission in a cell- and ligand-specific manner.¹ A variety of endogenous and exogenous ligands such as those in Figure 1 can activate AhR and induce the nuclear translocation of the ligand bound AhR. Within the nucleus, AhR-ligand complex heterodimerizes with aryl hydrocarbon receptor translocator (ARNT). The resulting heterodimer then binds to a consensus DNA sequence.^{2, 3} This triggers the expression of a number of genes, such as CYP1A1, CYP1A2, CYP1B1 and COX-2.⁴

Figure 1.

Representative AhR agonists.

The complexity of AhR cellular signaling networks and its crosstalk with other transcription factors present the major challenge in elucidating the physiological role of AhR. Nevertheless, accumulating evidence indicate that AhR has many important physiological functions in immune system, nerve system, metabolic regulation, host-microbiome interactions, and cancer progression,⁵ in addition to being involved in TCDD (1)-mediated toxicity.⁶ Beyond transcriptional regulation, AhR has also been reported as a ligand-dependent E3 ubiquitin ligase.^{7, 8} Selective AhR ligands not only can serve as molecular tools to elucidate the functions of AhR, but also have potential therapeutic benefits for a diverse range of diseases.^9–11

For example, TCDD (1) exposure was associated with a decreased risk of benign prostate hyperplasia (BPH).^{12, 13} It has been proposed that AhR agonists may have protective effect against BPH.¹⁴ However, the toxicity of TCDD (1) prevents its further development as potential therapeutics for the treatment of BPH. On the other hand, the tryptophan derivative 6-formylindolo[3,2-b]carbazole (FICZ, 2) is a potent endogenous ligand of AhR.^{10, 15, 16} To date, only limited studies have been reported on the structure and activity relationship (SAR) of analogues of FICZ (2).^17–21

We previously reported an efficient Pt-catalyzed tandem annulation method for the synthesis of various highly substituted 2,3’-diindolylmethanes.²¹ Based on this method, a collection of analogues including natural product malassezin were prepared as the agonists of AhR.²² The most potent malassezin analogue had an EC₅₀ of 55 nM. Herein, we report our studies on the SAR of FICZ (2), one of the endogenous ligands of AhR, and the discovery of more potent FICZ analogues as AhR agonists.

As illustrated in Scheme 1, using our Pt-catalyzed indole arylation method,²¹ we could easily introduce various R¹ substituent to the diindolylmethane products 8. Removal of the Boc-protecting group, acylation of diindolylmethane, followed by acid mediated cyclization produced indolo[3,2-b]carbazole 9. A nucleophilic substitution (S_N2) reaction between carbazole 9 and various alkyl halides furnished R² substituted esters, which were further converted to analogues of FICZ (2) following a sequence of reduction and oxidation.²³

Scheme1.

Synthesis of analogues of FICZ (2).

At first, we prepared alcohol 10f (R¹ = R² = H) to test its AhR activation with the well-documented ethoxyresorufin-O-deethylase (EROD) assay at three concentrations of compounds (0.13 nM, 3.2 nM and 80 nM).²⁴ Compared to known AhR agonist BNF (4), 10f is 5 fold and 12 fold more potent at concentrations of 0.13 nM and 3.2 nM, respectively.²⁵ At the maximum tested concentration of 80 nM, up to 59 fold increase over DMSO treatment was observed, while 11 fold increase was induced by BNF (4). In compounds 10a-e, the R² position was fixed with unsubstituted indole and a number of different substituents were introduced to the R¹ position. Changing the hydrogen to methyl group generated compound 10a, which displayed more potent activity than compound 10f at all three concentrations. When the hydrogen was replaced with ethyl group (10b), the stimulation of AhR activity was significantly decreased. Relatively bulky groups such as propyl group (10c), cyclopropyl methyl group (10d), and benzyl group (10e) could not be tolerated as these compounds had very minimal effect on the activity of AhR in the EROD assay.

Given the improved AhR activity of compound 10a, the SAR of this compound was further examined through the introduction of different substituents to R² position, while the methyl group in R¹ position was kept fixed. Our results indicate that the hydrogen in R² position is critical for activating AhR as all analogues 11a-d showed decreased potency compared to compound 10a. It is also worth noting that the corresponding primary alcohols of compounds 11a-d are not stable enough to be isolated and the reason is not clear. Therefore, we are not able to compare the activity of the corresponding alcohols of 11a-d with 10a, which has an alcohol on the R³ position.

To further evaluate the importance of aldehyde group on the R³ position, 10a was oxidized to furnish aldehyde 11e, which was 5 fold, 3 fold and 1.3 fold more potent at 0.13 nM, 3.2 nM and 80 nM concentrations, respectively, than 10a. We then performed the full dose response experiment for compound 11e to evaluate its AhR activity as shown in Figure 2. FICZ (2) was used as the positive control for comparison. While both FICZ (2) and 11e were effective in inducing CYP1A1 expression in a dose-dependent manner upon a 24 h treatment, compound 11e had EC₅₀ values of 0.26 nM and was 8 times more potent than FICZ (2). In fact, compound 11e could activate AhR at < 30 pM and achieve its maximal effects at around 3 nM in the EROD assay.

Figure 2.

Dose response study of compound 11e and FICZ (2).

To further confirm that analogues of FICZ (2) are inducers of monooxygenase enzyme CYP1A1 gene expression, compound 11e was selected for evaluating its ability to induce the mRNA level of CYP1A1 in HepG2 cell line using quantitative reverse transcription PCR (qRT-PCR). After treating the cell with 100 nM of 11e for 6 h, we observed a signficant increase of CYP1A1 mRNA level, while no effect on the mRNA of AhR was observed (Figure 3, GADPH was used as the control). Western blotting analysis showed that compound 11e, at concentrations as low as 3–10 nM, was effective in incresing the level of CYP1A1 protein in HepG2 cells, whereas BNF (4), a known AhR agonist, only slightly inceread the CYP1A1 protein level at 300 nM concentration (Figure 4). The up-regulation of CYP1A1 protein level by 11e was also dose-dependent.

Figure 3.

AhR and CYP1A1 mRNA level after treatment of 11e for 6 h.

Figure 4.

Dose-response of CYP1A1 protein level change after 24 h treatment.

In summary, we have prepared a series of indolo[3,2-b]carbazole derivatives as analogues of FICZ (2) and studied their AhR agonistic activity. Through systemic SAR optimization based on our previously developed synthetic method, compound 11e was identified as the most potent AhR agonist with a EC₅₀ value of 0.26 nM. Further mechanistic investigations confirmed that 11e effectively induced the CYP1A1 gene expression and protein up-regulation, which is a major outcome of AhR activation. Further optimization of compound 11e for disease-relevent in-vivo studies is ongoing and will be reported in due course.

Table 1.

The AhR activity of FICZ (2) analogues.^a

Comp	R1	R2	R3	EROD Activity (fold) at 3 concentrations
				0.13 nM	3.2 nM	80 nM
BNF (4)				0.8	1.1	11.2
10a		H	CH2OH	5.3	26.7	62.6
10b		H	CH2OH	1.4	3.5	31.8
10c		H	CH2OH	0.4	0.5	3.4
10d		H	CH2OH	0.3	0.5	2.6
10e		H	CH2OH	0.4	0.7	3.6
10f	H	H	CH2OH	3.8	13.4	59.2
11a			CHO	1.6	3.4	39.4
11b			CHO	1.4	4.5	25.1
11c			CHO	1.3	1.9	8.9
11d			CHO	1.0	4.2	27.5
11e		H	CHO	27.3	88.9	82.4
FICZ (2)	H	H	CHO	7.0	59.7	85.1

^aActivity fold was determined by EROD assay. HepG cells were treated with listed compounds or vehicle at three different concentrations (0.13nM, 3.2 nM, 80 nM) respectively for 24 h.