Iodine-catalyzed electrophilic substitution of indoles: Synthesis of (un)symmetrical diindolylmethanes with a quaternary carbon center

A novel, versatile approach for the synthesis of unsymmetrical 3,3'-diindolylmethanes (DIMs) with a quaternary carbon center has been developed via iodine-catalyzed coupling of trifluoromethyl(indolyl)phenylmethanols with indoles. In contrast to previously reported methods, the new procedure is characterized by chemoselectivity, mild conditions, high yields, and scalability to obtain gram amounts for biological studies. Selected compounds were found to display affinity for cannabinoid receptors, which are promising drug targets for the treatment of inflammatory and neurodegenerative diseases.


Introduction
Diindolylmethanes (DIMs) represent an important class of indole alkaloids, that are constituents of pharmaceuticals [1][2][3][4][5][6][7] and agrochemicals [8,9]. DIM derivatives possess a variety of biological activities (Figure 1) [10]. Unsubstituted DIM (I), for example, exhibits antimicrobial [5], anticancer [11][12][13], and anti-inflammatory effects ( Figure 1) [14]. There is preclinical evidence for activity against several types of cancer [15], and DIM has been clinically evaluated for the treatment of prostate cancer [16] and showed promise for the treatment of cervical dysplasia [17]. The related trisindoline (II) was reported to possess antibiotic activity [18], while DIM derivatives III and IV also showed anticancer activities (Figure 1). Owing to their exciting biological activities, DIM derivatives have recently received increasing attention from synthetic organic chemists, biologists, and pharmacologists. In general, DIMs can be synthesized via electrophilic substitution of indoles by aldehydes or ketones in the presence of conventional Lewis or Brønsted acids as catalysts [19]. This strategy is straightforward, but it always only provides symmetrical DIMs. The synthesis of unsymmetrical DIM derivatives, however, remains challenging, and merely sporadic examples are reported in literature [20,21].
Methods for the introduction of fluorinated groups into organic molecules are of high interest due to fluorine's unique physical and chemical properties, such as its small size, high electronegativity, and high C-F bond dissociation energy [22][23][24]. Organofluoro compounds developed as drug molecules often display increased metabolic stability and bioavailability compared to non-fluorinated analogs [25]. Considering the evergrowing demand for organofluorine compounds, the development of new methodologies that allow the incorporation of fluorine atoms into bioactive molecules is highly desired and will also be addressed herein.
Sasaki et al. reported the reaction of trifluoromethyl(indolyl)phenylmethanols with indoles in the presence of trifluoroacetic acid (TFA) and CHCl 3 ( Figure 2) [36]. Very recently, Ling et al. reported the same reaction in the presence of Ga(OTf) 3 in acetonitrile ( Figure 2) [37]. Although these methods are certainly useful, they have several undeniable drawbacks, including the use of heavy-metal catalysts and the necessity of employing indoles bearing bulky substituents at their 2-position (Ling et al.), or the need for chlorinated solvents (Sasaki et al.), as well as difficulty to scale up the reactions to a multigram scale, as well as a generally rather limited substrate scope. Therefore, finding a robust method with a broad substrate scope and functional group tolerance is highly desirable.
As part of our continuous efforts to prepare biologically active DIM derivatives [38], we herein report an innovative approach to synthesize unsymmetrical 3,3'-diindolylmethanes (DIMs) with a fluoromethyl-containing quaternary carbon center via an iodine-catalyzed coupling reaction of trifluoromethyl-(indolyl)phenylmethanol with indole derivatives. This method has also been extended to the synthesis of pentafluoro-ethylated and heptafluoro-propylated DIMs in excellent yields. Selected compounds were evaluated in radioligand binding studies for their affinities towards cannabinoid CB 1 and CB 2 receptors.

Results and Discussion
Optimization of the reaction The reaction conditions were optimized using 2,2,2-trifluoro-1-(5-methoxy-1H-indol-3-yl)-1-phenylethan-1-ol (1a, 5 mmol) and 1H-indole (2a, 5 mmol) as model substrates (Table 1). At first, the reaction was attempted in trifluoroethanol (TFE), and water, respectively, as these solvents had been utilized for the preparation of unsymmetrical DIMs from (1H-indol-3-yl)(phenyl)methanol by Xiao and co-workers [39,40]. However, no product was formed in either solvent even at high temperatures ( Table 1, entries 1-3). This is likely due to the steric hindrance of the CF 3 -substituted quaternary carbon atom in substrate 1a. Therefore, the solvent was changed to H 2 SO 4 (5%) in water (Table 1, entry 4) or glacial acetic acid (entry 5), and the reactions were performed at room temperature. While no reaction occurred in 5% H 2 SO 4 , traces of product were observed in acetic acid (entry 5). Therefore, the reaction mixture was gradually heated to 50 °C (Table 1, entry 6), 80 °C (entry 7), and  100 °C (entry 8). To our delight, the formation of the expected product was steadily increased to 32, 47, and 56%, respectively. Nevertheless, it was not possible to further increase the yield of the product using this solvent.
We further extended this protocol to the preparation of unsymmetrical pentafluoroethylated and heptafluoropropylated DIM derivatives ( Table 4). The (indol-3-yl)phenylmethanol derivative bearing a pentafluoroethyl residue (1j) was efficiently reacted with a series of indole derivatives (2d, 2e, 2h, 2j, and Table 4: Substrate scope of the reaction of 1j-l with trifluoromethyl(indolyl)phenylmethanols 1: modification of the trifluoromethyl group.
Compound 3e showed similar binding affinities at both CB 1 and CB 2 receptor. Therefore, it was selected to determine and compare its functional activity at both receptor subtypes. Compound 3ad was selected due to its high CB 2 receptor affinity and selectivity. It is well known that CB 1 receptors exhibit high constitutive activity [41]. Compound 3e reduced the basal activity of CB 1 receptors (Supporting Information File 1, Figure  S2A) but not that of CB 2 receptors indicating that this compound acts as an inverse agonist (EC 50 0.786 ± 0.233 µM) at CB 1 receptors (Supporting Information File 1, Figure S2B). This effect was less pronounced for 3ad. Non-transfected cells used as controls also did not show any effect after treatment with 3ad (Supporting Information File 1, Figure S2C). DIM was previously shown to be weak inverse agonist at CB 1 receptors which is consistent with our current findings for DIM derivatives 3ad and especially 3e [42]. Next, we investigated the antagonistic effect of 3e at CB 1 receptors (Supporting Information File 1, Figure S3A). Compound 3e blocked CB 1 receptor activation with an IC 50 value of 5.68 ± 0.54 µM, while it was weaker in inhibiting CB 2 receptor activation. Similarly, 3ad was also able to fully block CB 1 receptor activation (IC 50 value of 5.22 ± 0.68 µM). Our results indicate that the new DIM derivatives act as potent CB 1 receptor antagonists with inverse agonistic activity, i.e., they stabilize the inactive receptor conformation. Further optimization is warranted. This class of compounds also possesses potential for the development of CB 2selective or dual CB 1 /CB 2 -receptor antagonists.

Conclusion
The described novel and efficient synthetic protocol provides a convenient access to a wide range of unsymmetrical trifluoromethylated 3,3'-diindolylmethanes via I 2 -catalyzed Friedel-Crafts alkylation reaction of trifluoromethylated (indol-3-yl)-1-phenylethan-1-ols with substituted indoles. The method was also extended to the synthesis of pentafluoroethylated and heptafluoropropylated-DIMs. It constitutes an important addition to the active field of DIM syntheses facilitating the preparation of unsymmetrical quaternary DIMs without the need for chlorinated solvents, high temperatures, or heavy-metal catalysts. A broad range of substrates is tolerated and the reaction is suitable for large-scale preparation of the target compounds. The outlined methodology allows for the rapid generation of structurally diverse DIM derivatives to study structure-activity relationships, to optimize biological activity and other properties in order to prepare tool compounds and future drugs. Several compounds displayed micromolar binding affinities toward CB 1 and CB 2 receptors acting primarily as CB 1 receptor antagonists/inverse agonists. We are confident that our straightforward new approach will enable us and others to extensively investigate these bioactive molecules and their targets in future studies.

Supporting Information
Supporting Information File 1 Experimental and analytical data.