A rapid, efficient and versatile green synthesis of 3,3’ -diindolylmethanes

The natural product 3,3’ -diindolylmethane (DIM) exhibits anti-cancer and immunostimulatory properties. We report an operationally simple, efficient and versatile synthesis of DIM derivatives by reaction of indoles with aldehydes in the presence of sulfuric acid in water. Short reaction times of only 5 min, simple work-up procedure, avoidance of hazardous organic solvents, and excellent yields are apparent advantages of this method. The synthetic protocol tolerates a broad range of functional groups allowing fast and straightforward access to a large variety of DIM derivatives, including 20 new compounds not previously described in literature, which have potential as anti-cancer drugs.


Introduction
2][3] Specifically, diindolylmethanes 4 and diindolylethanes 5 are known to possess a wide variety of biological activities including anti-bacterial, 6 anti-microbial, 7 anti-fungal 8 and anti-tumor effects. 9For example, vibrindole A (Figure 1A, 1), 6 isolated from the culture medium of the marine bacterium Vibrio parahemolyticus, and trisindoline (Figure 1A, 2), obtained from the culture of a bacterium Vibrio sp.exhibit anti-biotic activity. 10Malassezin (Figure 1A, 3) isolated from the yeast Malassezia furfur 11 is an agonist of the aryl hydrocarbon receptor (AhR), 12 a ligand-activated transcription factor associated with gastric carcinogenesis.3,3'-Diindolylmethane (DIM, 4), which has a skeleton consisting of two indole groups bridged by a single carbon atom at the 3-and 3'-positions, is a metabolite produced from glucobrassicin, which is found in large amounts in cruciferous vegetables such as broccoli, burssel sprouts and cauliflower.After hydrolysis of glucobrassicin by myrosinase, indole-3-carbinol (I3C) are formed, which then dimerizes to 4, indolo [3,2-b]carbazole (ICZ, 5) and further products, in the presence of gastric hydrochloric acid (Figure 1B). 13t is estimated that approximately 10-20% of I3C is metabolized to 4, and in addition, more than 15 different oligomeric compounds are formed. 14Thus, 4 is a major product of I3C formed in vivo 15 and therefore the compound has been more extensively investigated than any other I3C metabolite. 168][19][20] It displays anti-inflammatory, 19 anti-angiogenic, 17 anticancer, 18,20 and anti-oxidant activities. 21DIM was previously reported to act as an AhR agonist 22 and to inhibit histone deacetylase-1 (HDAC-1). 23Recently DIM and its analogues have been identified and characterized as potent agonists of the immunostimulatory orphan G protein-coupled receptor GPR84. 24,25he goal of the present study was to develop a straightforward access to a broad range of synthetic derivatives and analogues of DIM for subsequent biological studies.
The majority of synthetic methods available for the preparation of DIM derivatives utilizes the Friedel-Crafts acylation reaction of indoles with various aldehydes or ketones in the presence of either a Lewis acid (e.g.AlCl3, BF3•Et2O), 26 protic acids (e.g.HCl, TFA), 26 metal salts, such as In(OTf)3, 26 Dy(Otf)3, 26 Ln(Otf)3, 26 CeCl3•7H2O, 26 RuCl3•3H2O, 26,27 or iodine. 26,27The use of solid acidic catalysts 28,29 such as Amberlyst-15, 26 montmorillonite clay K-10 26 and zeolites 26 has also been reported.Moreover, benzoic acid, 30 sodium dodecylsulfate (SDS), 31 oxalic acid or N-acetyl-N,N,N-trimethylammonium bromide (CTAB) in water 32 were employed for the transformation of indoles to DIMs.Recently, the reaction of indoles, with in situ generated aldehydes from the corresponding alcohols has been reported to obtain DIMs.Examples include an iodinecatalyzed one-pot reaction of benzylic alcohols with indoles, ruthenium(III) chloride trihydrate-catalyzed aerobic oxidation reaction of benzylic alcohols with indoles and RuCl2(PPh3)3/bis-[(2-diphenylphosphanyl)phenyl]ether-catalyzed reaction of benzylic alcohols with indole in the presence of large amount of tripotassium phosphate. 33,34Although the reported methodologies are useful, there are still certain drawbacks such as the requirement of expensive catalysts 26,35 long reaction times 36,37 formation of hazardous byproducts, harsh reaction conditions 38 and complex workup procedures to isolate the products of interest. 39In this context, the development of a facile and efficient procedure for the synthesis of diindolylmethanes (DIMs) is highly desirable.

Results and Discussion
Optimization of the reaction conditions.Organic reactions in water as a reaction medium offer many advantages, including low cost, safe handling and environment compatibility.Recently we developed a new procedure for the synthesis of DIMs in aqueous media under microwave irradiation at 100 o C. 25 No catalyst was used for the reaction of indoles with formaldehyde, while sodium dodecylsulfate (SDS) was employed as an emulsifier for the reaction of indoles with aliphatic and aromatic aldehydes. 25n continuation of our research program, we have now further improved the synthetic access to a large variety of target compounds by developing an operationally simple, fast and efficient green procedure for the synthesis of DIMs.
The reaction of 4-methoxyindole with benzaldehyde (Scheme 1) was tried under a variety of conditions (see Table 1), and considerable variation in yields was observed.

Scheme 1
Table 1.Optimization of the reaction conditions a a All the reactions were conducted with 4-methoxyindole (6a, 10 mmol), and benzaldehyde (7a, 5 mmol) in the presence of acid or additive at room temperature.b Isolated yield.
In order to test this idea, we decided to synthesize DIMs from indoles and aldehydes in the presence of sulfuric acid, which has not been explored well.To the best of our knowledge, only one previous method for the preparation of 3,3'-DIMs in the presence of sulfuric acid in water has been described. 15However, the published procedure was carried out under harsh conditions (reaction at 100 o C for 2.5 to 5 h) and suffered in several cases from low yields of less than 20%.Moreover, no efforts to optimize the reaction conditions had been undertaken and the scope of the reaction had not been explored.We therefore systematically studied the reaction conditions by exploring the transformation of 4-methoxyindole (6a, 10 mmol) with benzaldehyde (7a, 5 mmol) yielding 4,4'-dimethoxy-DIM (8a).The reaction was optimized by variation of type and amount of acid and solvent system.In Table 1, entries 1-4, various stoichiometries of sulfuric acid in water as a reaction medium were employed.In the presence of 2 mmol of sulfuric acid the reaction was slow and only 40% of product was isolated after 2 h, while increasing the amount to 5 or 10 mmol raised product formation to 87% and 95%, respectively.Reaction in dichloromethane (DCM) was rapid but only 30% of the desired product was obtained (entry 5).This study suggested that water as a reaction medium increased the yield.Reaction in the presence of SDS (entry 6) was very slow and only 30% of product was isolated after 12 h.The reaction was not initiated without adding any acid or additive (entry 7).Sulfuric acid was clearly superior to other acids, namely the Lewis acid BF3•Et2O (entry 8) or TFA (entry 9).We found sulfuric acid (10 mmol) in water to provide the optimal conditions for the synthesis of 8a from 4-methoxyindole (6a, 10 mmol) and benzaldehyde (7a, 5 mmol), which gave 8a in 95% yield.Substrate scope of the reaction and discussion.We then examined the substrate scope of these optimized reaction conditions with a variety of substituted indoles and aldehydes (see Trifluoroacetic acid (TFA) (5) DCM 24 h 25 electron-donating substituents or electron-withdrawing substituents on the indole or on the aromatic aldehydes did not significantly impact on the reaction.For example the reaction of 4-methoxybenzaldehyde (7b) or 4-fluorobenzaldehyde (7c) with various substituted indoles afforded the corresponding DIMs in excellent yields of 82 to 96% (Table 2, compounds 8a-8h).We observed that reactions of condensed aromatic aldehydes like naphthalene-1-carboxaldehyde (7d) or naphthalene-2-caboxaldehyde (7e) also proceeded smoothly and resulted in excellent yields of 83 and 89%, respectively (Table 2, compounds 8i and 8j).The synthesis of trisindolylmethane (TIM, 8k) a natural product isolated from the North Sea bacterium Vibrio parahaemolyticus Bio249 was explored. 45As indicated in Table 2, 8k was synthesized from 1H-indole (6b) by reaction with indol-3-carbaldehyde (7f) in a high yield of 90%.The efficiency of the new synthetic protocol was further examined by reacting heteroaromatic aldehydes.For example, the reaction of 2thiophenecarbaldehyde (7g) or 4-pyridinecarbaldehyde (7h) with indoles resulted in high yields of 77 to 94% (Table 2, compounds 8l-8r).In order to test the suitability of the procedure for large-scale synthesis, the reaction of 1H-indole (6b, 42 mmol) with 4-pyridinecarbaldehyde (7g, 21 mmol) was investigated.The reaction afforded 6.5 g of 8m in 94% yield without any significant loss in efficiency.Thus, this synthetic protocol can be used as a practical method to prepare DIM derivatives on a larger scale.
The scope of the reaction was further examined by reacting a variety of aliphatic aldehydes including cyclic aldehydes.The reaction of indoles with non-cyclic aldehydes, for example, propionaldehyde (7i) or isobutyraldehyde (7j), resulted in moderate to good yields of 61 to 72% (Table 2, compounds 8s-8u).On the other hand, the reaction of cyclic aldehydes, for example, the reaction of cyclopropanecarbaldehyde (7k) or cyclohexanecarbaldehyde (7l) with indoles gave excellent yields of 83 and 87%, respectively (Table 2, compounds 8v and 8w).The reaction of an aliphatic aldehyde containing a carboxylic acid ester group was also explored.As indicated in Table 2, product 8x was synthesized by reacting methyl 4-oxobutanoate (7m) with 4fluoroindole (6f).Methyl ester 8x was subsequently hydrolyzed by treatment with 2 N sodium hydroxide yielding the corresponding carboxylic acid derivative 8y.In order to compare the efficiency of the new synthetic procedure with that of previously reported reaction conditions, times and yields for the preparation of 3,3'-di-(1H-indolyl)phenylmethane (8b) were compared (Table 3).Moreover the present method used water as a reaction medium which made work-up easy compared to other published procedures (see entry 3, 4, 6, 9 10, 13, 14, and 15).The reaction mixture was washed with ethyl acetate, dried and evaporated to obtain the desired compounds, which were in most cases purified by recrystallization or by washing with nonpolar solvents.Thus, the present method clearly affords superior results with respect to the employed reagents, solvent, reaction time and eco-friendliness.CH3CN, LiClO4 2.0 h 80 26 Ionic liquid, Dy(OTf)3 1.0 h 98 50 AgOTf, 24 h 41 30 Benzoic acid, H2O, 80 o C 15 h 80 a Present method (in bold); b TCT, trichloro-1,3,5-triazine; c N-cetyl-N,N,N-trimethylammonium bromide (CTAB).

Conclusions
We have developed a simple, fast and efficient protocol for the synthesis of diversely substituted 3,3'diindolylmethanes.A series of 25 derivatives was obtained, 20 of which have not been previously reported in literature.The new synthetic protocol offers several advantages including short reaction times, high yields of products, eco-friendliness and simple experimental settings as well as isolation procedures.In addition, this method is highly feasible and applicable to a broad range of aromatic, heteroaromatic and aliphatic aldehydes.
It thus provides a convenient access to a large variety of DIM derivatives, which are of great interest due to their exciting biological activities.

Experimental Section
General.All materials were used as purchased (from Acros, Alfa Aesar, Grüssing, Merck, or Sigma-Aldrich, Germany).Thin-layer chromatography was performed using TLC aluminum silica gel 60 F254 sheets, or TLC aluminum RP silica gel 18 F254 sheets (Merck, Darmstadt, Germany).The purities of isolated products were determined by an LC-MS instrument coupled to electrospray ionization mass spectrometry (LC/ESI-MS) (Applied Biosystems API 2000 LCMS/MS, HPLC Agilent 1100) using the following procedure: the compounds were dissolved at a concentration of 1.0 mg/mL in acetonitrile, containing 2 mM CH3COONH4.Then, 10 µL of the sample was injected into an HPLC column (Phenomenex Luna 3µ C18, 50  2.00 mm).Elution was performed with a gradient of water:methanol (containing 2 mM CH3COONH4) from 90:10 to 0:100 starting the gradient immediately at a flow rate of 250 µL/min for 15 min followed by washing with 100 % methanol for another 15 min.UV absorption was detected from 200 to 600 nm using a diode array detector.The purity of the compounds was determined at 254 nm and was ≥ 95% for all products.
1 H-and 13 C-NMR data were measured in DMSO-d6 as a solvent.Chemical shifts are reported in parts per million (ppm) relative to the deuterated solvent (DMSO-d6), δ 1 H: 2.49 ppm, 13 C: 39.7 ppm, coupling constants J are given in Hertz and spin multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), sext (sextet), m (multiplet), br (broad).The purities of isolated products were determined by ESI-mass spectra obtained on an HPLC-MS instrument (LC-MS) using the same procedure as previously published.Melting points were measured on a melting point apparatus (Büchi melting point B-545) and are uncorrected.HRMS was recorded on a micrOTOF-Q mass spectrometer (Bruker) with ESI-source coupled with an HPLC Dionex Ultimate 3000 (Thermo Scientific) using an EC50/2 Nucleodur C18 Gravity 3 µm column (Macherey-Nagel).The column temperature was 425 o C. Ca. 1 µL of a 1 mg/mL solution of the sample in acetonitrile was injected and a flow rate of 0.3 mL/min was used.HPLC was started with a solution of acetonitrile in water (10:90), containing 2 mM CH3COONH4.The gradient was started after 1 min reaching 100% acetonitrile within 9 min and then flushed with this concentration for another 5 min.The infrared spectra were recorded as solid samples on an ALPHA-T (Bruker) with a Platinum ATR Module using Opus software.IR spectra were measured in the attenuated total reflection (ATR) mode in the region of 4000-385 cm -1 .

General procedure for the synthesis of 3,3'-diindolylmethanes 8a-8x
To the stirred mixture of the appropriate indole (3.1-6.7 mmol) and the appropriate aldehyde (1.5-3.3 mmol, 0.5 equiv.)dissolved in water (5 mL) concentrated sulfuric acid (1 equiv.) was added.The progress of the reaction was monitored immediately after the addition of H2SO4 by TLC using ethyl acetate: petroleum ether (20:80).After completion of the reaction as indicated by TLC, the aqueous mixture was dissolved in ethyl acetate and washed with brine.The organic layer was dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure.The resulting crude product was purified by recrystallization with an appropriate solvent system or by washing it with nonpolar solvents.Few compounds were purified by silica gel column chromatography using non-chlorinated solvent systems such as ethyl acetate: petroleum ether (b.p.42-62 °C) mixtures (10:90 to 20:80) as eluent to afford the pure diindolylmethane derivatives.

Table 2 .
Reaction of indole derivatives with aliphatic or aromatic aldehydes

Table 3 .
Comparison of reaction times and yields for the preparation of compound 8b from indole and benzaldehyde using different reaction conditions