molecules Article
Solvent-Free Addition of Indole to Aldehydes: Unexpected Synthesis of Novel 1-[1-(1H-Indol-3-yl) Alkyl]-1H-Indoles and Preliminary Evaluation of Their Cytotoxicity in Hepatocarcinoma Cells Graziella Tocco 1, * ID , Gloria Zedda 2 , Mariano Casu 3 , Gabriella Simbula 4,† and Michela Begala 1,† 1 2 3 4
* †
ID
Department of Life and Environmental Sciences, Unit of Drug Sciences, University of Cagliari, via Ospedale 72, 09124 Cagliari, Italy;
[email protected] Merck Millipore, 39 Route Industrielle de la Hardt, 67120 Molsheim, France;
[email protected] Department of Physics, University of Cagliari, 09042 Monserrato CA, Italy;
[email protected] Department of Biomedical Science, Oncology and Molecular Pathology Unit, University of Cagliari, 09124 Cagliari, Italy;
[email protected] Correspondence:
[email protected]; Tel.: +39-070-675-8711; Fax: +39-070-675-8553 These authors equally contributed to the work.
Received: 14 September 2017; Accepted: 13 October 2017; Published: 17 October 2017
Abstract: New 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles, surprisingly, have been obtained from the addition of indole to a variety of aldehydes under neat conditions. CaO, present in excess, was fundamental for carrying out the reaction with paraformaldehyde. Under the same reaction conditions, aromatic and heteroaromatic aldehydes afforded only classical bis (indolyl) aryl indoles. In this paper, the role of CaO, together with the regiochemistry and the mechanism of the reaction, are discussed in detail. The effect of some selected 3,30 - and 1,30 -diindolyl methane derivatives on cell proliferation of the hepatoma cell line FaO was also evaluated. Keywords: solvent-free reaction; 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles; hepatocarcinoma; cytotoxicity
1. Introduction Indole is one of the most versatile heterocyclic nuclei, identified as a pharmacophore in a large number of natural and synthetic biologically active molecules [1]. Due to its electron-rich character, indole promptly reacts with hard and soft electrophiles; thus, it is fair to consider it a privileged structure, quaintly referred to as the ‘lord of the rings‘ [2]. Today, considerable attention is focused on a number of 3,30 -diindolyl methanes (or bis (indolyl) methanes) (3,30 -BIMs), structurally dimers of the dietary component indole-3-carbinol (I3C), with whom they share the same ability to suppress proliferation and induce apoptosis in various cancer cells [3–5]. In view of their importance, many methods have been reported for the synthesis of 3,30 -BIM compounds, using a multitude of catalysts such as protic or Lewis acids, I2 , zeolite, K-10 clay, ZnO, water, gold(I)-complexes, oxalic acid dehydrate, cobalt nanocatalyst, nanoparticles, graphene oxide [6–17] and various reaction conditions (e.g., ionic liquids, flow chemistry, etc.) [18,19]. However, to the best of our knowledge, reports on the preparation of 1,30 -diindolyl methane derivatives are rare [20–23]. We report herein the first example of the synthesis of racemic 1-[1-(1H-indol-3-yl) alkyl]-1Hindoles through a direct solvent-free Mannich-type addition reaction of indole and aliphatic aldehydes (Scheme 1).
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Scheme Reaction between Scheme 1. 1. Reaction between indole indole 11 and and aliphatic aliphatic aldehydes aldehydes 2. 2. 0 -BIMs have several natural natural [24] [24] and have shown anti-cancer and synthetic synthetic 3,3 3,3′-BIMs shown important important anti-cancer Interestingly, several apoptosis by signaling signaling various properties and demonstrated the capacity to sensitize cancer cells to apoptosis proapoptotic genes and proteins [25–27]. In this regard, we present a preliminarily evaluation of the 0 - and 1,3 0 -BIM derivatives on the growth of the hepatoma cell line FaO. novel 3,3 FaO. 3,3′1,3′-BIM effect of these novel
2. Results and and Discussion Discussion 2. Results 0 -BIM derivatives, we In In the the quest quest to to develop develop aa simple simple and and eco-friendly eco-friendly protocol protocol to to 3,3 3,3′-BIM derivatives, we unexpectedly that, when the reaction observed, from the very beginning of our study, unexpectedly that, when the reaction was was carried carried 0 0 0 out with -bisindolyl methane (3,3 with formaldehyde formaldehyde or aliphatic aliphatic aldehydes, aldehydes, both both 3,3 3,3′-- and and 1,3 1,3′-bisindolyl (3,3′-- and 0 0 1,3 -BIM) derivatives were obtained. Conversely, aromatic aldehydes gave only classical 3,3′-BIMs. 3,3 -BIMs. 1,3′-BIM) To understand these unusual results, the reaction between indole 1, and formaldehyde 2a, derived To understand these unusual results, the reaction between indole 1, and formaldehyde 2a, derived from paraformaldehyde, paraformaldehyde, was was first first investigated investigated (Scheme (Scheme 2). 2).
Scheme 2. Reaction between indole 1 and formaldehyde 2a.
Surprisingly, apart from the expected 3,3′-diindolyl methane 3a, we observed, as shown in Surprisingly, apart from the expected 3,30 -diindolyl methane 3a, we observed, as shown in Scheme 2, the formation of indole-1-carbinol 5 [28] and 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a, Scheme 2, the formation of indole-1-carbinol 5 [28] and 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a, which was fully characterized via mass spectrometry (MS) and nuclear magnetic resonance (NMR) which was fully characterized via mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy experiments (spectra available in Supplementary Materials). The best results were spectroscopy experiments (spectra available in Supplementary Materials). The best results were achieved at 100 °C, and also observed an improvement in the yields when an excess of calcium oxide achieved at 100 ◦ C, and also observed an improvement in the yields when an excess of calcium oxide (CaO) was used. The reaction did not take place at all at room temperature, both with or without (CaO) was used. The reaction did not take place at all at room temperature, both with or without CaO. CaO. Similarly, when the operative temperature was fixed at 60 °C, traces of both isomers were Similarly, when the operative temperature was fixed at 60 ◦ C, traces of both isomers were detectable. detectable. Thus, it was speculated that CaO, which has recently been shown to catalyse some Thus, it was speculated that CaO, which has recently been shown to catalyse some Mannich reactions Mannich reactions for the preparation of lariat ethers [29], might affect our experiments. Hence, the for the preparation of lariat ethers [29], might affect our experiments. Hence, the role of CaO was role of CaO was explored, noticing that, when it was used in stoichiometric amounts or in large explored, noticing that, when it was used in stoichiometric amounts or in large excess, the reaction excess, the reaction seemed to proceed smoothly. Use of CaO in a catalytic amount (10 mol %), did seemed to proceed smoothly. Use of CaO in a catalytic amount (10 mol %), did not seem to produce not seem to produce any effect. Reasonably, the temperature and CaO might have a synergic effect any effect. Reasonably, the temperature and CaO might have a synergic effect in the reaction activation. in the reaction activation. We can plausibly assume that, in these conditions, the paraformaldehyde We can plausibly assume that, in these conditions, the paraformaldehyde is rapidly converted to is rapidly converted to free formaldehyde and the traces of formic acid produced during prolonged free formaldehyde and the traces of formic acid produced during prolonged heating are rapidly heating are rapidly neutralized by CaO. Following the experiment by means of gas neutralized by CaO. Following the experiment by means of gas chromatography–mass spectrometry chromatography–mass spectrometry (GC–MS) analysis (Figure 1), we also observed, from the very (GC–MS) analysis (Figure 1), we also observed, from the very beginning of the reaction and only in the beginning of the reaction and only in the presence of CaO, the formation of the indole-1-carbinol 5, presence of CaO, the formation of the indole-1-carbinol 5, which is usually obtained in a strong basic which is usually obtained in a strong basic condition [30,31]. condition [30,31].
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Figure 1. Gas chromatogram spectraofofcompounds compounds 4a and Figure 1. Gas chromatogram(GC) (GC)and andmass mass spectra 3a,3a, 4a and 5. 5.
We postulate that, in our experimental conditions, CaO induces a partial N–H proton abstraction
We postulate that, in our experimental conditions, CaO induces a partial N–H proton abstraction and, despite the modest ionic character of the N–Ca bond, formaldehyde is so reactive as to undergo a and, despite the modest ionic character of the N–Ca bond, formaldehyde is so reactive as to undergo nucleophilic attack, generating indole-1-carbinol 5 [28]. As a matter of fact, when DMSO was used as a nucleophilic attack, generating indole-1-carbinol 5 [28]. As a matter of fact, when4a DMSO was used polar aprotic solvent, the reaction afforded 1-[1-(1H-indol-3-yl) methyl]-1H-indole as a major Figure 1. Gas chromatogram (GC) and mass spectra of compounds 3a, 4a and 5. as polar aprotic solvent, the reaction afforded 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a as a major product in a high regioselective manner. product inInaaddition, high regioselective manner. an interesting improvement, especially for isomer was noticed when KOH We postulate that, in our yield experimental conditions, CaO induces a partial 4a, N–H proton abstraction was used instead of CaO.ionic Ayield possible explanation, apart fromfor theisomer stronger , and, despite modest character of the N–Ca bond, formaldehyde is so reactive as tonoticed undergowhen a of K+KOH In addition, an the interesting improvement, especially 4a,electropositivity was nucleophilic attack, generating indole-1-carbinol 5 [28]. As a matter of fact, when DMSO was used as might be thatofindole-1-carbinol when heatedapart in thefrom presence of KOH, electropositivity decomposes to regenerate was used instead CaO. A possible5,explanation, the stronger of K+ , might polar thereact reaction afforded 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a as 1). a major indole andaprotic HCHOsolvent, [30] again afford both 3,3′and 1,3′-BIM isomersto(Table be that indole-1-carbinol 5, that when heated intothe presence of KOH, decomposes regenerate indole and product in a high regioselective manner. 0 -BIM isomers (Table 1). HCHO [30] that react again to afford both 3,30 - and 1,3 In addition, an interesting yield improvement, especially for isomer 4a, was noticed when KOH Table 1. Base effect on reaction between indole 1 and formaldehyde 2a at 100 °C. was used instead of CaO. A possible explanation, apart from the stronger electropositivity of K+, ◦ C. Yields (%) 2a at to might be that 5, whenbetween heated inindole the presence KOH, decomposes regenerate Table 1. indole-1-carbinol BaseEntry effect onBase reaction 1 and of formaldehyde 100 Base/(1) Time (h) indole and HCHO [30] that react again to afford both 3,3′- and 1,3′-BIM 3a isomers 4a(Table 1). 1 6 N.I. a N.I. a(%) Yields Base/(1) Time (h) formaldehyde Entry a °C. Table 1.2BaseBase effect on reaction between indole at 100 CaO 0.1/1 6 1 and N.I. a 3a 2aN.I. 4a 3 CaO 1/1 6 50 (%) 8 Yields a a 1 6 N.I. N.I. Base Base/(1) Time (h) 4Entry 17/1 36 3a 65 N.I. a4a 25 2 CaOCaO 0.1/1 N.I. a a a 17/1 N.I. 5 1 CaOCaO- b 63 6 N.I. 3 1/1 50N.I. a 62 8 a 4 17/1 65N.I. a 37 25 CaO 0.1/1 61.53 N.I. 61 6 2 CaOKOH 1/1 5 6
b 17/1 3 N.I. a KOH 1/1 1.5 61 25 4 CaO 17/1 3 65 b a a Not b 17/1 3 N.I. 62 5 CaO Isolated. GC yields; DMSO was used as solvent. comprehension mechanism, 6 KOH of the 1/1 reaction1.5 61 we examined 37 a 3CaO
CaO GC 1/1 6 was used 50 as solvent. 8 Not Isolated. yields; b DMSO
62 37
To gain a better the possible role of compound 5. It did not demonstrate being a real bintermediate formation of 1-[1-(1H-indol-3-yl) a Not Isolated. GC yields; DMSO was usedinasthe solvent. To gain a better comprehension of the reaction mechanism, we examined the possible methyl]-1H-indole 4a. In fact, when 5 could react with indole 1 and CaO, only traces of the two isomers role of compound 5. It 3a). didInterestingly, not demonstrate being a realweintermediate in the formation To gain a better comprehension of the mechanism, examined 3a, the could possible role offrom a of were detected (Scheme thereaction classical bis (indolyl) methane derive compound 5. It did not demonstrate being a real intermediate in the formation of 1-[1-(1H-indol-3-yl) 1-[1-(1H-indol-3-yl)methyl]-1H-indole 4a. In fact, when 5 could react with indole 1 and CaO, only thermal-induced isomerisation of 4a (Scheme 3b) [20]. methyl]-1H-indole In fact, when 5 could react with 1 and CaO,the onlyclassical traces of the two isomers methane traces of the two isomers 4a. were detected (Scheme 3a). indole Interestingly, bis (indolyl) were detected (Scheme 3a). Interestingly, the classical bis (indolyl) methane 3a, could derive from a 3a, could derive from a thermal-induced isomerisation of 4a (Scheme 3b) [20]. thermal-induced isomerisation of 4a (Scheme 3b) [20].
Scheme 3. Investigation of the role of indole-1-carbinol 5 in the formation of compounds 3a and 4a. Scheme 3. Investigation of the role of indole-1-carbinol 5 in the formation of compounds 3a and 4a.
Scheme 3. Investigation of the role of indole-1-carbinol 5 in the formation of compounds 3a and 4a.
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With With the the perception that the reaction, if successful with formaldehyde, would allow similar Molecules 2017, 22, 1747 44ofof12 Molecules 2017, 22,the 1747 12 perception that reaction, if successful with formaldehyde, would allow similar 1,3′- 4 of 12 2017, 22, 1747 MoleculesMolecules 2017, 22, 1747 Molecules 2017, 22, 1747 Molecules 2017, 22,1747 1747 Molecules 2017, 22, 4 4ofof12412of 124 of 11 1,30 -diindolyl isomers when different carbonyl substrates were used, some other exploratory diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions Molecules 2017, 22, 1747 4 of 11 reactions With the perception reaction, ifif 2). successful formaldehyde, would allow similar With the perception that the reaction, successful with formaldehyde, would allow similar With thealdehydes perception thatthat the the reaction, if successful withwith formaldehyde, would allow similar with diverse ketones and were carried out (Table With the perception that the reaction, if successful with formaldehyde, would allow similar With the perception that the reaction, if successful with formaldehyde, would allow similar Molecules 2017, 22, 1747 4 of 11 With the perception that the reaction, if successful with formaldehyde, would allow similar Molecules 2017, 22, 1747 4 of 11 similar With theand perception that the reaction, if successful with formaldehyde, would allow with diverse ketones aldehydes were carried out (Table 2). 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory 1,3′-diindolyl isomers when different substrates were used, some other exploratory Molecules 22,isomers 1747 4 of 11 similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory 1,3′-diindolyl when different carbonyl substrates were used, some other exploratory With the2017, perception that the reaction, ifcarbonyl successful with formaldehyde, would allow 1,3′-diindolyl isomers when different carbonyl substrates were some other exploratory reactions with diverse ketones and aldehydes were carried out (Table 2). reactions with diverse ketones and aldehydes were carried out (Table 2). reactions with diverse ketones and aldehydes were carried out (Table 2).a used, Table 2. Reaction of indole 1 with aliphatic and aromatic aldehydes.
Molecules 2017, 22, 1747diverse ketones and aldehydes were carried out (Table 2). 4 of 11 reactions with a Molecules 2017, 22, 1747 4 of 11allow With the isomers perception thatwhen the reaction, if carbonyl successful withwere formaldehyde, similar 1,3′-diindolyl isomers different substrates were used, some other exploratory 1,3′-diindolyl when different carbonyl substrates used, somewould other exploratory
With the perception that the reaction, ifcarried successful with 2). formaldehyde, would allow similar reactions with diverse ketones and aldehydes were out(Table (Table 2).used, would Withwith the perception that the reaction, ifwere successful with formaldehyde, allow reactions diverse ketones and aldehydes out Molecules 2017, 22, 1747 of 11 similar 1,3′-diindolyl when different carbonyl substrates exploratory reactions with isomers diverse ketones and aldehydes werecarried carried outwere (Table 2). some 4 other
Table 2. Reaction of indole 1 with aliphatic and aromatic aldehydes.
a (%) a a Yields Table 2. of 11with aliphatic and aromatic aldehydes. Table 2.Reaction Reaction ofindole indole with aliphatic and aromatic aldehydes. Table 2. Reaction ofreaction, indole 1 with aliphatic and aromatic aldehydes. reactions with diverse ketones and aldehydes were carried out (Table 2). a With the diverse perception that the ifwith successful with formaldehyde, would similar Table 2. Reaction ofcarbonyl indole 1 Products with aliphatic and aromatic aldehydes. 1,3′-diindolyl isomers when different substrates were used, some exploratory a aotherallow Table Reaction ofindole indole aliphatic and aromatic aldehydes. reactions with diverse ketones and aldehydes were carried out (Table 2).allow Table 2.2.Reaction of 1 1with aliphatic and aldehydes. reactions with ketones and aldehydes were carried outaromatic (Table 2). With the perception that the reaction, if successful with formaldehyde, would similar [ref.] Entry Substrate Base Time (h) a exploratory With thereactions perception the2.reaction, ifofsuccessful with formaldehyde, would 1,3′-diindolyl isomers when different carbonyl substrates were used, somesimilar Table Reaction indole 1 with and aromatic aldehydes. Yields withthat diverse ketones and aldehydes werealiphatic carried out (Table 2). allow Yields (%) Yields (%) (%) Molecules 2017, 22, 1747 4other of 11 Yields (%) Products 3 4 3 4 Yields (%)Yields 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory Products Products (%) [ref.] Yields (%) a 1,3′-diindolyl isomers when different carbonyl substrates were used, Products some other exploratory
Products [ref.] a 4 of 11a [ref.] Entry Substrate Base Time (h) reactions with diverse and aldehydes were carried out (Table 2). Products Table 2. ketones Reaction of indole 11indole with and aromatic aldehydes. [ref.] Entry Substrate Base Time (h) Entry Substrate Base Time (h) of 2. Reaction 1aliphatic with aliphatic and aromatic aldehydes. Molecules 2017,Substrate 22, 1747 Products Table 2.Table Reaction of indole with aliphatic and aromatic aldehydes. [ref.] Time (h) Entryketones Substrate Base Time (h) Entry Base [ref.] Entry Substrate Base Time (h) [ref.] reactions with diverse and aldehydes were carried (Table 2).aromatic aldehydes. Entry Substrate Time (h) Products a reactions with diverse aldehydes carried outout (Table 444 similar 33 4 (%) 44 [ref.] Table 2.Base Reaction of (h) indole 1 with aliphatic and CH 3CH 2CHOketones 33 3332). 3 Yields With the perception that and the reaction, if were successful with formaldehyde, would4 4allow Molecules 2017, 22, 1747 4 of 11 Entry Substrate Base Time b 4 3 4 3 3 4 3[ref.] 1 CaO 5 5 aYields N.I. Products Yields 3 4 3 4(%) Products (%) [ref.] Products Yields (%) [ref.] 3 4 3 4 Table 2.Base Reaction of indole 1 (h) with aliphatic and aromatic aldehydes. 2b Substrate With the perception that the reaction, if successful with formaldehyde, would allow similar Entry Substrate Base Time 1,3′-diindolyl when different carbonyl substrates were used, some other exploratory Entry Substrate Time (h) Molecules 2017, 22,isomers 1747 4 of 11 Entry Base Time (h) 32CH 3CHO CH2CHO 2CHO CH Products Yields (%)3 [ref.] a 3CH CH CH N CH3 CH CHO 3 4 3 4 3 4 a bb4 Table 2. Reaction of indole 1 with aliphatic and aromatic aldehydes. 2 b 32CH 2CHO Table 2.Substrate Reaction of indole 1 with aliphatic and aromatic aldehydes. CaO 55 113CH 53 3CH CH CHO CH 3 4 4 CaO 5 N.I. N.I. N.I. Entry Base Time (h) CaO 5 1CH 5 Molecules 2017, 22,diverse 1747 4 of 11 2 CHO CaO 5 1 reactions 5 3that CH 2CHO CH b 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory b with ketones and aldehydes were carried out (Table 2). With the perception the reaction, if successful with formaldehyde, would allow similar 1 CH3CH 5[ref.] 2b CaOCaO 55 5 5 N.I. 2b Yields 2bCH 3 Products 4 3 5 5(%)5 4 bN.I. N.I. b CaO 121CHO N.I. 2b 3CH 2CHO 1CH3CH CaO 2CHO 2b NN 57 NH HN N b Entry Base ifCaO Time 2b 1when 5Products N.I. b b 2Substrate CHO 2 Withwith -2b2different 3.5CaO 1CH3CH 5 (h) 53 38 5 N.I. 2b the isomers perception the 2b reaction, successful with formaldehyde, would similar reactions diverse ketones and aldehydes were carried outwere (Table 2). some N 1,3′-diindolyl carbonyl other exploratory Products Yields (%) [ref.] Yields (%) [ref.] NH 3 used, 4N N 4 b Nallow 3CH CHO CHthat 2b32CH 2b1 1CH CaO 5 5 substrates 5 N.I. N.I. N[32] similar N 2CHO 57 CH Entry Substrate Base (h) 3CHO CH 2Time CHO 57 CH Entry Substrate Base Time (h) 3that CH 57 CH CaO 5 a With the perception the reaction, if successful with formaldehyde, would allow NH CH CHO NH HN NH HN 2.2CHO Reaction indole 1--with aliphatic aldehydes. 3 isomers 2 Table HN aromatic 2b CH 2of CHO 57 38 CH 1,3′-diindolyl when carbonyl substrates were used, some other exploratory 3.5 223CH 38 3CH CH 2CHO CHO 57 CH 38 4 3 4 with diverse ketones and aldehydes were carried out (Table 2). 33.53 and 4 3 4 3.5 2CH 2b 3CH 23different CH NH N 33CH 22CHO 57 CH HN NH 3 CH 2 CHO CH 2 reactions 3.5 57 [32] NH CHO CH HN N NH NH 5[32]57 b 38 NH NH 3.53.5 HN 2 2when 2b [32] - - 3.5 3.5 38 2b [32] 2b 12 Table CaO 5 N.I. 2 [32] 38 HN 3.5 2 38 a NH NH 57 [32] 38 NH 1,3′-diindolyl isomers different carbonyl substrates were used, some other exploratory 2b 2 3.5 57 [32] 38 HN NH HN aromatic 2. of indole 1 with and aldehydes. 2b 2b NH 2b2and [32][32] reactions with diverse ketones aldehydes werealiphatic carried out (Table 2). CH CH33CH 3CH 2CHO CH 2b 3Reaction CH CHO CH 2CHO CHO CH 2b 2b NH NH b [32] 2b N 5 NH Products Yields (%) b [32]38 CaO 5-5(h)N.I. 2 57 [32] NH 11 CaOand 5 [ref.] N.I.57 HN (TableNH 2with 3.53.5 38 HN 4 reactions diverse ketones aldehydes were carried out 2). a Entry Substrate Base Time N 2. Reaction of indole 1 with aliphatic and aromatic aldehydes. CH 3(CH CHO 43 2b 3 CH 2 CHO CH NH 2b2)4Table 2b N4 N 3 457 [32] Products Yields (%) NH [ref.] NH 3 3.5(h)34 2 3.5 3 38 HN a 4 4 Entry Substrate Base 44 4 4aldehydes. Table Reaction NN CH CHO CH 2b 2c 3CH (CH 2Time )24)of CHO NH 3(CH 2)indole 4CHO 1 with aliphatic and aromatic 32(CH 4CHO 43 CH 3CH (CH )44CHO CHO CH2. CH CHO (CH 43 43 CH 333CH 22) CHO CH N[32] 38 4 3 (CH 433 N N CH CH (CH 23) 4(CH CHO CH 3 3.5 3N57 4 b[32] 4 N NHNH 2)-4CHO --223))(CH 3.5 57 [32] 4 4 4 3.5 34 HN Products [ref.] - 3.5 43 43 [32]34 3 2. 3CH 24)CHO 4CHO 43 CH 3.5 3 CH 34 34 3.5 43 34 aYields N (%) 3.5 3 Entry 43 [32] HN -- 1---with 3322CHO 212 3.5 [32] 38 N 4 33(CH 43 Table Reaction of indole aliphatic and aromatic aldehydes. CaO 5 5 N.I. NH (CH 2 ) 4 CHO CH 3.5 43 [32] 3 Substrate 3.5 3 2b 2c [32] NH 2c (h)- 3.5 34 34 34 Base Time 2c 2c 2c [32] [32] 2c 4 2b 3.5 3332CHO NH N 4 2b 44 CH3CH 3(CH CHO CH 3.5 3 Products 43 34 2c2)34(CH 2c 2c Yields [ref.] [32][32] 34 4 NH 4 b[32] NH NH NH NH HN HN HN HN 4 4 3 N (%) HN 2c [32] 4 N NH 4 NH 1 CaO 5 5 N.I. 4 NH 2c 2 ) 4 CHO CH NH 3.5 43 [32] 34 NH Entry 3 CH Substrate Base Time- (h) NH HN HN NH N 4 4NH HN NH NH HN NH Products Yields (%) [ref.] 3 CH 2 CHO 2b NH 3.5 43 [32] 34 3 NH NH4 3 4 NH HN 3 3CH2CHO 2c CH N4 2 -2c Time 3.5 (h) 574 [32] 38 b Entry Substrate Base HN 3(CH2)4CHO CH NH 1
4
3132 41 2 32 243 43 3 543 4 5 54 4 54 665 65 5 65 76
CaO 5 3(CH 22)CHO 4CHO CH 2b 3.5 CH CH 2b 33CH CH - 2)5CHO 3(CH3.5 25 )3.5 5CHO 2c2CHOCH3(CH -CH CH3(CH CaO 2c2)5CHO
3
HN
4
5NH
HN HN
5
N4NH NH N 5NH N N
N CH3(CH (CH3.5 2)3.5 10CHO 2d2)4CHO N4 NH CH3(CH 5 NH NH HN -2)103CHO 45 [33] 22 10 HN N N [32] -CH 43 NH - 3.5 5 5 5 HN 4 3.5 NH34 10 2d 10 5 NH 2c52)4CHO CH3(CH CH3(CH 2e N 2e2)10CHO NH N [32] NH 5 10 HN 5 N NH NH 3.5 43 34 10 HN 10 3.5 5 5 4 4 54CHO CH CH 33(CH 222)))10 CHO NH HN HN 10 3(CH (CH CHO CH 10 10 N 1010 3 (CH 2 ) 10 CHO CH NH 10 2c N NH 2e N 3CH (CH 2) CHO CH3CH --3-(CH 3.5 45 [33] 22 CH )10 CHO NNH 2)210 N 10 NH 3.5 5 10 32(CH )10CHO CHO (CH ) 1010 CHO CH 3 (CH 2 NN 3.5 43 [32] 34 10 10 5 3.5 5 5 NH NH N HN 10 104 3(CH 2)10CHO-2d 3.5 3.5 10 10 10 HN -- 3.5 55CH 55 5 3CH (CH )10 CHO CH CH3(CH 10 55 10 3.510 2e 10 N 3.5 5252))1053CHO 2c 2)210 CHO N (CH 23)(CH 10CHO CH NH NH CH3(CH 10 5 5 5 - HN 5 2e 10 2e 3.5 3.5 2e 2e -2e 45 N[33] 22 1010 10 NNH 5 O 3.5 - HN O 2e N NH NH 3.5 5 5 CHO HN 4 3.5 5CH33(CH 5 NHNH NH 1010 - 2e 2e 3.55 NH 5 10 HN 10 101010 HN NH NH 2d HN HN NH HN 2e (CH 2 ) 5 CHO NH 2 ) 10 CHO CH NH 10 NH 2e N5NHN NH 2e O -NH HN HN HN 1010 NH NH22 NH 3.5 45- [33] 3.5 10NH 5 70 [32] NH HN HN 5 3.5 - 3.5 -NH NH 10 NH NH 70 [32] HN 10 NH 2d CH3(CH 2e62)5CHO6 O N NH NH22 3.5 45- [33] HNHN 5 NH NH O 5 26 CHO CH 3.5 10 70 [32] 10 O N (CH 2))10 5CHO CH33(CH 2f 2d 2f N O NH 3.5 5 O -- O NH 4510 O 3.5 [33] 22 NH NH 10 HN NH HN O HN 2e 5 O CH3(CH 3.5 OO 70 70 [32] 2d62)10CHO N 70 NHNH 3.5 10 - -5 70 10 NH 6 O 6 66 O --- O2f 3.5 3.5 [32]- -3.5 - 7070 HN 70 5 - HN 3.510 NH HN 3.5 3.5 NH 70 [32] 7070[32] 6 70 2)10CHO CH3(CH2e NH 2f - O -- - -- 3.5 -3.5 3.5 NH HN -- [32] - [32] [32] N10 [32] 3.5 HN NH 6 O 6762f 6 7 O 2f-- O2f 2f2f3.5 3.5 70 [32] 10 5 [32] [32] - 3.5 103.5 50 [9] - - 70 [32] 50 [9] [32] NH 3.5 2f 2e2)10 CHO CH3(CH NH O NH HN N NH NHO HN HN HN HN ONH - O2f2f 2f3.5 2g550 [9] HN3.5 7 2f -10 10 NH 10NH NH HN HN 2)10CHO 2f CHO3(CH O 2e NH HN N NH 2f O - O 2g 3.5 70-10 [32] O - NH 5-50 [9] HN NH NH HNHN 7 3.5 10 O O O 2g O O 2e O O O NH O NH -O 3.5 70 O O10-HN 3.5 50 [32] [9] -O7 O 8O2f 8OOO - OO 2g 3.55 [9] N b NN HN b 50 NH OO CaO 5NH N N.I. CaO - - - ---N.I. -50 -[9] H H O H 7 3.5 50 [9] H 7 3.5 50 [9] - 3.5 7 7 3.5 50 [9] 2f O 3.5 70 [32] O -- OOO 76 3.5 50- [9] -N.I. N -O - b- 50 7 O 50 [9] [9] --- 3.5 -- [9] [9] 7 O O 78O7 2g - 2g HN CaO 5 3.5 NH N - -50 - - - 3.5 3.5 5050 [9] 2g H H 2g O O O 6 3.5 70 [32] 2g O 2g O 2g O 2g N N OO O O 8O2f 2g CaO 76 -- O 2g 50- [32] [9] --N.I. b O 2g 3.5 NH OO O 2g O 2g 2g HN 5 H-H 3.5 70 O 2g O O2f ON N OO O OO CaO O 87 5 N.I.[9]b -O 3.5 50 NH 2g HN 2g O H H O O OO 2f NNN b N N 8 OO CaO 5 N.I. O N N - N 5 3.5NHN NH HN O-HHN 10 [13] 9O 8 9 O - O OO 3.5 - CaO - [9] ---b 10 bN.I.bb -- b50 - [13] CaOHN3.5 8O 82g 5 N 55 N.I. O HN H H 87 5CaO - N.I. N NN H b N.I.-8 b N OO CaO b N HH N H bN.I. CaO -- -- b 10 [13] N.I. - CaO 5 - -bN.I. 8 OOO 89OO8 8 O CaO CaO 5N3.5 N.I. O H 5 5H N.I. --HHH H H HH O O CaO O 2h N 2g 2h 7 3.5 50 [9] 88 CaO 2g 2g N.I. CaO 55 N.I. 2g 2g H NH HN NH H3.5 HN 9OO 2g - [9] 10 2g 2g 2g 3.5 ON 2h 78 - O 2g --OO 50 -- [13] O b 2g N 2g CaO 5 N.I. O O HN O 2g O H ONH 9 - O O 3.5 -O OOO 10 [13] OH O O 2h O O O O 2g O O O ONH N N HN O O O CaO O 2g 8 5 N.I. b O H H O 2h O 10 10 O 10 9 OOO 10 - -- -- HN 3.5 10 [13]-- -- - O O N N 98 -10 10 NH 99 10 CaO -O 3.53.5 - 3.5 5 [32] 53.5 - N.I. -5 [32] 9O 2g -b -[13] 3.5 3.5 - 10 10 H H 9 3.5 [13] 9 3.5 - [13] O [13] 9 3.5 10 [13] N -3.5 -b - 9 3.5 N O 9 O 910 10 [13]-10 8 CaO 5 N.I. [13] 3.5 5 [32] - [13] O [13] 10 O OO 2h H O 2g H [13] 2h O 2h 2h 2h O O O O 99 3.5 -- OO2j2h 10- [13] -5 [32]2h NH NH NH HN HN 2h 3.5 O 2h HN HN 2j NH- NH HN 10 3.5 O 2g [13] 2h O 2h NH NH NH HN HN HN NH NH HN HN O O O ONH HN O - NH O O 2h HN O 9 - 2j 3.5 10 [13] 10 5 [32] O O NH HN O O NHNH HNHN O O OO O2j O O -OOO 9 3.5 10 [13] O O 2h O NO 2 NH HN O OO NO O 2 O O O O NH HN 2j 11O -- NO 2 NO 3.5 10- [13] - [34]- 70 [34] 2 109 3.5 5 -[32] NO 2 - 3.5 70 NH HN 2h O O3.5 10 OO 11O 10 3.5 5 [32] [32] -- - 10 3.5 5 [32] O NO 2 3.5 55[32] 10 3.5 NH 10 -- --- HN3.5 5 [32] O 2h 11 3.5 - - - --70 [34] NO 2 10 3.5 [32] ---- 2k 2k 10 3.5 [32] 10 3.5 55[32] O 10 3.5 10 - NO 2 - 3.5 5 [32] - 5 5[32] NH HN O O O 2j N NN 10 5 [32] N O11 3.5 O NO 2 NH H 70 [34] H HH O 2j OO2k HN O 2j N2j2j3.5 N O NO 2 2j NH HN O 10 5 [32] NO 10 3.5 5 [32] NO O 2 2 H O O NH H HN HN 11 - 2j 3.5 - NH 70 [34] NH O2j2k2j HN NH HN OO22, 1747 2j 2017, Molecules of 12 12 NH Molecules 55 of 2j1747 NNH HN N HN 2j2017,O22, NO NH NH HN HN 10 3.5 5 [32] 2 O H- NH O HNH 2k O O b [34] b [34] 2j NO N 2NO 2 N 10 3.5 5 [32] O 2j 12 3.5 N.I. 12 3.5 N.I. NO 2 HNHN O HNH NH NO 2 H O O NO 11 3.5 70 [34] N.I. 11 -3.5 -NO 270- [34] - b [34] 12O2j2 3.5 NO NO22 NO 2 NO 2 2l NO 2 2l NH HN O O NO O NO O 11 3.5 70 [34] O 2j 2 HN b HN NH 2 NH 11 70- [34] - [34] 2k O - NO 2 2l 3.5 12 2k N.I. NO HN3.5 NO-2222NO NO NNH2N NO 2 NO 2 NO 2 7070 [34] NH NO b [34] 70 O 3.5NH3.5NH - O -2k H H HN NO NO 2 2k 12 11 O 3.5- -N.I. 11 NO22222 2l- 3.5 11 - NO 70 [34] 11 3.5 70 [34] NO 11 3.5 NO 2 N 22NO N N NO [34] O 2N O HN NH [34] O O NO H H H H NO222k NO 11 -2k 2k 70 [34] 2 2k 3.5 2l NO 2 N-N b [32] N NO N N NO N N HN NO 2 2k13 O NH2 N -N 3.5 N.I.b [32]N 13 3.5 - [34] N.I. O 2 11 3.5 70 H H H H H H H H O b H H 12 -3.5 - N.I.- N.I. [34]b [34] N.I. - b [32] 12 3.5 13 2k 3.5 NO 2 NNO 2 O N NO 2 O H H 2k O b [34] NO 2 2m 2 2m NO3.5 b 12 N.I. NO N N 2 13 3.5 N.I. [32] NO 22l NO NO2222 NO 2 HN HN NO NH NH 2l OO H H 12 O 3.5 N.I. b [34] O NO 2 HN NH HN NO 2 2l NO 2NH 13 N.I.b [32] 12 - 2m 3.5 N.I. [34] HN NONO NH 2 N.I. 12 OOO 3.5 N.I.bb b [34] b 2 HN NH N.I. 2 2m NO NO 2 b 12 3.5 2l 12 N.I. [34] 12 NO 2 HN 12 - 3.5 3.53.5 NO 2 N.I. [34] NH HN [34] NH O NO 2 2m2l HN NO 2-2lNO NH 12 N.I. bb [34] - [34] NO2222 2l 3.5 HN NO 2 2l NO 2 -NO 2 NH HN 13 - NO N.I. [32]b 2l 3.5 NHO2l HN NH HN O HN NH NH HN 13 3.5 N.I. [32] NH NO 2 2l NO 2 NO 2 NH 13 3.5 N.I.b [32] HN O O NO 2 2m NO 2 NO O NO2222 O NO b [32] O HN NH O NO 2 2m 13 3.5 N.I. 13 3.5 N.I.b [32] NO 2 2m HN NH HN NH 13 13 3.5N.I.b [32] - N.I. bb b [32] N.I. 3.5 N.I. NO13 13 3.5 - b [32] 2 2m b [32] 3.5 13 N.I. - 3.5 3.5 13 N.I. NO 2 2m [32] HN NH [32] NO 2 2m HN NH NO 2 2m NO HN NO2222 2m NH HN NO NH HN NH 2 2m 2m HN Molecules 2017, 22, NO 1747 5 of 11 NH 2m HN NH
14
14
-
O O
2n
2n
O O O
O O O
- 3.5
--
2n 2n
3.5 3.53.5
S
O O O
O
16
5 [8]--
-
-
[8] 55 [8]
-- 5 [8]
-
73 73 [34] [34]
--
88 88 [32] [32]
--
NH NH NH
S
152o 15
S S S
-
S S S
3.5 -2o 2o
3.5 3.5
HN
O
-
NH
HN HN HN
73 [34] --
NH NH NH
3.5 88 [32] O O O O O O 2p HN NH 16 3.5 16 3.5 O O a Operative temperature: O 100 2p °C. All the reactions did not occur at room temperature; b Not Isolated. 2p HN NH HN NH HN NH GC yields. O
aa
34
22
5
-
-
-
-
-
-
-
-
O O O
HN HN NH HN
HN
O
15
O O O O
14 14
38
NH
O
O O O
O
N.I. b
N.I. 4 4 434 53[32] 34 43 [32] NH38 b 34 57 N 45 N.I. N 5[32]NH
- 3.5 3.5 22 NH 2b CH CHO 4 CH 45 [33] 45 [33]22 422CHO NH HN 2b -32(CH 33CH 55 5N45 5 CHCH 3) (CH 2))55CHO CHO CH23CH NN NH 3(CH 2)25)2d CHO CH 5CHO 45 (CH 5CaO CHO CH 53.5 5[32] N.I. b 22 3 (CH 2 )53CHO CH (CH 45 45 N N 5 4 NH -2d 57 2d HNHN 3.5 3.5 45 [33] 22 5 5 NH 3(CH 2)-53.5 CHO 45 45 22 [33] 4CHO CH NN N5[33] 38 44 CH CH 3CH (CH 5CHO 3.54 NH HN 22 22 2b HN 4422)CHO 3.5 22 - --- --- 3.5 3.5 45 [33] 4CH3(CH 5 NHNH 5 5N 5 N 3(CH 2)25)CHO 4545 2b 3CH N NH HN 2)2d 5CHO 4 CH --2d 43 34 2d [33] 4 3(CH 3.5 3.54 NH NH 2d 3.5 [33] 2222 22 NH 2d [33] 2)35(CH CHO CH NH NH N [32] 4 3.5 2d 3.5 57 [32] 38 HN N 5 NH 5 3.5 5 45 [33] NH 2c42)CHO HN HN NH 2d 2d - (CH 4CHO CH [33] 5 5 NH HN 3CH CH33(CH HN 3.5 45[33] [33]22 22 4CH 2d NNH NH 2b NH NH NH 2)5CHO NH HN 43 5 5 5NH5NHNH NH 34 [33] HN HN HN 4 3.5 57 38 N HN NH 5NH HN NH 2d --2d NH 10NH 3.5 455 [32] [33] NH NH 22 HN 4 CH3(CH 2c2)5CHO 2b NH 10
HN
4
Operative temperature: temperature: 100 100 °C. °C. All All the the reactions reactions did did not not occur occur at at room room temperature; temperature; bb Not Not Isolated. Isolated. Operative
It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, GC yields. yields. GC
-
NO 2
O
12
-
3.5
NO 2 2l
HN
-
3.5
NO 2 2m
HN
Molecules 2017, 22, 17472017, 22, 1747 Molecules
1414
Entry
14
O
O
O
O
15
15
15
15
16
16
O
O
O 2n
O
2n
2n
3.5-
-
O
O
2o
2o
O 162p O 2p
5 of 11
HN
-
- 3.5- -
-2o2o
O
O O
-2p
-
3.5
3.5-
- 3.5
2p
HN
-
HN NH
3.5S 3.5 3.5
3.5
3.5
3.53.5 HN
NH
HN NH HN
5 [8]
NH
5 of 11
5 of 11
-
O
O
4
- [34] - 73 73 [34]
-
NH NH
O
-
5 [8] -
-
(%) -[ref.] 5 [8]Yields
3
4
NH
-
HN NH HN
O O
S
NH
S
HN HN
3
NH HN
S
5 [8]
-Products - 5 [8]
HN
5 of 10
5 of 11
-
O
Time (h)
Base 3.5
2o
16 O
-
OO
O
3.5
3.5 3.5
O O
2p
a
-
2n2n
S S
O
O
OO
O O 15 15 S 2o S
S
16
-
14 O Substrate
N.I.b [32]
NH
O
14
O
-
O Table 2. Cont. O O O O
O
O O
-
NO 2
Molecules 2017, 22, 1747 13
Molecules 2017, 22, 1747
N.I. b [34]
NH
O
Molecules 2017, 22, 1747
-
NH
-
-
- 88 [32]
88 [32] -
NH
-
73 [34]
73 [34] -
-
- -[34] 73
73 [34] 88 [32] 88 [32]
-
-
-
- [32] 88
-
HN NH HN NH b Not Isolated. b Not Isolated. 100 °C. the reactions not occur room temperature; 16 a aOperativeOatemperature: - All 100 3.5All 88 [32] Operative temperature: °C. thedid reactions didatnot occur at room temperature; Operative temperature: 100 °C. All the reactions did not occur at room temperature; b Not Isolated.b a Operative temperature: 100 °C. All the reactions did not occur at room temperature; Not Isolated. GC yields. GC yields. ◦ C. All the b Not 2p Operative temperature: 100 reactions did not occur at room temperature; Isolated. GC yields. GC yields. HN NH
GC yields.
Operative thethe reactions did not occur at room temperature; Isolated. ItItwas immediately evident100 that°C. theAll reaction was highly chemoselective for aldehydes; in fact,b Not Ittemperature: was immediately that was highly chemoselective aldehydes; in fact, was immediately evident thatevident the reaction wasreaction highly chemoselective for aldehydes;for in fact, It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, such as 2-octanone, 2-hexanone, 4′-methylacetophenone 1-(p-methoxyphenyl)ketones asevident 2-octanone, 2-hexanone, 4′-methylacetophenone and 1-(p-methoxyphenyl)GC yields. ketones such as such 2-octanone, 2-hexanone, 4′-methylacetophenone andand 1-(p-methoxyphenyl)It ketones was immediately that the reaction was highly chemoselective for aldehydes; ketones 2-hexanone, 4′-methylacetophenone andafforded 1-(p-methoxyphenyl)2-propanone did notsuch react at2-octanone, all. Besides, only aliphatic aldehydes afforded and 3,3′- and 2-propanone did asnot react at all. Besides, only aliphatic aldehydes 2-propanone did not react at all. Besides, only aliphatic aldehydes afforded bothboth 3,3′- 3,3′andboth 0 2-propanone did not react2-hexanone, at all. Besides, 4 only aliphatic aldehydes afforded both1-(p-methoxyphenyl)-23,3′- and fact, ketones such as 2-octanone, -methylacetophenone and a
in 1,3′-isomers, while aromatic and heteroaromatic ones produced only (indolyl) methanes. whileand aromatic and heteroaromatic ones produced only aryl bis aryl (indolyl) aryl methanes. 1,3′-isomers, while aromatic heteroaromatic ones produced only bis bis (indolyl) methanes. It was1,3′-isomers, immediately evident that reaction was highly chemoselective for aldehydes; in0 fact, 1,3′-isomers, while aromatic andthe heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly, the reactions efficiently proceeded under neat conditions [35] yields seemed be3,30 - to the reactions efficiently proceeded under neat conditions [35] and the to yields be 1,3 -isomers, propanone didInterestingly, not at all. Besides, only aliphatic aldehydes afforded both and Interestingly, the react reactions efficiently proceeded under neat conditions [35] andand the the yields seemed beto seemed Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be ketones such as by 2-octanone, 2-hexanone, 4′-methylacetophenone and positively affected by the absence absence CaO,which which demonstrated importance mainly for1-(p-methoxyphenyl)the for the positively affected by theofofabsence of CaO, which demonstrated itsmainly importance mainly positively affected the CaO, demonstrated its its importance for the positively affected by the absence of CaO, which only demonstrated its importance mainly for the while2-propanone aromatic and heteroaromatic ones produced bis (indolyl) aryl methanes. Interestingly, didactivation. not react at all. Besides, only aliphatic aldehydes afforded paraformaldehyde activation. Remarkably, thereactions reactions almost not occur or took place very slowly, paraformaldehyde Remarkably, the almost diddid not occur or took place very slowly, paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very both slowly, 3,3′- and paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very slowly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively with or without when a solvent (toluene, CH 3CN, CHCl 3, 3THF, DMF) was employed [35]. with or without CaO, when a solvent (toluene, CH 3CN, CHCl 3, THF, DMF) was employed [35]. with or without CaO, when a solvent (toluene, CH CN, CHCl 3 , THF, DMF) was employed [35]. 1,3′-isomers, with while aromatic and aheteroaromatic produced onlywasbis (indolyl) or without CaO, when solvent (toluene, CHones 3CN, CHCl 3, THF, DMF) employed [35]. aryl methanes. Mechanistically, we presume (Scheme 4)4)(Scheme that two reaction pathways canpathways be conceived the Mechanistically, we presume (Scheme that two reaction pathways can be conceived for the Mechanistically, we presume 4) that two reaction canbefor beconceived conceived for the affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde Mechanistically, we presume (Scheme 4) that two reaction pathways can for the Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be formationformation of 3,3′3,3′- and 1,3′-BIMs isomers. Both routes share thethe same well-known 2-azafulvene formation of and 1,3′-BIMs isomers. routes share same well-known 2-azafulvene of of 3,3′isomers. Both routes share the samewell-known well-known 2-azafulvene formation 3,3′-and and1,3′-BIMs 1,3′-BIMsBoth isomers. Both routes share the same 2-azafulvene activation. Remarkably, the reactions almost did not occur or took place very slowly, withfor or without positively affected by the absence of CaO, which demonstrated its importance mainly the intermediate [20–23,36,37] which may add to nucleophilic species. Specifically, if the path a could bepath intermediate [20–23,36,37] which may add to nucleophilic species. Specifically, if the path a the could be intermediate [20–23,36,37] species.Specifically, Specifically,if ifthe a could intermediate [20–23,36,37]which whichmay mayadd addto to nucleophilic nucleophilic species. path a could be be merely assumed as aa Friedel ––Crafts alkylation, thethe path b the isalmost presumably apresumably Mannich-type merely as Friedel Crafts alkylation, path b is presumably a Mannich-type activation. Remarkably, the reactions did not occur or took place very slowly, merely asas aCH – Crafts alkylation, path b is a Mannich-type CaO,paraformaldehyde when a assumed solvent (toluene, CN, CHCl , THF, DMF) was employed [35]. merelyassumed assumed aFriedel Friedel – Crafts alkylation, the path b is presumably a Mannich-type 3 3 N-aminoalkylation. In respect, the different reactivity of of aliphatic and aromatic aldehydes seems N-aminoalkylation. In this this respect, the different reactivity aldehydes seems N-aminoalkylation. InIn respect, the different reactivity ofand aliphatic andaromatic aromatic aldehydes seems[35]. N-aminoalkylation. this respect, the different reactivity of aliphatic and aldehydes seems with or without CaO, when athis solvent (toluene, CH 3aliphatic CN, CHCl 3aromatic , THF, DMF) was employed Mechanistically, presume (Scheme that two reaction pathways plausible given theirwe intrinsically diverse steric and4) electronic features, and associated with thecan be conceived for the plausible plausible given their intrinsically diverse steric andsteric electronic features, and associated with thewith given diverse and electronic electronic features, andassociated associated with plausible giventheir theirintrinsically intrinsically diverse steric features, and thethe 0 - and of 0 presume Mechanistically, we (Scheme Both 4) thatroutes two reaction be conceived for the modest nucleophilicity the indole nitrogen. formation ofnucleophilicity 3,3 1,3 isomers. share pathways the samecan well-known 2-azafulvene modest of the-BIMs indole nitrogen. modest nucleophilicity the indolenitrogen. nitrogen. modest nucleophilicity ofofthe indole formation of 3,3′- and 1,3′-BIMs isomers. Both routes share the same well-known 2-azafulvene intermediate [20–23,36,37] which may add to nucleophilic species. Specifically, if the path a could intermediate [20–23,36,37] which may add to nucleophilic species. Specifically, if the path a could be be merely assumed as a Friedel–Crafts alkylation, the path b is presumably a Mannich-type merely assumed as a Friedel–Crafts alkylation, the path b is presumably a Mannich-type N-aminoalkylation. In this respect, aliphatic and aromatic aldehydes N-aminoalkylation. In this respect,the thedifferent different reactivity reactivity ofofaliphatic and aromatic aldehydes seemsseems plausible given their intrinsically diverse steric and electronic features, and associated with the modest plausible given their intrinsically diverse steric and electronic features, and associated with the nucleophilicity of the indole nitrogen. modest nucleophilicity of the indole nitrogen.
Scheme 4. Proposed reaction mechanism.
Scheme 4. Proposed mechanism. Scheme 4.reaction Proposed reaction mechanism.
Scheme4.4.Proposed Proposed reaction Scheme reactionmechanism. mechanism.
Electron-impact (EI) mass spectra allowed us to characterize and differentiate both isomers easily (Figure 2).
-diindolyl isomers when different carbonyl substrates were used, some other exploratory Electron-impact (EI) were masscarried spectraout allowed ctions with diverse ketones and aldehydes (Table us 2). to characterize and differentiate both isomers easily (Figure 2). Table 2. Reaction of indole 1 with aliphatic and aromatic aldehydes. a Molecules 2017, 22, 1747
ntry 1 2
3
4
5
6
7
8
9
Substrate CH3CH2CHO 2b CH3CH2CHO 2b CH3(CH2)4CHO 2c
Base
Time (h)
CaO
5
Products 3
4
5
N.I. b
57 [32]
38
6 of 10
N
-
-
3.5
NH
HN
NH
Figure 2. Electron-impact4 mass spectrometry (EI-MS) spectra of compounds 4b and 3b. N 3.52. Electron-impact mass spectrometry 43 [32]of compounds 34 Figure (EI-MS) spectra 4b and 3b. NH
HN
4
In fact, in the mass spectra of all 1,30 -diindolyl NH alkane isomers, the formation of the [M-116]+ In fact, in the mass spectra of all 1,3′-diindolyl alkane isomers, the formation of the [M-116]+ ion, presumably due to N–C bond cleavage, is the most favored fragmentation process (RA % 100), 5 CH3(CH2)5CHO ion, presumably due to N–C bond cleavage, is the most favored fragmentation process (RA % 100), 0 + N while in- the case 3.5 of 3,3 -isomers, the formation of [M-116]45ion [33] (RA %225), related to the rupture 2d while in the case of 3,3′-isomers, the formation of [M-116]+ ion (RA % 5), related to the rupture of the NH of the more stable C–C bond, is suppressed in favor of m/z 245 ion (RA % 100), generated by the HN 5 more stable is suppressed in favor of m/z 245NH ion generated by the radical of radical lossC–C of Rbond, substituent. MS/MS experiments are (RA now%in100), progress to investigate theloss most Rcharacteristic substituent.fragmentation MS/MS experiments areNMR nowanalysis in progress to 1 H, investigate thegHSQC, most characteristic 13 C, COSY, 10 pathways. included gHMQC and CH3(CH2)10CHO 1N 13C, COSY, gHSQC, 3.5 10 5 fragmentation pathways. NMR analysis included H, gHMQC and ROESY (spectra 1,30 -diindolyl 2e ROESY (spectra available in Supplementary Materials), and confirmed the structure of NH HN 10 available in Supplementary Materials), and confirmed the structure of 1,3′-diindolyl alkane isomers. NH alkane isomers. Recent studies have reported on the pleiotropic protective properties the chronic liver O Recent studies have reported on the pleiotropic protective properties on theon chronic liver injuries injuries steatohepatitis [38] and hepatocarcinoma [39,40], and on the multiple anti-tumour activities, steatohepatitis [38] activities, including 3.5 and hepatocarcinoma [39,40], and - on the multiple 70 [32] anti-tumour including the apoptotic, anti-proliferative and anti-angiogenetic effects [24], of 3,3′-bisindolylmethane, 0 2f the apoptotic, anti-proliferative and anti-angiogenetic effects [24], of 3,3 -bisindolylmethane, the parent NH one of the most abundant dietary compounds derived from HN the parent compound 3,3′-BIMs and compound of 3,30 -BIMsofand one of the most abundant dietary compounds derived from Brassica-genus O Brassica-genus vegetables. Hence, we decided to evaluate effect our 1,3′ on 0 -BIMs vegetables. Hence, we decided to evaluate the effect of our the novel 1,30of and 3,3novel onand cell3,3′-BIMs proliferation O 3.5 50 [9] cell proliferation of cell the line rat hepatoma cell line their FaO effects by comparing their effects to0 -bisindolylmethane those induced by of the rat hepatoma FaO by Ocomparing to those induced by 3,3 2g 3,3′-bisindolylmethane 3a and indole-3-carbinol (I3C), the natural active precursor of 0 3a and indole-3-carbinol (I3C), the natural active precursor of 3,3 -bisindolylmethane. In experiments, O 3,3′-bisindolylmethane. In of experiments, carried out were on a treated selectionwith of compounds, FaO cells were carried out on a selection compounds, FaO cells increasing concentrations of O N b N CaO 5 - 4b as well N.I. -of I3C for 24 h. As shown treated with increasing concentrations of 3b, 4a and as 400 µM H H 3b, 4a and 4b as well as 400 µM of I3C for 24 h. As shown in Figure 3, the hepatoma cells were 2g in Figure 3, the hepatoma cells were highlyeffect susceptible toBIMs. the anti-proliferative effect of these BIMs. highly susceptible to the anti-proliferative of these In particular, compounds 4a, 3b and O O In particular, compounds 4a, 3b and 4b exhibited a concentration-dependent growth inhibitory 4b exhibited a concentration-dependent growth inhibitory effect in FaO cells similar to that observed effect in-FaO cells similar to that observed after well-characterized BIM 3a [38,39]. after treatment with the well-characterized BIMtreatment 3a- [38,39].with10the 3.5 [13] O
2h
O
10
O
3.5
2j
HN
O
-
5 [32]
-
-
70 [34]
-
NH
3.5
2k
N H
O
12 NO 2 2l
O
-
NO 2
NO 2
11
NH
HN
O
13
Yields (%) [ref.] 3 4
N H NO 2
3.5 N.I. b [34] Figure 3. Effect of 400 µM I3C and increasing concentrations of the indole derivatives (A) 3a, 4a and Figure 3. Effect of 400 µM I3C and increasing concentrations of the indole derivatives (A) 3a, 4a and HN NH (B) 3b, 4b on FaO cell viability, determined by neutral red uptake (NRU) assay after 24 h of treatment. NO 2 determined by neutral red uptake (NRU) assay after 24 h of (B) 3b, 4b on FaO cell viability, Data are expressed as percentage of viable cells assessed by neutral red. treatment. Data are expressed as percentage of viable cells assessed by neutral red. -
3.5
-
N.I.b [32]
-
More importantly, importantly, our our novel novel derivatives, derivatives,as as well wellas as3a, 3a, were were 2020- to to 4-fold 4-fold more more potent potentthan thanI3C I3C More NO 2 2m in suppressing the viability of FaO cells with an IC value ranging from 50–100 µM after treatment in suppressing the viabilityHNof FaO cells with an IC5050value ranging from 50–100 µM after treatment NH with 4a 4a and and from from 20–100 20–100 µM µM and and 25–100 25–100 µM µM after after treatment treatment with with 3b 3b and and 4b, 4b, respectively. ItIt was was with noteworthy that all the tested BIMs induced a significant inhibition of growth of hepatoma cells at noteworthy that all the tested BIMs induced a significant inhibition of growth of hepatoma cells at lower concentrations than I3C. Furthermore, 3b was more effective in inducing loss of viability at very low doses.
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3. Materials and Methods 3.1. Chemistry All starting materials were purchased from commercial sources (Sigma-Aldrich, Milan, Italy) and AlfaAesar Thermo Fisher Scientific, Karlsruhe, Germany with purity ≥98%) and used without further purification. Reaction progress was monitored by thin layer chromatography (TLC) using Aldrich silica gel 60 F254 (0.25 mm) with detection by ultra-violet (UV) light. Chromatography was performed using Aldrich silica gel (60–120) mesh size with freshly distilled solvents. 1 H- and 13 C-NMR were recorded by Varian 400 and 500 MHz (Varian Medical Systems, Palo Alto, CA, USA) using tetramethylsilane as the internal standard. Microanalysis for carbon, nitrogen and hydrogen (CHN) were performed by a Carlo Erba 1106 Elemental Analyzer (Milan, Italy). GC–MS: Low resolution mass spectra were carried out on a Saturn 2000 ion-trap coupled with a Varian 3800 gas chromatograph (Varian, Walnut Creek, CA, USA) operating under EI conditions (electron energy 70 eV, emission current 20 µA, ion-trap temperature 200 ◦ C, manifold temperature 80 ◦ C, automatic gain control (AGC) target 21,000) with the ion trap operating in scan mode (scan range from m/z 40–400 at a scan rate of 1 scan·s−1 ). Aliquot of 1 µL of solutions 1.0 × 10−5 M in chloroform have been introduced into the gas chromatographer inlet. A Varian factor four low-bleed/MS capillary column (VF-5ms 30 m, 0.25 mm i.d., 0.25 µm film thickness) (Varian) was used. The oven temperature was programmed from 150 ◦ C (held for 2 min) to 310 ◦ C at 30 ◦ C/min (held for 2 min). The temperature was then ramped up to 350 ◦ C at 20 ◦ C/min. The transfer line was maintained at 250 ◦ C and the injector port 30/1 split) at 270 ◦ C. High-resolution mass spectra were obtained from the Department of Environmental Health Sciences Mass Spectrometry Laboratory of Mario Negri Institute for Pharmacological Research, Milan. High-resolution mass spectrometry (HRMS) experiments were carried out on a Finnigan LTQ hybrid ion trap/orbitrap. 3.2. Experimental Procedures Synthesis of 1-[1-(1H-indol-3-yl) methyl]-1H-indole (4a) Indole 1 (8.54 mmol), paraformaldehyde (10.25 mmol) and CaO (145.18 mmol) were blended with a magnetic stirrer for the period indicated (TLC) at 100 ◦ C. After reaction, the crude mixture was extracted with CH2 Cl2 and the obtained extract was passed through celite. The concentrated filtrate was flash chromatographed (hexane/ethyl acetate 3/1) on silica gel, obtaining the desired product. Typical procedure for 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles (4b–e) A mixture of indole 1 (8.54 mmol) and the appropriate aldehyde (10.25 mmol) was stirred for the period indicated (TLC) at 100 ◦ C. After reaction, the crude mixture was flash chromatographed (hexane/ethyl acetate 10/1, 5/1 or 3/1) on silica gel, obtaining the desired product. Synthesis of 3-(1-(1H-indol-3-yl) dodecyl)-1H-indole (3e) A mixture of indole 1 (8.54 mmol) and dodecanal 2e (10.25 mmol) was stirred for the period indicated (TLC) at 100 ◦ C. After reaction, the crude mixture was washed with CH2 Cl2 and filtered through celite. The concentrated filtrate was flash chromatografied (hexane/ethyl acetate 10/1) on silica gel, obtaining the desired product. 3.3. Biology Cell Line and Culture Rat FaO cell line was supplied by the Interlab Cell Line Collection (Servizio Biotecnologie, IST, Genova, Italy). FaO cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM plus
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Glutamax I, Invitrogen S.r.l., Milano, Italy) supplemented with penicillin, streptomycin and 10% heat-inactivated fetal calf-serum (FCS) (Invitrogen) in a humidified atmosphere of 5% CO2 /95% air, at 37 ◦ C. Indole 3-carbinol (I3C) and compound 3a, 3b, 4a and 4b were dissolved in DMSO and were added to the culture media to the final concentrations specified in the text. Control cells were treated with an equivalent amount of the solvent alone. Cell Viability Cell viability was determined by the uptake of neutral red by lysosome of viable cells. Determination of viability of adherent cells by NRU assay was performed according to Borefreund and Puerner [40]. The value obtained for treated cells was expressed as percentage of the value obtained in control cells. All experiments were performed in triplicate. Statistical Analysis Instant software (GraphpAd Prism 5, GraphPad Software Inc., San Diego, CA, USA) was used to analyse data. One-way analysis of variance (ANOVA) with post hoc analysis using Tukey’s multiple comparison test was used for parametric data. The results of multiple observations were presented as the means ± S.D. of three experiments. A p value of
