INTRODUCTION

1-Aryl-2,3,4,9-tetrahydro-1H-β-carboline derivatives are indole fused piperidines containing tricyclic alkaloids found in mammalian tissues [1] and cells of plant origin [2], which possess various biological activities including anticancer, antimicrobial, antithrombotic, antimalarial, antitubercular, antileishmanial, anti-HIV and antiviral [3]. A natural tetrahydro-β-carboline alkaloid tetrahydroharman(e) has been extracted from Guiera senegalensis, an African plant [4]. The synthesis of this type of compounds has been achieved by the Pictet-Spengler reaction [5]. This reaction involves the condensation of an aryl ethyl amine such as tryptamine with aryl aldehydes to form the imine intermediates, which upon auto endo cyclization results in the formation of 1-aryl-2,3,4,9-tetrahydro-1H-β-carbolines [6]. Since the development of this reaction [7], optimization studies including catalysts, reaction conditions and starting substrates have been tried with some success. Various catalyst systems such as Bronsted acids [8], Lewis acids [9], DES [10], enzymatic [11], organocatalysts [12], RTILs [13], and microwaves [14] were successfully employed. Even though all these systems were fruitful in terms of short reaction times and good product yields, there still is a need for the development of efficient and ecofriendly methodologies.

In this connection, we have selected the acidic clay montmorillonite as catalyst for the synthesis of 1-aryl-2,3,4,9-tetrahydro-1H-β-carboline derivatives. This catalyst proved to be useful for the synthesis of important series of compounds includinbg β-acetamido ketones [15], amidoalkyl naphthols [16], γ-lactones [17], enamines [18], and porphyrins [19]. Montmorillonite is a monoclinic zeolite with molecular formula Al2Si4O10(OH)2nH2O. This compound is useful as an efficient and versatile acid catalyst for a wide range of organic reactions[20]. Such acid-treated clays act as solid acid heterogeneous catalysts [21] containing acidic sites that are associated with interlamellar regions. The interlamellar acidic sites on the surface area are very much influenced by the addition of water and heating up to around 100°C and act as a very strong catalytic acid [22]. Hence, the aqueous mediated organic reactions are well catalyzed by the increased surface area with swelling properties. Along with pure montmorillonite, we have also prepared and investigated the effect of some transition metal ion (Sc3+, Ti4+, V3+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+) paired montmorillonites as catalysts in the synthesis of 1-aryl-2,3,4,9-tetrahydro-1H-β-carbolines. The main advantage of these catalysts is that they direct product specific reactions and form the title compounds rather than forming unnecessary byproducts. Moreover, these heterogeneous catalysts can be easily removed from the reaction medium and can be reused further. Additional advantages are that these catalysts are costeffective, strongly acidic, non-corrosive, and provide high product yield under mild operating conditions.

EXPERIMENTAL

Materials and Methods

Reagents and chemicals were purchased from Sigma-Aldrich, and reactions were carried out sonochemically in Power Sonic 405 micron process controlled benchtop ultrasonic reactor at 40 KHz frequency and 350Woutput power. The melting points of the products were determined in open capillaries by using EZ-Melt automated melting point apparatus. The IR spectra were recorded on Bruker Alpha-Eco ATR-FTIR interferometer equipped with ZnSe crystal and presented as absorbance versus wavenumber (cm-1). The NMR spectra were recorded in CDCl3 on Bruker NMR spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR, and referenced to TMS internal standard. The mass spectra were measured using JEOL GCMATE II GC-MS spectrometer. Elemental analyses were carried out using Thermo Finnigan analyzer.

Chemical Synthesis

Tryptamine (1), 4-chlorobenzaldehyde (2a) and 10 mol% of Fe(III)-Mmntmorillonite K-10 were dissolved in distilled water in RB flask, and heated ultrasonically at 80°C for 2 h. The progress of reaction was monitored by thin layer chromatography (TLC). Upon completion of reaction, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and filtered. The resulting filtrate was evaporated under reduced pressure to obtain crude product (Scheme 1). Then, the product was purified by column chromatography on silica gel using ethyl acetate and hexane (1:2) mixture to obtain pure 1-(4-chlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline (3a) and other synthesized compounds 3b – 3l as confirmed by spectral data and elemental analyses.

Scheme 1.
scheme 1

Synthesis of 1-substituted-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indoles 3a – 3l.

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1-(4-Chlorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline (3a): Yield: 95%; White solid; MP: 205 – 207°C; IR (ZnSe): 3297 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.45 (s, 1H), 7.40 – 7.37 (m, 3H), 7.25 – 7.04 (m, 5H), 5.26 (s, 1H), 3.95 – 3.92 (m,1H), 3.22 – 3.19 (m, 1H), 2.69 – 2.88 (m, 2H), 1.69 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 139.24, 136.46, 136.26, 134.76, 129.24, 128.83, 127.53, 122.12, 121.96, 119.25, 118.96, 114.04, 111.12, 107.53, 62.04, 43.44, 26.82 ppm; LCMS m/z: 282 (M+). Anal. calcd. for C17H15ClN2 (%): C, 72.21; H, 5.35; N, 9.91; Found: C, 72.15; H, 5.34; N, 9.87.

1-(4-Bromophenyl)-2,3,4,9-tetrahydro-1H-β-carboline (3b): Yield: 88%; Yellow solid; MP: 198 – 199°C; IR (ZnSe): 3285 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.35 (s, 1H),7.54 – 7.50 (d, 2H), 7.47 – 7.33 (m, 4H), 7.25 (s, 2H), 5.53 (s, 1H), 3.28 – 3.23 (m, 2H), 2.86 – 2.81 (m, 2H), 1.75 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 140.27, 137.06, 134.55, 132.67, 131.49, 129.84, 127.38, 126.94, 125.76, 122.88, 120.49, 119.03, 112.26, 109.83, 58.01, 46.35, 22.46 ppm; LCMS m/z: 326 (M+). Anal. calcd. for C17H15BrN2 (%): C, 62.40; H, 4.62; N, 8.56; Found: C, 62.35; H, 4.60; N, 8.53.

1-(2-Fluorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline (3c): Yield 98%; Pale yellow solid. 1H NMR (400 MHz, CDCl3) δ : 7.78 (br s, 1H), 7.54 – 7.59 (m, 1H), 7.27 – 7.33 (m, 1H), 7.19 – 7.24 (m, 1H), 7.07 – 7.18 (m, 5H), 5.56 (s, 1H), 3.26 – 3.32 (m, 1H), 3.10 – 3.17 (m, 1H), 2.76 – 2.84 (m, 2H), 1.93 (br s, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ : 160.6, 135.9, 132.7, 129.6, 129.4, 128.8, 127.3, 124.2, 121.7, 119.3, 118.2, 115.6, 110.7, 110.6, 50.4, 41.8, 22.6 ppm; LCMS m/z: 266 (M+). Anal. calcd. for C17H15FN2 (%): C, 76.67; H, 5.68; N, 10.52; Found: C, 76.64; H, 5.67; N, 10.49.

1-(4-Fluorophenyl)-2,3,4,9-tetrahydro-1H-β-carboline (3d): Yield: 89%; Pale yellow solid; MP: 183 – 188°C; IR (ZnSe): 3346 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.43 (s, 1H), 7.47 – 7.41 (m, 4H), 7.25 – 7.19 (d, 2H), 6.80 – 6.77 (d, 2H), 5.83 (s, 1H), 3.44 – 3.41 (m, 2H), 2.85 – 2.82 (m, 2H), 1.75 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 164.27, 141.35, 136.60, 134.29, 130.47, 128.22, 123.15, 121.93, 120.53, 118.88, 115.04, 114.31, 112.26, 110.07, 60.85, 45.94, 22.26 ppm; LCMS m/z: 266 (M+). Anal. calcd. for C17H15FN2 (%): C, 76.67; H, 5.68; N, 10.52; Found: C, 76.62; H, 5.67; N, 10.48.

1-Phenyl-2,3,4,9-tetrahydro-1H-β-carboline (3e): Yield: 93%; White solid; mp 206 – 207°C; IR (ZnSe): 3290 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): _ 12.45 (s, 1H), 7.42 – 7.36 (m, 4H), 7.23 – 7.05 (m, 5H), 5.26 (s, 1H), 3.96 – 3.93 (m, 1H), 3.22 – 3.19 (m, 1H), 2.65 – 2.83 (m, 2H), 1.64 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 139.28, 136.48, 136.26, 134.74, 129.23, 128.45, 127.56, 122.34, 121.95, 119.19, 118.92, 114.12, 111.22, 107.52, 62.08, 43.41, 26.76 ppm; LCMS m/z: 248 (M+). Anal. calcd. for C17H16N2 (%): C, 82.22; H, 6.49; N, 11.28; Found: C, 81.26; H, 6.34; N, 11.25.

2-(2,3,4,9-Tetrahydro-1H-β-carbolin-1-yl)phenol (3f): Yield: 84%; Yellow solid; MP: 213 – 214°C; IR (ZnSe): 3277 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.65 (s, 1H), 7.83 – 7.79 (m, 4H), 7.63 (s, 1H), 7.44 (s, 1H), 6.95 (s, 1H), 6.88 (s, 1H), 5.43 (s, 1H), 3.96 (s, 1H), 3.08 – 3.04 (m, 2H), 2.85 – 2.80 (m, 2H), 1.89 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 158.47, 138.53, 134.67, 131.03, 130.44, 127.83, 126.36, 122.35, 120.28, 119.67, 118.84, 118.45, 111.55, 108.26, 56.67, 45.23, 20.84 ppm; LCMS m/z: 264 (M+). Anal. calcd. for C17H16N2O (%): C, 77.25; H, 6.10; N, 10.06; Found: C, 77.21; H, 6.09; N, 10.04.

4-(2,3,4,9-Tetrahydro-1H-β-carbolin-1-yl)phenol (3g): Yield: 89%; Pale yellow solid; MP: 191–193°C; IR (ZnSe): 3318 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.36 (s, 1H), 7.95 (d, 2H), 7.35 (d, 2H), 6.95 – 6.91 (m, 4H), 5.43 (s, 1H), 4.23 (s, 1H), 3.05 – 3.03 (m, 2H), 2.88 – 2.87 (m, 2H), 1.93 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 153.36, 140.87, 136.54, 132.46, 130.62, 127.44, 121.37, 120.45, 118.44, 115.74, 110.27, 108.36, 60.14, 45.25, 22.07 ppm; LCMS m/z: 264 (M+). Anal. calcd. for C17H16N2O (%): C, 77.25; H, 6.10; N, 10.60; Found: C, 77.20; H, 6.08; N, 10.56.

1-(p-Tolyl)-2,3,4,9-tetrahydro-1H-β-carboline (3h): Yield: 89%; Pale yellow solid; MP: 135 – 137°C; IR (ZnSe): 3344 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.25 (s, 1H), 7.57 – 7.52 (m, 2H), 7.46 – 7.39 (m, 3H), 7.33 – 7.26 (m, 3H), 12.25 (s, 1H), 5.36 (s, 1H), 3.44 – 3.40 (m, 2H), 3.23 – 3.19 (m, 2H), 2.44 (d, 2H), 2.29 (s, 1H) 1.92 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 138.43, 137.58, 136.24, 134.14, 130.46, 129.35, 126.75, 122.83, 120.25, 116.47, 113.55, 108.44, 64.29, 51.13, 22.80, 21.52 ppm; LCMS m/z: 262 (M+). Anal. calcd. for C18H18N2 (%): C, 82.41; H, 6.92; N, 10.68; Found: C, 82.34; H, 6.89; N, 10.64.

1-(4-Methoxyphenyl)-2,3,4,9-tetrahydro-1H-β-carboline (3i): Yield: 91%; Pale yellow solid; MP: 204 – 206 °C; IR (ZnSe): 3296 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.45 (s, 1H), 8.12 (d, 2H), 7.53 (d, 2H), 6.91 – 7.34 (m, 4H), 5.42 (s, 1H), 3.87 (s, 3H), 3.34 (m, 1H), 3.09 (m, 1H), 2.65 – 2.87 (m, 2H), 1.98 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 159.49, 136.58, 134.97, 132.29, 130.04, 127.56,121.84, 120.55, 118.35, 113.93, 111.24, 109.18, 62.23, 55.96, 45.27, 22.05 ppm; LCMS m/z: 278(M+). Anal. calcd. for C18H18N2O (%): C, 77.67; H, 6.52; N, 10.06; Found: C, 77.62; H, 6.49; N, 10.04.

2-Methoxy-4-(2,3,4,9-tetrahydro-1H-β-carbolin-1-yl)-phenol (3j): Yield: 85%; Yellow solid; MP: 185 – 186°C; IR (ZnSe): 3335 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.36 (s, 1H), 7.83 – 7.79 (m, 3H), 7.64 (s, 1H), 7.45 (s, 1H), 7.25 (s, 1H), 7.04 (s, 1H), 5.27 (s, 1H), 4.26 – 4.21 (m,1H), 3.95 (s, 3H), 3.13 – 3.08 (m, 2H), 2.65 – 2.60 (m, 2H), 1.99 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 156.41, 145.52, 138.44, 136.23, 133.35, 128.07, 121.86, 120.99, 119.18, 118.80, 117.32, 116.23, 112.35, 109.71, 62.24, 56.83, 43.42, 22.75 ppm; LCMS m/z: 294(M+). Anal. calcd. for C18H18N2O2 (%): C, 73.45; H, 6.16; N, 9.52; Found: C, 73.39; H, 6.13; N, 9.49.

N,N-Dimethyl-4-(2,3,4,9-tetrahydro-1H-β-carbolin-1-yl)aniline (3k): Yield: 87%; Yellow solid; MP: 204 – 205°C; IR (ZnSe): 3359 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.38 (s, 1H), 7.76 (d, 2H),7.25 (d, 2H), 6.85 – 6.81 (m, 4H), 5.65 (s, 1H), 3.44 – 3.40 (m, 2H), 3.25 (s, 6H), 2.71 – 2.68 (m, 2H), 2.03 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 152.25, 138.66, 135.27, 132.03, 128.24, 127.85, 122.35, 120.43, 118.57, 112.74, 111.46, 108.03, 60.24, 44.57, 40.07, 22.85 ppm; LCMS m/z: 291(M+). Anal. calcd. for C19H21N3 (%): C, 78.32; H, 7.26; N, 14.42; Found: C, 78.26; H, 7.24; N, 14.38.

1-(4-Nitrophenyl)-2,3,4,9-tetrahydro-1H-β-carboline (3l): Yield: 95%; Yellow solid; MP: 172 – 173°C; IR (ZnSe): 3308 (-NH) cm–1; 1HNMR (400 MHz, CDCl3): δ 12.26 (s, 1H), 8.33 (m, 2H), 7.54 (m, 1H), 7.33 – 7.28 (m, 4H), 5.45 (s, 1H), 3.45 – 3.39 (m, 2H), 2.93 – 2.87 (m, 2H), 1.96 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 146.85, 145.47, 136.23, 134.17, 128.24, 123.56, 120.57, 118.95, 110.24, 109.63, 62.27, 46.43, 24.25 ppm; LCMS m/z: 293(M+). Anal. calcd. for C17H15N3O2 (%): C, 69.61; H, 5.15; N, 14.33; Found: C, 69.59; H, 5.14; N, 14.30.

Antimicrobial Activity Assay

The antimicrobial activity of compounds 3a – 3l against bacteria Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and fungi Aspergillus niger (A. niger) and Helminthosporium oryzae (H. oryzae) was tested by disc diffusion and agar dilution methods, respectively. The bacterial microorganisms were cultured on nutrient broth at 37°C and fungal microorganisms were cultured on potato dextrose agar at 28°C for 18 – 24 h. The microbial growth inhibition was evaluated and compared to standard bactericide penicillin and standard fungicide griseofulvin at three concentrations (100, 50, 25 ppm) [23, 24].

RESULTS AND DISCUSSION

Chemistry

Experimental conditions for the synthesis of 1-aryl-2,3,4,9-tetrahydro-1H-β-carbolines (3a – 3l) from tryptamine (1) and substituted benzaldehydes (2a – 2l) were standardized with illustrative reaction of tryptamine (1) and 4-chloro benzaldehyde (2a) with respect to variable reaction conditions including catalyst selection, concentration, effective reaction temperature, energizing the reaction with conventional heating, microwave irradiation and ultrasonication.

Synthesis of metal ion exchanged montmorillonites. Montmorillonite was purchased from Sigma-Aldrich, and then transition metal ion-exchanged montmorillonites were prepared by thorough mixing of montmorillonite with aqueous solutions of selected metal salts for 4 h at room temperature, with subsequent filtration. The solid product was washed repeatedly with deionized water, dried under vacuum at room temperature to retain their surface properties intact, and ground to get fine powder [25].

Optimization of catalyst. The effective catalyst was optimized by microwave synthesis of 3a in aqueous medium by screening the efficacy of initial montmorillonite and some of ion-exchanged derivatives paired with transition metal cations (Table 1).

Table 1. Catalyst Optimization for Synthesis of Compound 3a
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The Lewis acidic strength of these catalysts grows with increase in the charge to radius ratio of exchanged cation and followed trends of the Ahrland-Chatt-Davies (ACD) and Pearson’s hard and soft acid classification of metal ions in the acidity and turnover of the catalytic activity. In comparison, the yield of compound 3a obtained using montmorillonite paired class A metal ions (Sc3+, Ti4+, V3+, Cr3+, Fe3+) is higher compared to montmorillonite paired with borderline metal ions (Mn2+, Co2+, Ni2+, Cu2+, Zn2+), which is also higher than the yield obtained with pure montmorillonite. Hence, Fe(III)-montmorillonite catalyst produced compound 3a in higher yields and was currently identified as catalyst of interest.

Optimization of catalyst concentration. In developing Fe(III)-montmorillonite catalyst, we have optimized its concentration using microwave synthesis of 3a loaded with 1.0, 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mol% of catalyst under otherwise the same reaction conditions described above and obtained 60, 64, 67, 70, 85, 75 and 74% yield, respectively (Table 2). Hence, 10 mol % is identified as effective concentration to produce 3a in good yield, as no further improvement was observed at higher concentration (12.5 and 15.0%).

Table 2. Optimization of Catalyst Concentration for Synthesis of Compound 3a
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Method optimization. After selection and optimization of the concentration of Fe(III)-montmorillonite catalyst, we have checked the effect of energizing the reaction in different modes including the conventional heating, microwave irradiation and ultrasonication. It was established that the reaction with Fe(III)-montmorillonite catalyst produced the title compounds without any byproducts by conventional heating method. The better product yields were obtained under microwave irradiation conditions. However, ultrasonication of the reaction produced title compounds in higher yields as compared to the other two methods. The results of this investigation are summarized in Table 3. Hence, ultrasonication is identified as the best mode of energy supply for promoting this reaction.

Table 3. Optimization of Energy Source for Synthesis of Selected Compounds
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Optimization of the reaction temperature for synthesis of compound 3a. We have optimized the reaction temperature at which the substrates are converted completely to products in high yields. We have studied the ultrasonicated reaction at seven different temperatures in the range of 60 – 90°C as shown in Table 4. The effective temperature was found to be 80°C for synthesis of compound 3a in good yield at short reaction times.

Table 4. Optimization of Reaction Temperature for Ultrasonic Synthesis of Compound 3a
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As the ultrasonic synthesis of compounds 3a – 3l from 1 and 2a – 2l (containing both electron-withdrawing and electron-donating substituents) under catalytic action of Fe(III)-montmorillonite has been found effective, a deeper understanding of the reaction mechanism is an important task to discuss herein. As the reaction seems to be a classic example of Schiff base formation from primary amines and aldehyde, the hypothesis of extended possibility derived from Schiff bases of aryl substituted ethanamines to generate tetrahydro-β-carbolines via 6-endo cyclization has been paved the way for their successful synthesis. Here the transition state energy of the intermediate has also enables it to produce stable Pictet-Spengler products either simply staying as N-(2-arylethyl) Schiff bases or forming unwanted byproducts [26]. In this context, tryptamine and aldehyde will get condensed to produce imine, which on cyclization by α-carbon attack on imino carbon (6-endo cyclization) generates tetrahydro-β-carbolines. Here, Fe(III)-montmorillonite catalyst makes Fe(III) ion and carbonyl oxygen of substrate to bound electrostatically and promotes the reaction to form an imine. Similarly, Fe(III) ion and imine were also bound electrostatically and facilitated the 6-endo cyclization, followed by proton migration to form the title products.

Antimicrobial Activity:

Compounds 3a – 3l were screened for their antibacterial activity against the growth of S. aureus (Gram positive) and E. coli (gram negative) at different concentrations (100, 50, 25 ppm) by disk diffusion method and were found to be effective against both bacteria with reference to Penicillin. Further, their antifungal activity was evaluated against the growth of A. Niger and H. oryzae fungi at different concentrations (100, 50, 25 ppm) with reference to griseofulvin. The results expressed in terms of the zone of inhibition (presented in Tables 5 and 6) were found consistent with respect to cultures as well as concentrations. Compounds 3b, 3f, 3g, 3i, 3j and 3k substituted with bromo, 2-hydroxy, 4-hydroxy, 4-methoxy, 2-methoxy-4-hydroxy and 4-N,N-dimethyl amino groups, respectively, are found more potent as compared to other. Theoretically, the structure – activity relationship of the above compounds shows that all groups substituted in the aforementioned active compounds are potent to inhibit the growth of test microorganisms and, hence, they have successfully improved the activity of basic 2,3,4,9-tetrahydro-1H-β-carboline molecule.

Table 5. Antibacterial Activity (Zone of Inhibition, %) of Compounds 3a – 3l (μg/ mL)
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Table 6. Antifungal Activity (Zone of Inhibition, %) of Compounds 3a – 3l (μg/ mL)
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Compounds 3b, 3f, 3g, 3i, 3j and 3k substituted with bromo, 2-hydroxy, 4-hydroxy, 4-methoxy, 2-methoxy-4-hydroxy and 4-N,N-dimethyl amino groups, respectively, are found to effectively inhibit the growth of microbial cultures of E. coli, S. aureus, A. niger, and H. oryzae in good comparison to the standard drugs.