Abstract
Three new series of bis-indole derivatives were synthesized based on p-phenylenediamine (2–4, 5 and 6) and 4,4′-ethylenedianiline moieties (7–9) using facile and efficient condensation of three positional isomeric indole-carboxaldehyde derivatives (1a–c) with bifunctional amines upon microwave irradiation. The symmetric dimeric indole derivatives 2–4 as well as non-symmetric analogues 5 and 6 were obtained by in situ condensation of the respective positional 3-, 2- and 5-isomeric indole-carboxaldehydes with p-phenylenediamine, while compounds 7–9 resulted from respective condensation based on 4,4′-ethylenedianiline. Structures of the obtained compounds were deduced by advanced spectroscopic methods (1H NMR, 13C NMR and MS). In agar diffusion assay, derivative 6 showed moderate antibacterial activity against various Gram positive and negative bacteria, while derivative 7 displayed moderate activity against several Gram positive bacteria. However, in Resazurin assay employing the human cervix carcinoma cell line (KB-3-1), derivatives 2–9 turned out to be inactive.
1 Introduction
Indole derivatives occur in nature as secondary metabolites from diverse natural sources including microorganisms and plants [1], [2], [3], [4], [5], [6]. The indole nucleus has been reported to play an important role as a therapeutic agent in medicine, exhibiting antimicrobial [7], [8], antiinflammatory [9], [10], antioxidant [11], [12], [13], anticancer [14], [15], [16], [17], antibiotic [2] and anti-HIV [18], [19] activities. Bis-indole alkaloids obtained either synthetically [e.g. N′-(1H-indol-3-yl)methylene-2-(1H-indol-3-yl)acetohydrazide (I) and N′,N″-(1,4-phenylenebis(methan-1-yl-1-ylidene))bis(1H-indole-2-carbohydrazide) (II)] [20], [21] or from nature [e.g. nortopsentins A–C (IIIa–c) and 2,5-bis(3-indolylmethyl)-pyrazine (IV)] [22] showed broad pharmacological activities as well [23], [24], [25], [26], [27], [28], [29], [30].
The aim of the present investigation was to design new bis-indole derivatives in the search for high bioactivities and less toxicity, employing microwave irradiation based on the Schiff base principle as a facile and efficient method. In accordance with that, six symmetric (2–4, 7–9) and two non-symmetric (5 and 6) dimeric bis-indole derivatives (Fig. 1) were obtained by individual condensation of indole-3/2/5-carbaldehydes (1a/1b/1c) with the bifunctional aromatic amines p-phenylenediamine and 4,4′-ethylenedianiline.
Chemical structures of the synthesized bis-indole derivatives 2–9.
2 Results and discussion
2.1 Chemistry
As a trial to find out the optimal reacting conditions for Schiff base condensation, the reaction of indole-3- carboxaldehyde (1a) with p-phenylenediamine was selected as a model reaction using microwave irradiation, which resulted in compound 2 (Scheme 1). Methanol – as a polar protic solvent – showed higher efficiency and yield of compound 2 compared to acetonitrile and dioxane (Table 1). On the other hand, trifluoroacetic acid showed high catalytic activity. However, it is difficult to be removed from the final product. Therefore, glacial acetic acid was selected as a preferable catalytic agent. The optimal reaction time and temperature were realized to be 30 min and 65°C, respectively, affording 98% yield of bis-indole 2. Applying the discussed optimal conditions, the corresponding bis-indoles 3–9 were synthesized using indole-carboxaldehydes (1a–c) and bifunctional amines (p-phenylenediamine or 4,4′-ethylenedianiline) with excellent yield (Table 2).
Model reaction of indole-3-carboxaldehyde (1a) and p-phenylenediamine via microwave irradiation.
Model reaction and optimization studies for the synthesis of bis-indole 2.
| Entry | Catalyst (drops) | Solvent | Temp. (°C) | Time (min) | Power (W) | Yield (%) |
|---|---|---|---|---|---|---|
| 1 | CF3COOH | CH3CN | 100 | 15 | 50 | 65 |
| 2 | CF3COOH | CH3CN | 80 | 20 | 50 | 69 |
| 3 | CH3COOH | Dioxane | 100 | 20 | 50 | 76 |
| 4 | CH3COOH | MeOH | 65 | 30 | 50 | 98 |
| 5 | CH3COOH | MeOH | 100 | 20 | 50 | 91 |
Physical properties of bis-indole derivatives 2–9.
| Cpd. | M.f. | Mol. wt. | Appearance | M.p. (°C) | Yield (%) |
|---|---|---|---|---|---|
| 2 | C24H18N4 | 362 | Yellow | 291–294 | 98 |
| 3 | C24H18N4 | 362 | Yellow | 300–302 | 98 |
| 4 | C24H18N4 | 362 | Yellow | 276–278 | 80 |
| 5 | C24H18N4 | 362 | Yellow | 255–257 | 85 |
| 6 | C24H18N4 | 362 | Yellow | 237–239 | 87 |
| 7 | C32H26N4 | 466 | Lemon yellow | 216–218 | 91 |
| 8 | C32H26N4 | 466 | Yellow | 233–235 | 95 |
| 9 | C32H26N4 | 466 | Faint pink | 257–259 | 88 |
M.f., Molecular formula; M.p., Melting point.
The molecular weight and formula of structure 2 were confirmed by electrospray ionization-mass spectrometry (ESI-MS) spectrum analysis displaying a molecular ion peak at m/z=363 [M+H]+, and the corresponding molecular formula C24H18N4 was established based on a high-resolution electron impact mass spectroscopy (HR-EI-MS) spectrum. In the 1H NMR spectrum, the formyl group of the indole 1a disappeared, while a singlet with integral 2H was formed at δ=8.79 ppm, and their corresponding carbons (C-8/8′) were visible at δ=154.8 ppm, confirming the newly formed imine functional groups in 2. The remaining NMR patterns for the 3-substituted indole moieties in 2 remained at their previous positions, including the 2NH proton signals (δ=11.77 ppm) and those for the residual 14 aromatic protons (δ=8.45–7.21 ppm). The 13C NMR spectrum displayed 11 carbon signals that were classified into four sp2 quaternary carbons, two of which were attached to nitrogen (δ=150.4 and 137.6 ppm). Six aromatic methine carbons were resonated between δ=133.7 and 122.0 ppm (Table 3). Moreover, the downfield methine carbon at δ=154.8 ppm was assigned to the imine carbon. Finally, a full assignment of bis-indole 2 was further confirmed by 1H,1H correlation spectroscopy (COSY) and heteronuclear multiple-bond correlations (HMBCs) (Supplementary Information, Figs. S3–S5).
13C (125 MHz) and 1H (500 MHz) NMR data of bis-indoles 2 and 3 in [D6]DMSO.
| No. | 2 | 3 | ||
|---|---|---|---|---|
| δC | δH (mult, J in Hz) | δC | δH (mult, J in Hz) | |
| 2/2′ | 133.7 | 8.01 (d, 2.6) | 136.3 | – |
| 3/3′ | 115.6 | – | 110.3 | 7.08 (m) |
| 3a/3′a | 125.2 | – | 128.0 | – |
| 4/4′ | 122.4 | 8.43 (m) | 122.0 | 7.66 (dd, 8.1, 1.2) |
| 5/5′ | 121.3 | 7.21 (td, 7.4, 1.2) | 120.2 | 7.05 (m) |
| 6/6′ | 123.2 | 7.25 (ddd, 8.2, 7.1, 1.4) | 124.7 | 7.24 (ddd, 8.2, 7.1, 1.2) |
| 7/7′ | 112.4 | 7.50 (m) | 112.7 | 7.48 (dq, 8.3, 0.9) |
| 7a/7′a | 137.6 | – | 138.5 | – |
| 8/8′ | 154.8 | 8.79 (s) | 151.4 | 8.69 (s) |
| 1″/4″ | 150.4 | – | 149.6 | – |
| 2″, 6″/3″, 5″ | 122.0 | 7.30 (s) | 122.5 | 7.42 (s) |
N,N′-(1,4-Phenylene)bis[1-(1H-indol-2-yl)methanimine] (3) was obtained by condensation of indole-2-carboxaldehyde (1b) with p-phenylenediamine. Structure 3 was confirmed by ESI-MS and HR-EI-MS exhibiting the same molecular mass and formula as 2. According to the 1H NMR spectrum, a characteristic singlet for the newly created 8/8′-imine functional group was resonated at δ=8.69 (C-8/8′: 151.4) ppm, while the remaining 1H NMR spectral patterns of 2-substituted indole systems showed no significant change. Additionally, the 13C NMR spectrum of 3 exhibited 11 carbon signals, which were classified into four sp2 quaternary carbons, three of which were attached to nitrogen (δ=149.6, 138.5 and 136.3 ppm), and the last quaternary one was resonated at δ=128.0 ppm. Six aromatic methine carbons were resonated at δ=124.7–110.3 ppm (Table 3). Moreover, the downfield methine carbon at δ=151.4 ppm was assigned to the imine carbon. Structure 3 was further fully assigned utilizing 1H,1H COSY and HMBCs (Supplementary Information, Figs. S11–S13).
Furthermore, indole-5-carboxaldehyde (1c) was condensed with p-phenylenediamine to form the corresponding bis-indole derivative 4, having the same molecular formula of 2 and 3. The 1H and 13C NMR spectra of 4 revealed the presence of the methine groups signals at δ=8.70 and 159.1(C-8/8′) ppm with higher chemical shifts than those shown for compounds 2 and 3, which might be attributed to its less influence by the neighboring indole NH groups.
The unsymmetrical bis-indole derivative 5 was formed by the one-pot condensation reaction of indole-3-carboxaldehyde (1a), p-phenylenediamine and indole-2-carboxaldehyde (1b) in the 1:1:1 ratio. The NMR spectra of 5 exhibited the presence of two imine groups at δ=8.78, and 8.69 ppm, and also the respective imine carbons were resonated at δ=154.8 and 151.4 ppm. Its ESI-MS spectrum displayed a molecular ion peak at m/z=363 [M+H]+, and the HR-EI-MS spectrum showed a molecular ion peak at m/z=362.15026, which corresponded to the molecular formula C24H18N4. Besides, N-{4-[(-(1H-indol-3-yl)methylene)amino]phenyl}-1-(1H-indol-5-yl)methanimine (6) was synthesized by the one-pot condensation reaction of indole-3-carboxaldehyde (1a), p-phenylenediamine and indole-5-carboxaldehyde (1c) in the 1:1:1 ratio. Its ESI-MS spectrum displayed a molecular ion peak at m/z=363 [M+H]+ and 361 [M–H]−, and also the HR-EI-MS spectrum was compatible with the molecular formula C24H18N4 (m/z=362.15034 [M]+).
4,4′-Ethylenedianiline was condensed with indole-3-carboxaldehyde (1a) under the same optimal reaction conditions to form a newly symmetrical bis-indole derivative 7, as cited in Scheme 2.
Reaction of indole-3-carboxaldehyde (1a) and 4,4′-ethylenedianiline.
The structure of 7 was supported by the following evidence. The 1H NMR spectrum showed a broad doublet signal at δ=11.75 ppm due to 2NH, and a singlet at δ=8.72 ppm which was assigned to the imine group. Signal integration between δ=8.39 and 7.21 ppm corresponded to 18 aromatic protons present in the molecule. In the aliphatic region, a singlet signal at δ=2.92 ppm due to 2CH2 was observed. The 13C NMR spectrum displayed a signal at δ=155.2 ppm which was assigned to the imine carbon, and a signal at δ=37.2 ppm due to 2CH2. The ESI-MS spectrum analysis of 7 showed a molecular ion peak at m/z=421 [M–2Na+H]−, in agreement with the proposed empirical formula.
Likewise, 4,4′-ethylenedianiline was condensed with 1b and 1c to form the corresponding bis-indole derivatives 8 and 9, respectively. Both ESI-MS and MALDI-MS spectra of 8 and 9 showed a molecular ion peak at m/z=467 [M+H]+.
2.2 Biological activity
Bis-indole derivatives 2–9 were subjected to cytotoxicity assay against the human cervix carcinoma cell line (KB-3-1) Resazurin assay [31], [32] in comparison with (+)-griseofulvin as a reference cytotoxic compound. However, none of the tested compounds displayed toxicity against this cell line. Furthermore, compounds 2–9 were submitted to antibacterial assay by the agar diffusion method [33] employing Gram positive bacteria, namely Bacillus subtilis, Micrococcus luteus and Staphylococcus warneri, as well as Gram negative bacteria, namely Escherichia coli and Pseudomonas agarici. Activities were compared to gentamycin as positive control, while dimethyl sulfoxide (DMSO) was used as negative control. Interestingly, compound 6 showed activity against all bacterial test strains, delivering inhibition diameters of 7–14 mm, with the best activity achieved against B. subtilis. In addition, compound 7 exhibited a moderate activity against E. coli, M. luteus and S. warneri with inhibition diameters of 7–8 mm. Furthermore, moderate activities were found for derivatives 3–5 and 9 against B. subtilis, with the best inhibition diameter of 12.5 mm for derivative 5. Compound 2 gave an inhibition zone with 7 mm diameter for S. warneri and compound 5 gave 8 mm for P. agarici; both results were in the low to moderate range of activity. It should be noted that out of eight derivatives tested, five displayed inhibition zones against B. subtilis, ranging from 6.5 to 14 mm, while other test strains showed much less overall susceptibility towards test compounds (Table 4).
Zones of inhibition of compounds 2–9 against bacterial strains (∅: mm).a
| No. | Conc. (mg mL−1) | E. coli | P. agarici | B. subtilis | M. luteus | S. warneri |
|---|---|---|---|---|---|---|
| 2 | 20 | – | – | – | – | 7 |
| 3 | 10 | – | – | 6.5 | – | – |
| 4 | 20 | – | – | 10 | – | – |
| 5 | 20 | – | 8 | 12.5 | – | – |
| 6 | 10 | 8 | 7 | 14 | 7 | 7 |
| 7 | 10 | 7 | – | – | 8 | 7.5 |
| 8 | 5 | – | – | – | – | – |
| 9 | 10 | – | – | 7 | – | – |
| Gentamycinb | 0.4 | 18 | 20 | 23 | 18 | 16 |
| DMSO | – | – | – | – | – |
a(–) no activity, breference.
3 Materials and methods
3.1 Analytical methods
Melting points were determined on a BÜCHI Melting Point B-540 apparatus (BÜCHI, Germany). NMR spectra (1H NMR, 13C NMR, COSY, heteronuclear multiple-quantum coherence and heteronuclear multiple-bond correlation) were measured on a Bruker Avance DRX 500 MHz (125 MHz for 13C NMR) spectrometer (Bruker, USA) with tetramethylsilane as the internal standard. Chemical shifts are reported relative to residual solvent peaks ([D6]DMSO: 1H: 2.50 ppm, 13C: 39.5 ppm). ESI mass spectra were recorded using an ion trap mass spectrometer equipped with a standard ESI/APCI source. Samples were introduced by direct infusion with a syringe pump. Nitrogen served both as the nebulizer gas and the dry gas. Nitrogen was generated by a nitrogen generator. Helium served as cooling gas for the ion trap and collision gas for MSn experiments. High-resolution mass spectra (HRMS) were performed using a Fourier transform ion cyclotron resonance mass spectrometer equipped with a 7.0 T, 160 mm bore superconducting magnet, infinity cell, and interfaced to an external (nano) ESI or matrix-assisted laser desorption/ionization (MALDI) ion source (Bruker Daltonik GmbH, Bremen, Germany). Nitrogen served both as the nebulizer gas and the dry gas for ESI. Nitrogen was generated by a nitrogen generator. Argon served as cooling gas in the infinity 3 cell and collision gas for MSn experiments. Scan accumulation and Fourier transformation were performed with XMASS NT 7.08 on a PC Workstation; for further data processing Data Analysis 3.4 was used. The microwave-assisted reactions were carried out in a CEM Discover, single-mode cavity with focused MW heating (MW power supply 0–300 W, IR temperature sensor, open or closed vessel mode, pressure range 0–20 bar, 10 mL or 80 mL vials). Starting materials, reagents, and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The purity of the synthesized compounds was investigated by thin-layer chromatography (TLC), performed on Merck precoated silica gel 60 F254 aluminum sheets with a solvent mixture of dichloromethane–methanol (95:5) as eluent. Spots were visualized under a UV lamp at 254 and 366 nm and again after spraying with anisaldehyde-H2SO4 reagent and heating to accelerate the reaction.
3.2 General synthetic procedure
A mixture of indole-carboxaldehyde (1a–c) (2 mmol) and bifunctional amine (p-phenylene-diamine or 4,4′-ethylenedianiline) (1 mmol) in methanol (3 mL) in the presence of four drops of glacial acetic acid was placed into a 10 mL vial and stirred at room temperature for 2 min. Irradiation was done by a microwave for 30 min at 65°C and 50 W. The progress of the reaction was monitored by TLC. After completion of the reaction, the obtained solid was filtered, washed with dichloromethane followed by methanol and dried to afford the title compounds 2–9.
3.2.1 N,N′-(1,4-Phenylene)bis[1-(1H-indol-3-yl)-methanimine] (2) [34]
1H and 13C NMR data (see Table 3). – MS [(+)-ESI]: m/z=363 [M+H]+. – HRMS (EI): m/z=362.15144 (calcd. 362.15260 for C24H18N4, [M]+).
3.2.2 N,N′-(1,4-Phenylene)bis[1-(1H-indol-2-yl)-methanimine] (3) [35]
1H and 13C NMR data (see Table 3). – MS [(+)-ESI]: m/z=363 [M+H]+.
3.2.3 N,N′-(1,4-Phenylene)bis[1-(1H-indol-5-yl)-methanimine] (4)
1H NMR (500.14 MHz, [D6]DMSO, 25°C): δ=11.45–11.40 (br m, 2H, 1/1′-H), 8.70 (s, 2H, 8/8′-H), 8.12–8.10 (m, 2H, 4/4′-H), 7.82 (dd, J=8.5, 1.5 Hz, 2H, 6/6′-H), 7.54–7.50 (m, 2H, 7/7′-H), 7.46–7.44 (m, 2H, 2/2′-H), 7.34 (s, 4H, 2″, 6″/3″, 5″-H), 6.59 (ddd, J=3.0, 1.9, 0.8 Hz, 2H, 3/3′-H). – 13C NMR (125.76 MHz, [D6]DMSO): δ=159.1 (C-8/8′), 147.8 (C-1″/4″), 136.1 (C-7a/7′a), 126.2 (C-3a/3′a), 125.9 (C-2/2′), 125.0 (C-5/5′), 121.7 (C-4/4′), 120.1 (C-2″, 6″/3″, 5″), 120.3 (C-6/6′), 119.1 (C-7/7′), 100.6 (C-3/3′). – MS [(+)-ESI]: m/z=363 [M+H]+.
3.2.4 N-{4-[(-(1H-Indol-2-yl)methylene)amino]phenyl}-1-(1H-indol-3-yl)methanimine (5)
1H NMR (500.14 MHz, [D6]DMSO, 25°C): δ =11.78–11.74 (m, 2H, 1/1′-H), 8.78 (s, 1H, 8′-H), 8.69 (s, 1H, 8-H), 8.44–8.40 (m, 1H, 4′-H), 8.01 (s, 1H, 2′-H), 7.65 (dd, J=8.0, 1.2 Hz, 1H, 4-H), 7.49 (tt, J=9.0, 1.0 Hz, 2H, 7/7′-H), 7.42 (s, 2H, 2″/6″-H), 7.29 (s, 2H, 3″/5″-H), 7.26–7.18 (m, 3H, 6/5′/6′-H), 7.08 (dq, J=3.9, 1.1 Hz, 2H, 3/5-H). – 13C NMR (125.76 MHz, [D6]DMSO): δ=154.8 (C-8′), 151.4 (C-8), 150.4 (C-4″), 149.6 (C-1″), 138.5 (C-7a), 137.6 (C-7′a), 136.3 (C-2), 133.7 (C-2′), 128.0 (C-3a), 125.2 (C-3′a), 124.7 (C-6), 123.2 (C-6′), 122.5 (C-2″/6″), 122.4 (C-4′), 122.0 (C-4/3″/5″), 121.3 (C-5′), 120.2 (C-5), 115.6 (C-3′), 112.7 (C-7), 112.4 (C-7′), 110.3 (C-3). – MS [(+)-ESI]: m/z=363 [M+H]+. – HRMS (EI): m/z=362.15026 (calcd. 362.15260 for C24H18N4, [M]+).
3.2.5 N-{4-[(-(1H-Indol-3-yl)methylene)amino]phenyl}-1-(1H-indol-5-yl)methanimine (6)
1H NMR (500.14 MHz, [D6]DMSO, 25°C ): δ =11.83–11.79 (br m, 1H, 1′-H), 11.49–11.44 (br m, 1H, 1-H), 8.83 (s, 1H, 8′-H), 8.74 (s, 1H, 8-H), 8.49–8.43 (m, 1H, 4′-H), 8.15 (d, J=1.5 Hz, 1H, 4-H), 8.05 (d, J=2.9 Hz, 1H, 2′-H), 7.86 (dd, J=8.6, 1.5 Hz, 1H, 6-H), 7.58–7.52 (m, 2H, 7/7′-H), 7.49 (t, J=2.8 Hz, 1H, 2-H), 7.37 (s, 2H, 2″/6″-H), 7.33 (s, 2H, 3″/5″-H), 7.29 (ddd, J=8.1, 7.0, 1.4 Hz, 1H, 6′-H), 7.24 (ddd, J=8.1, 7.1, 1.2 Hz, 1H, 5′-H), 6.63 (ddd, J=3.2, 2.0, 0.9 Hz, 1H, 3-H). – 13C NMR (125.76 MHz, [D6]DMSO): δ=161.2 (C-8), 154.8 (C-8′), 150.4 (C-4″), 149.8 (C-1″), 138.2 (C-7a), 137.6 (C-7′a), 133.7 (C-2′), 128.4 (C-3a), 128.1 (C-2), 127.1 (C-5), 125.2 (C-3′a), 123.8 (C-4), 123.2 (C-6′), 122.2 (C-4′), 122.0 (C-6), 121.3 (C-5′), 121.2 (C-7), 115.6 (C-3′), 112.4 (C-7′), 102.8 (C-3). – MS [(+)-ESI]: m/z=363 [M+H]+. – MS ((–)-ESI): m/z (%)=361 [M–H]−. – HRMS (EI): m/z=362.15034 (calcd. 362.15260 for C24H18N4, [M]+).
3.2.6 N,N′-[Ethane-1,2-diylbis(4,1-phenylene)]bis[1-(1H-indol-3-yl)methanimine] (7)
1H NMR (500.14 MHz, [D6]DMSO, 25°C): δ=11.75 (d, J=3.2 Hz, 2H, 1/1′-H), 8.72 (s, 2H, 8/8′-H), 8.39 (d, J=7.8 Hz, 2H, 4/4′-H), 7.99 (d, J=2.7 Hz, 2H, 2/2′-H), 7.49 (d, J=8.0 Hz, 2H, 7/7′-H), 7.25 (dd, J=7.7, 5.5 Hz, 6H, 6, 6′/3″, 5″/3′″, 5′″-H), 7.23–7.18 (m, 2H, 5/5′-H), 7.18–7.13 (m, 4H, 2″, 6″/2′″, 6′″-H), 2.92 (s, 4H, 7″/7′″-H). – 13C NMR (125.76 MHz, [D6]DMSO): δ=155.2 (C-8/8′), 151.4 (C-1″/1′″), 138.4 (C-4″/4′″), 137.6 (C-7a/7′a), 133.7 (C-2/2′), 129.6 (C-3″, 5″/3′″, 5′″), 125.2 (3a/3′a), 123.2 (C-6/6′), 122.3 (C-4/4′), 121.3 (C-5/5′), 121.1 (C-2″, 6″/2′″, 6′″), 115.6 (C-3/3′), 112.4 (C-7/7′), 37.2 (C-7″/7′″). – MS [(–)-ESI]: m/z=421 [M–2Na+H]−.
3.2.7 N,N′-[Ethane-1,2-diylbis(4,1-phenylene)]bis[1-(1H-indol-2-yl)methanimine] (8)
1H NMR (500.14 MHz, [D6]DMSO, 25°C): δ=11.74 (br s, 2H, 1/1′-H), 8.65 (s, 2H, 8/8′-H), 7.65 (d, J=7.1 Hz, 2H, 4/4′-H), 7.48 (d, J=1.2Hz, 2H, 7/7′-H), 7.32 (d, J=2.1 Hz, 4H, 3″, 5″/3′″, 5′″-H), 7.28 (d, J=2.5 Hz, 4H, 2″, 6″/2′″, 6′″-H), 7.20–7.17 (m, 2H, 6/6′-H), 7.15–7.11 (m, 2H, 3/3′-H), 7.05 (d, J=1.1 Hz, 2H, 5/5′-H), 2.95 (s, 4H, 7″/7′″-H). – 13C NMR (125.76 MHz, [D6]DMSO): δ=151.4 (C-8/8′), 149.4 (C-1″/1′″), 139.8 (C-4″/4′″), 138.4 (C-7a/7′a), 136.2 (C-2/2′), 129.8 (C-3″, 5″/3′″, 5′″), 128.0 (3a/3′a), 124.7 (C-6/6′), 122.0 (C-4/4′), 121.4 (C-2″, 6″/2′″, 6′″), 120.2 (C-5/5′), 112.6 (C-7/7′), 110.2 (C-3/3′), 37.1 (C-7″/7′″). – MS [(+)-ESI]: m/z=467 [M+H]+. – MS ((+)-MALDI): m/z=467 [M+H]+.
3.2.8 N,N′-[Ethane-1,2-diylbis(4,1-phenylene)]bis[1-(1H-indol-5-yl)methanimine] (9)
1H NMR (500.14 MHz, [D6]DMSO, 25°C): δ=11.46 (d, J=2.9 Hz, 2H, 1/1′-H), 8.68 (s, 2H, 8/8′-H), 8.13 (d, J=1.7 Hz, 2H, 4/4′-H), 7.84 (dd, J=8.6, 1.6 Hz, 2H, 6/6′-H), 7.55 (d, J=8.5 Hz, 2H, 7/7′-H), 7.49 (t, J=2.8 Hz, 2H, 2/2′-H), 7.34–7.32 (m, 4H, 3″, 5″/3′″, 5′″-H ), 7.25–7.22 (m, 4H, 2″, 6″/2′″, 6′″-H), 6.62 (d, J=2.5 Hz, 2H, 3/3′-H), 2.98 (s, 4H, 7″/7′″-H). – 13C NMR (125.76 MHz, [D6]DMSO): δ=161.4 (C-8/8′), 150.4 (C-1″/1′″), 139.0 (C-4″/4′″), 138.2 (C-7a/7′a), 129.6 (C-3″, 5″/3′″, 5′″), 128.3 (3a/3′a), 128.0 (C-2/2′), 127.1 (C-5/5′), 123.8 (C-4/4′), 122.0 (C-6/6′), 121.2 (C-2″, 6″/2′″, 6′″), 121.1 (C-7/7′), 102.8 (C-3/3′), 37.1 (C-7″/7′″). – MS [(+)-ESI]: m/z=467 [M+H]+. – MS ((+)-MALDI): m/z=467 [M+H]+.
3.3 Pharmacology In vitro
3.3.1 cytotoxicity assay
KB 3-1 cells were cultivated as a monolayer in Dulbecco’s modified Eagle medium with glucose (4.5 g L−1), l-glutamine, sodium pyruvate and phenol red, supplemented with 10% of KB-3-1 and fetal bovine serum (FBS). Cells were maintained at 37°C and 5.3% CO2-humidified air. One day prior to the test, cells of 70% confluence were detached with 0.05%/0.02% of trypsin-ethylendiamine tetraacetic acid solution in Dulbecco’s phosphate buffered saline and placed in sterile 96-well plates in a density of 10 000 cells in 100 μL medium per well. The dilution series of the compounds were prepared from stock solutions in DMSO of concentrations of 100 μm, 50 μm or 25 μm. The stock solutions were diluted with culture medium (10% FBS [KB-3-1]) down to the pm range and dilutions added to the wells. Each concentration was tested in six replicates. Dilution series were prepared by pipetting liquid from well to well, with the control containing the same concentration of DMSO as the first dilution. After incubation for 72 h at 37°C and 5.3% CO2-humidified air, 30 μL of aqueous Resazurin solution (175 μm) was added to each well. The cells were incubated under the conditions mentioned above for further 5 h and fluorescence was measured (λex=530 nm, λem=588 nm). Results were given as IC50, which were calculated as the average of two determinations by the sigmoidal dose-response curve fitting model using Graph Pad Prism software version 4.03 [31], [32].
3.3.2 Agar plate diffusion assay
By using a sterile pipette, 0.2 mL of the broth culture of each test bacterium was added to a sterile petri dish containing a nutrient broth agar or trypticase soy broth layer. Sterile paper disks of diameter 6 mm were impregnated with 10 or 20 mg mL−1 of pure compounds dissolved in DMSO, dried under sterile conditions and disks were placed on agar plates pre-inoculated with broth cultures of the Gram positive bacteria B. subtilis, M. luteus or S. warneri or Gram negative bacteria E. coli or P. agarici. Plates were incubated for 24 h at 30–37°C and diameters of inhibition zones measured by a ruler in mm. Gentamycin at 0.4 mg mL−1 was used as positive control for each plate with test strain [33], and DMSO as negative control.
Supplementary information
NMR spectra and other supplementary data associated with this article are given as Supplementary Information available online (https://doi.org/10.1515/znb-2017-0039).
Acknowledgments
The authors are grateful to the NMR and MS Departments at Bielefeld University for spectral measurements. The authors would also like to thank Carmela Michalek for biological activity testing and Marco Wißbrock and Anke Nieß for technical assistance. This research work has been financed by the German Academic Exchange Service (DAAD) with funds from the German Federal Foreign Office in the frame of the Research Training Network “Novel Cytotoxic Drugs from Extremophilic Actinomycetes” (Project ID57166072).
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