Abstract
The naturally occurring oxygenated bibenzyls lunularin and m-O-methyllunularin were prepared in a modular synthesis in four steps from two appropriate iodophenols and trimethylsilylacetylene utilizing microwave-assisted Sonogashira couplings as the crucial steps. The antimicrobial activity of the resulting natural products was evaluated in an agar diffusion assay.
1 Introduction
Lunularin (1a), its m-O-methyl ether (1b) (Fig. 1), as well as several related bibenzyls were isolated from liverworts (Hepaticaceae), algae, and higher plants such as Hydrangea or Morus macroura [1–5]. In addition, numerous dimers of these compounds were isolated from liverworts. The bibenzyls show a broad variety of pharmacological effects like antibiotic, antifungal, as well as plant growth inhibitory activities. Lunularin (1a) is a metabolite of lunularic acid (Fig. 1), formed by enzymatic decarboxylation. Biosynthesis of lunularin is starting from phenylalanine and three units of malonyl-CoA and leads via prelunularic acid and lunularic acid to lunularin [6].
Lunularin and related compounds.
Until now, several total syntheses of these natural polyketides were published. The first total syntheses of lunularin were described by Huneck in 1976 [7] and Gorham in 1977 [1] using a Wittig reaction as key step. A similar approach was used by Sawada et al. in 2012 [8]. In 2000, our group described a synthesis by a palladium-catalyzed cross-coupling of styrene derivatives and phenyl triflates [9]. These published syntheses require five or six steps and lead to the target compounds in poor to moderate total yields.
This prompted us to work out a novel, modular synthesis of oxygenated bibenzyls utilizing iodoarenes as building blocks for both aromatic rings and trimethylsilylacetylene as the central C2 building block. Both C,C coupling reactions were to be performed by transition metal-catalyzed coupling reactions. The Sonogashira coupling [10] is an important reaction in the synthesis of natural products, with a wide area of applications [11–14]. In the last years, microwave-assisted Sonogashira couplings have been included in modern organic synthesis [15].
2 Results and discussion
2.1 Chemistry
4-Iodophenol (2) was reacted in a microwave reactor with trimethylsilylacetylene (3) under Sonogashira conditions (CuI, tertiary amine, Pd catalyst) to give the alkyne 4. The Sonogashira reaction could be catalyzed by bis(triphenylphosphine)palladium(II) dichloride or 1,1′-[bis(diphenylphosphino)ferrocene]palladium(II) dichloride, with either catalyst excellent yields were obtained (Scheme 1).
Synthesis of lunularin (1a) and m-O-methyllunularin (1b).
Cleavage of the trimethylsilyl group of 4 was first attempted with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in acetonitrile/water (19:1) [16], but this reaction led to a hydration of the alkyne function to give 4-hydroxyacetophenone (8) in 29 % yield (Scheme 2). Alkaline cleavage of the trimethylsilyl group with methanolic K2CO3 led to the terminal alkyne 5in about 25 % yield [17]. Best results for the cleavage of the trimethylsilyl group were obtained with tetra-n-butylammonium fluoride (TBAF) in methanol, which led to a quantitative desilylation of 4 (for related Sonogashira couplings with phenoxide-mediated in situ-desilylation, see Ref. [18]). The first two steps of this approach gave almost quantitative yields, so the crude products can as well be used for further conversion without purification by column chromatography.
In a second Sonogashira reaction under the same conditions, arylalkyne 5 was reacted with 3-iodophenol (6a) or 3-iodoanisole (6b) to give the diarylalkynes 7a and 7b. As by-product of the Sonogashira reaction, a trace amount of the 1,3-diyne 9was obtained as a product of a Glaser coupling of 5[19] (Scheme 2). This dimer was difficult to separate from 7a/7b by column chromatography, but after hydrogenation (see below), it was much easier to separate the resulting diarylbutane derivative 10.
Side reactions of the lunularin synthesis.
Subsequent hydrogenation of 7a/7b under Pt catalysis gave the target compounds 1a and 1b. Lunularin (1a) was obtained in an overall yield of 57 %, its methyl ether 1b in 34 % yield.
2.2 Antimicrobial evaluation
The antimicrobial activity of target compounds 1a and 1b was evaluated in an agar diffusion assay [20] against four bacteria (Gram-negative: Escherichia coli and Pseudomonas marginalis; Gram-positive: Staphylococcus equorum and Streptococcus entericus) and four fungi (the yeasts Candida glabrata and Yarrowia lipolytica, the mold Aspergillus niger, and the dermatophyte Hyphopichia burtonii) (Table 1). Lunularin (1a) showed weak antibiotic activity against Gram-positive bacteria and antifungal activity in this assay, compared to the reference substances tetracycline (antibacterial) and clotrimazole (antifungal). Astonishingly, m-O-methyllunularin (1b) showed a different antimicrobial spectrum and showed significantly higher antifungal activity than lunularin.
Agar diffusion assay (50 μg per disc; diameter of inhibition, in mm).
| E. coli | P. marginalis | S. equorum | Str. entericus | Y. lipolytica | C. glabrata | A. niger | H. burtonii | |
|---|---|---|---|---|---|---|---|---|
| 1a | 0 | 0 | 11 | 9 | 7 | 7 | 0 | 6 |
| 1b | 0 | 0 | 10 | 10 | 15 | 12 | 10 | 10 |
| Tetracycline | 32 | 28 | 28 | 34 | 0 | 0 | 0 | 0 |
| Clotrimazole | 0 | 0 | 11 | 12 | 22 | 18 | 20 | 25 |
3 Conclusion
We have worked out a novel, modular four-step synthesis of oxygenated bibenzyls starting from readily available building blocks (iodoarenes, trimethylsilylacetylene) utilizing two consecutive microwave-assisted Sonogashira couplings. This protocol does not require protective groups for phenolic residues and should be applicable to the synthesis of numerous related natural products and related compounds. In the agar diffusion assay, m-O-methyllunularin (1b) showed a remarkable antifungal activity.
4 Experimental section
4.1 General information
All solvents used were of HPLC grade or p.a. grade and/or purified according to standard procedures. Chemical reagents were purchased from Sigma Aldrich (Schnelldorf, Germany) and Acros (Geel, Belgium). Microwave-assisted reactions were carried out in a microwave reactor Discover SP (CEM GmbH, Kamp-Lintfort, Germany). IR spectra: Perkin-Elmer FT-IR Paragon 1000 (Perkin Elmer, Rodgau, Germany); MS: Hewlett Packard MS-Engine, electron ionization (EI) 70 eV, chemical ionization (CI) with CH4 (300 eV); GC-MS: Shimadzu GC17A, carrier gas: He, column: fused silica capillary column 30 m, detector: MS: EI (70 eV) (Shimadzu Europa GmbH, Duisburg, Germany). NMR: Avance III HD 400 MHz Bruker BioSpin (1H, 400 MHz; 13C, 100 MHz); Avance III HD 500 MHz Bruker BioSpin (1H, 500 MHz; 13C, 125 MHz) (Bruker, Billerica, MA, USA); melting points: Büchi Melting Point B-540 (not corrected) (Büchi Labortechnik AG, Flawil, Switzerland); flash column chromatography (FCC): silica gel 60 (230–400 mesh; E. Merck, Darmstadt, Germany).
4.2 4-((Trimethylsilyl)ethynyl)phenol (4)
4-Iodophenol (2) (440 mg, 2.0 mmol) and trimethylsilylacetylene (0.42 mL, 3.0 mmol) were dissolved in 10 mL of diethylmethylamine and 10 mL of dimethylformamide, and then, 60 mg (0.30 mmol) of CuI and 70 mg (0.10 mmol) of PdCl2(PPh3)2 were added. The solution was stirred in a microwave reactor at 60 °C for 20 min (100 W) under N2 atmosphere. The solvent was evaporated, and the residue was dispersed in 20 mL aqueous 2 M HCl and extracted with diethyl ether (3 × 20 mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated, and the residue purified with FCC (isohexane-ethyl acetate 3:1) to give 380 mg (100 %) of 4 as a brown oil. – 1H NMR (400 MHz, CDCl3): δ = 0.23 (s, 9 H, 3 CH3), 6.76 (d, J = 8.8 Hz, 2 H, aromat. CH), 7.34 (d, J = 8.8 Hz, 2 H, aromat. CH). – 13C NMR (125 MHz, CDCl3): δ = 0.19 (3 CH3), 92.12 (quat. C), 105.39 (quat. C), 114.98 (quat. C), 115.48 (2 aromat. CH), 133.51 (2 aromat. CH), 156.44 (quat. C). These data are in accordance with those published in Ref. [12]. – GC-MS (EI): m/z (%) = 190 (23) [M]+, 175 (100). – HRMS: m/z = 190.0814 (calcd. 190.0815 for C11H14O2Si, [M]+). – IR (KBr): ν (cm−1) = 3322, 2959, 2155, 1662, 1607, 1508, 1437, 1251, 1230, 1167, 1100, 867, 840.
4.3 4-Ethynylphenol (5)
4 (380 mg, 2.0 mmol) was dissolved in 20 mL methanol, and 1.0 g (2.0 mmol) of TBAF was added. The suspension was stirred for 12 h at ambient temperature, and then, the solvent was evaporated. The residue was dissolved in 20 mL of 2 M aqueous HCl and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over Na2SO4, and the solvent was evaporated. The residue was purified by FCC (isohexane-ethyl acetate 2:1) to give 230 mg (97 %) of 5 as a pale brown oil. – 1H NMR (400 MHz, [D4]methanol): δ = 4.24 (s, 1 H, CH), 4.32 (s, 1 H, OH), 6.78 (m, 2 H, aromat. CH), 7.35 (m, 2 H, aromat. CH). – 13C NMR (125 MHz, CDCl3): δ = 79.07 (CH), 87.87 (quat. C), 116.81 (quat. C), 119.25 (2 aromat. CH), 137.50 (2 aromat. CH), 161.77 (quat. C). – GC-MS (EI): m/z (%) = 118 (100) [M]+. – IR (KBr): ν (cm–1) = 3264, 1657, 1603, 1582, 1512, 1361, 1280, 1170, 838. These data are in accordance with those published in Ref. [21].
4.4 4-Hydroxyacetophenone (8)
4 (380 mg, 2.0 mmol) was dissolved in acetonitrile/water (19:1), and 304 mg (2.0 mmol) of DBU was added. The solution was stirred for 1 h at 70 °C in a microwave reactor (210 W). The solvent was evaporated, and the residue was purified by FCC (isohexane-ethyl acetate 2:1) to give 70 mg (29 %) of 4-hydroxyacetophenone as a brown solid. – 1H NMR (400 MHz, CDCl3): δ = 2.57 (s, 3 H, CH3), 5.90 (s, 1 H, OH), 6.89 (d, J = 8.8 Hz, 2 H, aromat. CH), 7.91 (d, J = 8.8 Hz, 2 H, aromat. CH).
4.5 3-[(4-Hydroxyphenyl)ethynyl]phenol (7a)
5 (210 mg, 1.78 mmol), 3-iodophenol (6a) (440 mg, 2.0 mmol), CuI (60 mg, 0.30 mmol), and PdCl2(PPh3)2 (70 mg, 0.10 mmol) were reacted according to the procedure described for 4 to give 228 mg (61 %) of 7a as a pale-brown solid after purification by FCC (isohexane/ethylacetate 3:1); m.p. 172 °C. – 1H NMR (400 MHz, [D4]methanol): δ = 3.94 (s, 2 H, 2 OH), 6.79 (m, 3 H, aromat. CH), 6.90–7.23 (m, 2 H, aromat. CH), 7.38 (m, 3 H, aromat. CH). – 13C NMR (125 MHz, [D4]methanol): δ = 88.32 (quat. C), 90.03 (quat. C), 115.36 (quat. C), 116.41 (aromat. CH), 116.53 (2 aromat. CH), 118.79 (aromat. CH), 123.71 (aromat. CH), 126.12 (quat. C), 130.53 (aromat. CH), 134.09 (2 aromat. CH), 158.54 (quat. C), 159.20 (quat. C). – GC-MS (EI): m/z (%) = 210 (100) [M]+, 181 (12), 152 (19). – HRMS: m/z = 210.0706 (calcd. 210.0681 for C14H10O2, [M]+).
4.6 4-((3-Methoxyphenyl)ethynyl) phenol (7b)
5 (235 mg, 1.9 mmol), 6b (468 mg, 2.0 mmol), CuI (60 mg, 0.30 mmol), and PdCl2(PPh3)2 (70 mg, 0.10 mmol) were reacted according to the procedure described for 4 to give 158 mg (36 %) of 7b as a pale-brown oil after purification by FCC (isohexane-ethyl acetate 4:1). – 1H NMR (400 MHz, CDCl3): δ = 3.83 (s, 3 H, OCH3), 6.82 (d, J = 8.5 Hz, 2 H, aromat. CH), 6.88 (dd, J = 2.0 Hz, J = 7.8 Hz, 1 H, aromat. CH), 7.04 (s, 1 H, aromat. CH), 7.11 (d, J = 7.8 Hz, 1 H, aromat. CH), 7.25 (dd, J = 7.8 Hz, J = 7.8 Hz, 1 H, aromat. CH), 7.43 (d, J = 8.5 Hz, 2 H, aromat. CH). – 13C NMR (100 MHz, CDCl3): δ = 55.31 (OCH3), 87.88 (quat. C), 89.19 (quat. C), 114.65 (aromat. CH), 115.23 (quat. C), 115.61 (2 aromat. CH), 116.15 (aromat. CH), 124.05 (aromat. CH), 124.58 ( quat. C), 129.39 (aromat. CH), 133.30 (2 aromat. CH), 156.08 (quat. C), 159.31 (quat. C). – GC-MS (EI): m/z (%) = 224 (100) [M]+, 181 (21), 152 (19). – HRMS: m/z = 224.0817 (calcd. 224.0837 for C15H12O2, [M]+).
4.7 Lunularin (1a)
7a (204 mg, 0.97 mmol) was dissolved in 25 mL of methanol, 40 mg of platinum on carbon (5 %) was added, and the mixture was stirred under H2 atmosphere for 12 h. The catalyst was filtered off and the solvent was evaporated; the residue was purified by FCC (isohexane-ethyl acetate 2:1) to give 200 mg (96 %) of 1a as a pale brown solid; m.p. 125 °C (Ref. [7], 106 °C). – 1H NMR (400 MHz, CD2Cl2): δ = 2.73 (s, 4 H, 2 CH2), 5.35 (s, 2 H, 2 OH), 6.57 (s, 1 H, aromat. CH), 6.63 (m, 1 H, aromat. CH), 6.66 (d, J = 7.8 Hz, 2 H, 2 aromat. CH), 6.92 (m, 1 H, aromat. CH), 6.95 (d, J = 7.8 Hz, 2 H, aromat. CH), 7.04 (dd, J = 8.1 Hz, J = 8.1 Hz, 1 H, aromat. CH). – 13C NMR (100 MHz, CD2Cl2): δ = 35.91 (CH2), 37.12 (CH2), 111.88 (aromat. CH), 114.23 (2 aromat. CH), 114.54 (aromat. CH), 119.95 (aromat. CH), 128.59 (aromat. CH), 128.70 (2 aromat. CH), 133.02 (quat. C), 143.90 (quat. C), 153.21 (quat. C), 155.02 (quat. C). – GC-MS (EI): m/z (%) = 214 (10) [M]+, 122 (2), 107 (100). All spectroscopic data were in full accordance with those published in Refs. [4, 8, 9].
4.8 4-((3-Methoxyphenyl)ethyl)phenol; (m-O-methyllunularin) (1b)
7b (148 mg, 0.63 mmol) was dissolved in 2 mL of ethyl acetate and 25 mL of methanol, 50 mg of platinum on carbon (5 %) was added, and the mixture was stirred under H2 atmosphere for 12 h. The catalyst was filtered off, the solvent was evaporated, and the residue was purified by FCC (isohexane-ethyl acetate 2:1) to give 142 mg (99 %) of 1b as a pale-brown oil. – 1H NMR (400 MHz, [D4]methanol): δ = 2.80 (m, 4 H, 2 CH2), 3.72 (s, 3 H, CH3), 4.92 (s, 1 H, OH), 6.71 (d, J = 8.4 Hz, 2 H, aromat. CH), 6.67–6.75 (m, 3 H, aromat. CH), 6.97 (d, J = 8.4 Hz, 2 H, aromat. CH), 7.14 (dd, J = 7.6 Hz, J = 7.6 Hz, 1 H, aromat. CH). – 13C NMR (100 MHz, CDCl3): δ = 39.75 (CH2), 41.05 (CH2), 57.11 (CH3), 113.86 (aromat. CH), 116.72 (aromat. CH), 117.59 (2 aromat. CH), 123.53 (aromat. CH), 131.75 (aromat. CH), 132.05 (2 aromat. CH), 135.51 (quat. C), 146.36 (quat. C), 157.97 (quat. C), 162.59 (quat. C). – GC-MS (EI): m/z (%) = 228 (27) [M]+, 121 (14), 107 (100). – HRMS: m/z = 228.1141 (calcd. 228.1150 for C15H16O2, [M]+). The 13C NMR data were in full accordance with those published in Ref. [2].
4.9 4,4′-(Buta-1,3-diyne-1,4-diyl) diphenol (9)
This compound was obtained as by-product of the syntheses of 7a and 7b. – GC-MS (EI): m/z (%) = 234 (100) [M]+, 176 (13). The spectroscopic data are in full accordance with those published in Ref. [22].
4.10 4,4′-(Butane-1,4-diyl)diphenol (10)
This compound was obtained as by-product of the synthesis of 1a and 1b, after not extensively purified samples of 7a/7b had been subjected to catalytic hydrogenation. – 1H NMR (400 MHz, CD2Cl2): δ = 1.62 (t, J = 7.0 Hz, 4 H, 2 CH2), 2.57 (t, J = 7.0 Hz, 4 H, 2 CH2), 5.05 (s, 2 H, 2 OH), 6.75 (d, J = 8.1 Hz, 4 H, aromat. CH), 7.02 (d, J = 8.1 Hz, 4 H, aromat. CH). – 13C NMR (100 MHz, CD2Cl2): δ = 31.7 (2 CH2), 35.16 (2 CH2), 115.27 (4 aromat. CH), 129.75 (4 aromat. CH), 135.39 (quat. C), 154.14 (quat. C). – GC-MS (EI): m/z (%) = 242 (10) [M]+, 212 (27), 165 (15), 107 (100). – HRMS: m/z = 242.1305 (calcd. 242.1307 for C16H18O2, [M]+).
4.11 Agar diffusion assay [20]
The bacteria and fungi were cultivated on an AC agar (Sigma). The substances were placed on the agar on 6-mm paper discs, each impregnated with 50 μg of the tested compound or 50 μg of the reference drug. The bacteria media were incubated for 24 h at 32 °C, the fungi media for 48 h at 28 °C. The diameters of the zones of inhibition (mm) were registered manually. Tests were performed in duplicate.
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