Bioactive Coumarin Derivatives from the Fern Cyclosorus interruptus

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material
• Extraction and isolation
• Antibacterial activity
• KB cell activity
• Results and Discussion
• Acknowledgements
• References

Abstract

Three new coumarin derivatives, compounds 1 - 3, three new furanocoumarins, compounds 4 - 6, and a novel dioxocane derivative, compound 7, were isolated from the fern Cyclosorus interruptus (Willd.) H. Itô. Based on spectrometric and spectroscopic analysis (FAB or EI mass spectrometry as well as 1D and 2D NMR experiments) their structures were characterised as 5,7-dihydroxy-6-methyl-4-phenyl-8-(3-phenylpropionyl)-1-benzopyran-2-one (1), 5,7-dihydroxy-6-methyl-4-phenyl-8-(3-phenyl-trans-acryloyl)-1-benzopyran-2-one (2), 5,7-dihydroxy-8-(2-hydroxy-3-phenylpropionyl)-6-methyl-4-phenyl-1-benzopyran-2-one (3), 8-benzyl-5,8-dihydroxy-6-methyl-4-phenylfuro[2,3-h]-1-benzopyran-2,9-dione (4), 8-benzyl-5,8β,9β-trihydroxy-6-methyl-4-phenyl-8,9-dihydro-furo[2,3-h]-1-benzopyran-2-one (5), 8-benzyl-5,8β,9α-trihydroxy-6-methyl-4-phenyl-8,9-dihydro-furo[2,3-h]-1-benzopyran-2-one (6) and 5,11-dihydroxy-6-methyl-4-phenyl-11-(1-phenylmethyl)-7,10-dioxocane [5,6-h]-1-benzopyran-2,12-dione (7). For these compounds we propose the trivial names interruptins A - F. Compounds 1, 5/6 and 7 showed antibacterial activity while compounds 1 and 2 were cytotoxic to a KB cell line.

Introduction

Continuing our phytochemical studies aimed at the discovery of novel bioactive agents from natural sources, we have investigated the leaves of Cyclosorus interruptus (Willd.) H. Itô (Thelypteridaceae), one of the plants used in the traditional medicine of Papua New Guinea (PNG). The leaves of this fern are used in folk medicine for the treatment of cough and burns, as well as against malaria and general sickness [1], [2], [3].

Materials and Methods

General

UV spectra were taken in MeOH on a Kontron-Uvikon 930 spectrophotometer and the optical rotations were measured in MeOH with a Perkin-Elmer model 241 polarimeter. The NMR spectra were recorded on Bruker AMX 300 (operating at 300.13 MHz for ¹H and 75.47 MHz for ¹³C) and DRX 500 (operating at 500.13 MHz for ¹H and 125.9 MHz for ¹³C) spectrometers. Compounds were measured in CDCl₃ (1, 2 and 4), CD₃OD (3) and DMSO-d ₆ (5, 6 and 7). As internal standard the resonances of the residual undeuterated solvents were used. FABMS of the compounds were obtained in the positive mode on a ZAB 2-SEQ (VG) spectrometer at 8.3 KeV, using 3-nitrobenzyl alcohol as matrix (3, 4 and 7), while EIMS were obtained on a Hitachi-Perkin-Elmer-RMUGM mass spectrometer at 70 eV (1, 2, 5 and 6). TLC was performed on precoated silica gel 60 F₂₅₄ plates (Merck) and detection was achieved by spraying with vanillin-H₂SO₄, followed by heating.

Plant material

Leaves of Cyclosorus interruptus (Willd.) H. Itô were collected in the Central province in Papua New Guinea in September 1996. The plant was identified by Pius Piskaut (University of Papua New Guinea) and Dr. M. M. J. van Balgooy, Rijksherbarium, Leiden, The Netherlands, where a voucher specimen with the identification number ETH 96/10 is deposited.

Extraction and isolation

Air-dried leaves (1.7 kg) were ground and extracted with CH₂Cl₂ by percolation at room temperature to give 62.9 g dried extract. The remaining plant material was then extracted with MeOH, giving 260.1 g lyophilised extract and MeOH-H₂O (7 : 3), yielding further 98.6 g crude extract.

An aliquot (44 g) of the dichloromethane extract was subjected to flash chromatography on silica gel (particle size 40 - 60 μm, 150 g) using a step gradient of hexane with increasing concentrations of EtOAc and EtOAc with increasing concentrations of MeOH to give 11 fractions (B1 - B11). Fraction B5 (6.1 g, eluted with hexane-EtOAc [6 : 4]) was subjected to open column chromatography over silica gel using the same step gradient and giving compound 1 (11.6 mg, eluted with hexane-EtOAc [1 : 1]) and 2 (17.3 mg, eluted with hexane-EtOAc [65 : 35]), which was pure by TLC (silica gel, solvent system: hexane-EtOAc [1 : 1], R_f = 0.3 and 0.5, respectively). Fraction B6 (3.2 g, eluted with hexane-EtOAc [4 : 6]) was subjected to MPLC (Büchi 681 pump) with a step gradient (hexane-EtOAc [7 : 3] to MeOH, flow 15 ml/min) on a Büchi MPLC column, 45 × 3.5 cm, packed with silica gel (Merck, 15 μm, 225 g), to give 10 subfractions (B6.1 - 10). Subfraction B6.6 (eluted with hexane-EtOAc [1 : 9]) was separated by open column chromatography using a gradient (hexane-EtOAc [1 : 1] to MeOH) and giving a mixture of compounds 5 and 6 (9.6 mg, eluted with hexane-EtOAc [1 : 1]). Subfraction B6.7 (eluted with EtOAc-MeOH [9 : 1]) was separated by RP-MPLC (Büchi MPLC column, 50 × 2 cm, packed with RP18 silica gel, 15 - 35 μm, 220 g, with acetonitrile-H₂O [4 : 6] as eluent, flow 8.2 ml/min) to give a mixture of compound 3 and an unknown pollution (2.3 mg) as well as the pure compound 7 (1.7 mg), both eluted with acetonitrile-H₂O [4 : 6].

Fraction B9 (5.0 g, eluted with EtOAc-MeOH [9 : 1]) was partitioned between hexane and methanol. The methanol fraction was subjected to open column chromatography over silica gel (particle size 60 - 200 μm, 280 g) using a step gradient of hexane-EtOAc followed by EtOAc with increasing concentrations of MeOH giving 14 fractions (B9.1 - 14). Fraction B9.11 (eluted with EtOAc-MeOH [93 : 7]) was subjected to open column chromatography over Sephadex-LH 20 (53 g) to elute compound 4 (1.2 mg) with pure methanol.

Antibacterial activity

The antibacterial assays of the crude plant extracts, fractions and isolates were carried out by the bioautographic agar overlay technique [4]. As test organisms B. cereus (ATCC 10702), S. epidermidis (ATCC 12228) and M. luteus (ATCC 9341) were used. The MIC values of the pure compounds were determined by the broth doubling dilution method [5] using a modified procedure. Bacterial suspensions were obtained from overnight cultures in BBL nutrient broth (Becton & Dickinson Co. 11479) cultivated at 37 °C and diluted to ca. 10⁵ cells/ml in fresh medium. The isolates were dissolved in MeOH to 1 mg/ml as stock solutions. The required amount of stock solution was pipetted into the wells of the first column of a 96-well tissue culture plate (Falcon) and dried. The sample was redissolved in 50 μl DMSO, 50 μl sterile BBL nutrient broth and 100 μl dilute culture suspension. Twofold dilutions were made in 100 μl volumes of dilute bacterial suspensions. The plates were kept in a moist atmosphere at 37 °C for 20 h. After incubation, 10 μl of 0.25 % aqueous solution of thiazolyl blue tetrazolium bromide were added in each well and incubated for 4 h to detect living bacteria as violet turbid solutions. Chloramphenicol was used as a positive control. All pure compounds were tested within the range of 64 - 0.5 ppm.

KB cell activity

Inhibition of KB cell growth (cell line ATCC CCL 17; human nasopharyngeal carcinoma) was tested in 96-well plates, with dilutions between 0.1 and 10 ppm [6]. Compounds showing no activity at 10 ppm were considered as not active.

Interruptin A (1): yellow-white powder, [α]²⁵ _D: 0.0° (c 0.05, MeOH), m.p. 143 - 145 °C. For ¹³C- and ¹H-NMR data see Tables [1] and [2]. UV (MeOH): λ_max = 417, 283, 214 nm. EIMS: m/z (rel. int.) = 400 [M]⁺ (36), 382 (7), 295 (100), 91 (24).

Interruptin B (2): yellow powder, [α]²⁵ _D: 0.0° (c 0.10, MeOH), m.p. 222 - 224 °C. For ¹³C- and ¹H-NMR data see Tables [1] and [2]. UV (MeOH): λ_max = 331, 328, 223 nm. EIMS: m/z (rel. int.) = 398 [M]⁺ (11), 321 (8), 294 (4), 266 (3), 91 (4), 44 (100).

Interruptin C (3): For ¹³C- and ¹H-NMR data see Tables [1] and [2]. UV (MeOH): λ_max = 360, 290 nm. FABMS: m/z (rel. int.) = 441 [M + 2H + Na]⁺ (25), 417 [M + H]⁺ (54), 295 (80). Optical rotation and m.p. could not be measured, because the compound was instable and completely decomposed.

Interruptin D (4): small white crystals, m.p. 181 - 183 °C. For ¹³C- and ¹H-NMR data see Tables [1] and [2]. UV (MeOH): λ_max = 400 (sh), 372, 288, 207 nm. FABMS: m/z (rel. int.) = 415 [M + H]⁺ (100), 397 (5), 295 (20). Optical rotation could not be measured, because of the small amount.

Interruptins E and F (5 and 6; mixture): m.p. 118 - 120 °C. For ¹³C- and ¹H-NMR data see Tables [1] and [2]. UV (MeOH): λ_max = 331, 268, 210 nm. EIMS: m/z (rel. int.) = 398 [M - H₂O]⁺ (100), 321 (82), 294 (36). Optical rotation of the mixture was not measured.

Interruptin G (7): m.p. 162 - 164 °C. For ¹³C- and ¹H-NMR data see Tables [1] and [2]. UV (MeOH): λ_max = 407, 375, 290, 208 nm. FABMS: m/z (rel. int.) = 460 [M + 2H]⁺ (6), 398 (66), 395 (100). Optical rotation could not be measured, because of the small amount.

Results and Discussion

After different chromatographic separations, the plant extract afforded three coumarin derivatives (1 - 3), three furanocoumarins (4 - 6) and a novel dioxocane-coumarin (7).[]

Interruptin A (1) was obtained as a yellow-white powder. The molecular formula C₂₅H₂₀O₅ was assigned on the basis of EIMS (m/z 400 [M]⁺) and ¹³C-NMR. A fragment at m/z 295 [M - 105]⁺ suggested the presence of an ethyl-substituted aromatic rest, while m/z 382 showed the loss of water [M - H₂O]⁺. The ¹H-NMR spectrum (in CDCl₃, Table [2]) exhibited protons of two CH₂ groups at δ 3.29 (2H, t, J = 7.6 Hz, H-2′) and δ 2.96 (2H, t, J = 7.6 Hz, H-3′). A resonance of a methyl group was recorded at δ 2.28 (3H, s, CH₃) while a single proton was shown at δ 5.95 (1H, s, H-3). The other signals were overlapping aromatic signals (δ 7.1 - 7.4). The ¹³C-NMR spectra (Table [1]) exhibited 21 signals due to 25 carbons, including one methyl, eleven CH, two CH₂ and eleven quaternary carbons. The correlations of ¹H-¹H and ¹H-¹³C were deduced by ¹H-¹H COSY and HSQC experiments, which pointed to two aromatic rings and three other fragments separated by quaternary carbons: R_q-CH₂-CH₂R_q′; CH₃R_q″; R_q‴-CH=R_q‴. The interconnection of these fragments was established through HMBC experiments (Fig. [1]). The phenyl-acryloyl substituent showed nearly identical ¹³C shifts as found in literature [7], [8], [9]. Even with 2D NMR it was not possible to assign exactly the quaternary carbons C-4, C-4a, and C-8a, and the position of the phenyl ring. These positions had to be assigned by comparing the shifts with literature data [10], [11], [12]. Compounds with a phenyl substituent at C-4 show ¹³C-shifts of 112 - 114 ppm for C-3 and 155 - 156 ppm for C-4 [10], [11], [12], while a substituent at C-3 shows ¹³C-shifts of 122 ppm for C-3 and 139 ppm for C-4 [13]. The comparison showed clearly that the phenyl substitution must be at C-4. According to our ¹H-, ¹³C-NMR and MS analyses and the corresponding data given in the literature for similar compounds [10], [11] we propose the structure 5,7-dihydroxy-6-methyl-4-phenyl-8-(3-phenylpropionyl)-1-benzopyran-2-one for the compound (1). A very similar structure, without the double bond at C-3/C-4 and without a methyl group at C-6 has been found in the fern Pityrogramma calomelanos (L.) Link [7].

Interruptin B (2) was obtained as a yellow powder. Its molecular formula was assigned on the basis of EIMS and ¹³C-NMR as C₂₅H₁₈O₅ (m/z 398 [M]⁺). Ions at m/z 91 and 321 [M - 77]⁺ indicated the presence of unsubstituted aromatic rings. Ions at m/z 294 [M + H - 105]⁺ are due to the loss of a phenylethyl moiety and pointed to the same basic structure as compound 1, while m/z 266 [M - H - 131]⁺ confirmed the presence of a side chain with a double bond. The ¹H- and ¹³C-NMR data of 2 (Tables [1] and [2]) were similar to 1, except those recorded for the aliphatic moiety and the presence of a trans-configured double bond (J = 15.5 Hz, H-2′ and H-3′). Therefore upfield shifts of the neighboured protons and carbons could be observed. ¹³C shifts of the carbons C-2′ and C-3′ correspond to shifts of this structure element given in [14]. The structure of compound 2 was finally confirmed by 2D NMR spectroscopic methods as 5,7-dihydroxy-6-methyl-4-phenyl-8-(3-phenyl-trans-acryloyl)-1-benzopyran-2-one.

Interruptin C (3) was identified as the 2′-hydroxy derivative of 1. Its molecular formula was assigned on the basis of FABMS and ¹³C-NMR as C₂₅H₂₀O₆ (m/z 417 [M + H]⁺ and m/z 441 [M + 2H + Na]⁺). The presence of the ion at m/z 295 [M - 121]⁺) confirmed fragmentation of a side chain with a hydroxy substituent and a basic structure similar to 1. All the signals in the ¹H-NMR and ¹³C-NMR spectra were close to those recorded for 1, except for C-2′ and C-3′, which showed ¹H shifts like compounds with an analogous substitution [15]. The structure of compound 3 was confirmed by 2D NMR spectroscopic methods as 5,7-dihydroxy-8-(2-hydroxy-3-phenylpropionyl)-6-methyl-4-phenyl-1-benzopyran-2-one. Compound 3 is very unstable and was polluted with decomposition products. Therefore physical data (except for UV) were not recorded. Because of its rapid decomposition (after 24 h the pure compound 3 was completely decomposed) biological assays could not be performed either.

Interruptin D (4) was obtained as small white crystals. Its molecular formula was assigned by FABMS and ¹³C-NMR as C₂₅H₁₈O₆ (m/z 415 [M + H]⁺ and 397 [M + H - H₂O]⁺). The occurrence of an ion at m/z 295 indicated the presence of a coumarin derivative with the same basic structure as described for compounds 1 - 3. The signals recorded in the NMR spectra were very similar to those of compounds 1 - 3, except for the signal for a quaternary carbon at position 2′, a different coupling of the protons at C-3′, and small differences in the chemical shifts. Due to MS data, the presence of a second hydroxy group at C-2′ was excluded, and ring closure from C-2′ to C-7 was supposed, building a furanocoumarin. The NMR shifts of this furan ring region were nearly identical to data found in literature [16], [17]. The structure of compound 4 was then confirmed by 2D NMR spectroscopic methods as 8-benzyl-5,8-dihydroxy-6-methyl-4-phenylfuro[2,3-h]-1-benzopyran-2,9-dione.

Interruptin E and F (5 and 6) were obtained as a mixture, showing only one spot on TLC (Solvent: EtOAc-MeOH [9 : 1]; R_f = 0.7) and the same molecular formula C₂₅H₂₀O₆, deduced from the EIMS and ¹³C-NMR. In the EIMS only the ion at m/z 398 [M - H₂O]⁺ could be found, while [M]⁺ was not detectable. This could be explained through the instability of the vicinal hydroxy groups, which can easily split off an H₂O molecule. From the presence of the ion at m/z 294 [M - 122]⁺, it could be supposed, that they have the same basic structure as the other compounds isolated. ¹³C-NMR spectra showed the presence of a quaternary carbon at C-2′ and of a tertiary carbon at C-1′ for each compound, which proved the compounds to be similar to compound 4, without a carbonyl at C-1′. This was also confirmed by NMR spectra, which showed great similarity to the corresponding spectra of the other compounds. Protons of the furan ring and the benzyl substituent showed the same shifts as reported in literature for such structural elements [15]. The structures of compounds 5 and 6 were finally confirmed by 2D NMR spectroscopic methods as 8-benzyl-5,8β,9β-trihydroxy-6-methyl-4-phenyl-8,9-dihydrofuro[2,3-h]-1-benzopyran-2-one (5) and 8-benzyl-5,8β,9α-trihydroxy-6-methyl-4-phenyl-8,9-dihydrofuro[2,3-h]-1-benzopyran-2-one (6).

The probable protonation of the carbonyl group in compound 4 at C-1′ building α- and β-hydroxy groups, could be an explanation for the presence of both stereoisomers 5 and 6, which could not be separated with usual chromatographic methods.

Interruptin G (7) was identified as an ethylene-dioxo derivative with the molecular formula C₂₇H₂₂O₇ (m/z 460 [M + 2H]⁺). A fragment at m/z 398 [M - 60]⁺ suggested the presence of an ethylene-dioxo substituent. In the ¹H-NMR spectrum this compound showed two triplets at 3.49 ppm (2H, J = 5.2 Hz) and 3.41 ppm (2H, J = 5.3 Hz) corresponding to the CH₂ protons at C-10′ and C-11′. The ¹³C and ¹H shifts of these two C-atoms (60.2 and 72.3 ppm) are in accordance with values found in the literature for this type of compounds [18], [19]. The structure of compound 7 was then confirmed by 2D NMR spectroscopic methods as 5,11-dihydroxy-6-methyl-4-phenyl-11-(1-phenylmethyl)-7,10-dioxocane[5,6-h]-1-benzopyran-2, 12-dione.

Except for interruptin D (4), which showed extremely low antibacterial activity, the other tested compounds showed moderate to strong biological activity (either antibacterial or cytotoxic). Interruptin A and B (1 and 2) showed moderate activity against KB cells. These compounds do not contain a third ring in the basic structure, indicating that the absence of this structure element is necessary for the cytotoxicity. Ring closure (4 - 7) decreases the activity against KB cells significantly.

Interruptin A (1) showed strong antibacterial activity against Bacillus cereus and Staphylococcus epidermidis. It is even more active than the reference compound chloramphenicol, while interruptin B (2) showed no activity at 64 ppm. This clearly indicates that the mechanism of action of the compounds in bacteria and KB cells is not general toxicity. The other tested compounds showed moderate antibacterial activity, compared to interruptin A (1).

Since we found furanocoumarin derivatives and their corresponding ring-opened structures, it would be interesting to know more about the biosynthesis of these compounds in ferns.[]

Acknowledgements

This work was supported by the Swiss National Science Foundation. We thank Pius Piskaut (University of Papua New Guinea) for collection, Dr. M. Baltisberger (ETH Zurich) and Dr. M. M. J. van Balgooy (Rijksherbarium, Leiden, The Netherlands) for identification of plant material. Thanks are also due to Dr. O. Zerbe (ETH Zurich) for assisting in the NMR measurement, Mr. R. Häfliger and Mr. O. Greter (ETH Zurich) for recording the mass spectra, Mr. M. Wasescha (ETH Zurich) for KB cell assays and Dr. E. Zass (ETH Zurich) for performing literature searches.

References

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Prof. Dr. O. Sticher

Institute of Pharmaceutical Sciences

ETH Zurich

Winterthurerstr. 190

8057 Zürich

Switzerland

Email: sticher@pharma.anbiethz.ch

Phone: +41 1 635 6882

Fax: +41 1 635 6050

References

• 1 Holdsworth  D K,, Rali  T.. A survey of medicinal plants of the southern highlands, Papua New Guinea.  International Journal of Crude Drug Research. 1989;;  27 1-8
  PubMedSearch in Google Scholar
• 2 Holdsworth  D K.. A phytochemical survey of medicinal plants in Papua New Guinea. Part I.  Science in New Guinea. 1974;;  2 142-54
  PubMedSearch in Google Scholar
• 3 Webb  L J.. Some new records of medicinal plants used by the aborigines of tropical Queensland and New Guinea.  Proceedings of the Royal Society of Queensland. 1959;;  71 103-10
  PubMedSearch in Google Scholar
• 4 Rios  J L,, Recio  M C,, Villar  A.. Screening methods for natural products with antimicrobial activity: a review of literature.  Journal of Ethnopharmacology. 1988;;  23 127-49
  CrossrefPubMedSearch in Google Scholar
• 5 Frost  J A.. Testing for resistance to antimicrobial drugs. In: Methods in Practical Laboratory Bacteriology,. Chart H, editor 73-82, CRC press, Boca Raton, Ann Arbor, London, Tokyo,; 1994
  Search in Google Scholar
• 6 Swanson  S M,, Pezzuto  J M.. Bioscreening technique for cytotoxic potential and ability to inhibit macromolecule biosynthesis. In: Drug Bioscreening. Drug Evaluation Techniques in Pharmacology,. Thompson EB, editor 273-7, VCH, New York, Weinheim, Basel, Cambridge,; 1990
  Search in Google Scholar
• 7 Asai  F,, Iinuma  M,, Tanaka  T,, Mizuno  M.. Complex flavonoids in farinose exudate from Pityrogramma calomelanos. .  Phytochemistry. 1991;;  30 3091-3
  CrossrefPubMedSearch in Google Scholar
• 8 Hufford  C D,, Oguntimein  B O.. Dihydrochalcones from Uvaria angolensis. .  Phytochemistry. 1980;;  19 2036-8
  CrossrefPubMedSearch in Google Scholar
• 9 Kaouadji  M.. Two C-methyl-C-prenyldihydrochalcones from Platanus acerifolia. .  Phytochemistry. 1989;;  28 3191-2
  CrossrefPubMedSearch in Google Scholar
• 10 Palmer  C J,, Josephs  J L.. Synthesis of the Calophyllum coumarins. Part 2.  Journal of the Chemical Society. Perkin Transactions. 1995;;  1 3135-52
  PubMedSearch in Google Scholar
• 11 Gustafson  K R,, Bokesch  H R,, Fuller  R W,, Cardellina  J H, II,, Kadushin  M R,, Soejarto  D D et al.. Calanone, a novel coumarin from Calophyllum teysmannii. .  Tetrahedron Letters. 1994;;  35 5821-4
  CrossrefPubMedSearch in Google Scholar
• 12 Mata  R,, Calzada  F,, Del Rosario Garcia  M.. Chemical studies on Mexican plants used in traditional medicine, VI. Additional new 4-phenylcoumarins from Exostema caribaeum. .  Journal of Natural Products. 1988;;  51 851-6
  CrossrefPubMedSearch in Google Scholar
• 13 Fukai  T,, Zeng  L,, Nishizawa  J,, Wang  Y-H,, Nomura  T.. Four isoprenoid-substituted flavonoids from Glycyrrhiza aspera. .  Phytochemistry. 1994;;  36 233-6
  CrossrefPubMedSearch in Google Scholar
• 14 Barrero  A F,, Herrador  M M,, Arteaga  P,, Rodriguez-Garcia  I,, Garcia-Moreno  M.. Resorcinol derivatives and flavonoids of Ononis natrix subsp. ramosissima. .  Journal of Natural Products. 1997;;  60 65-8
  CrossrefPubMedSearch in Google Scholar
• 15 Gomez-Garibay  F,, Chilpa  R R,, Quijano  L,, Pardo  J SC,, Castillo  T R.. Methoxy-furan auranols with fungistatic activity from Lonchocarpus castilloi. .  Phytochemistry. 1990;;  29 459-63
  CrossrefPubMedSearch in Google Scholar
• 16 Li  X-C,, Lining  C,, Wu  C D.. Antimicrobial compounds from Ceanothus americanus against oral pathogens.  Phytochemistry. 1997;;  46 97-102
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Prof. Dr. O. Sticher

Institute of Pharmaceutical Sciences

ETH Zurich

Winterthurerstr. 190

8057 Zürich

Switzerland

Email: sticher@pharma.anbiethz.ch

Phone: +41 1 635 6882

Fax: +41 1 635 6050